Title of Invention	A METHOD OF UTILIZING RDL1 GENE TO PROMOTE SEED ENLARGEMENT AND COTTON FIBER ELONGATION
Abstract	Use of the plant RDL1 gene or RDL1 protein in improving the properties of crop seeds and the method thereof. Specifically, a gene engineering method of utilizing the plant RDL1 gene to promote seed enlargement and cotton fiber elongation. Also, the vector and the host cell comprising the RDL1 gene, and the method for preparing the transgenic plant, and obtaining the seed with improved properties using thereof are provided. The transgenic plant comprising the RDL1 gene, and the hybrid progeny obtained by crossing the transgenic plants and the non-transgenic plants or other transgenic plants. The method can increase the seed size, the seed weight, the seed fiber and/or the seed fiber intensity, and is valuable for improving the crop yield and properties, and has widely application prospect.

Title of Invention

A METHOD OF UTILIZING RDL1 GENE TO PROMOTE SEED ENLARGEMENT AND COTTON FIBER ELONGATION

Abstract

Use of the plant RDL1 gene or RDL1 protein in improving the properties of crop seeds and the method thereof. Specifically, a gene engineering method of utilizing the plant RDL1 gene to promote seed enlargement and cotton fiber elongation. Also, the vector and the host cell comprising the RDL1 gene, and the method for preparing the transgenic plant, and obtaining the seed with improved properties using thereof are provided. The transgenic plant comprising the RDL1 gene, and the hybrid progeny obtained by crossing the transgenic plants and the non-transgenic plants or other transgenic plants. The method can increase the seed size, the seed weight, the seed fiber and/or the seed fiber intensity, and is valuable for improving the crop yield and properties, and has widely application prospect.

Full Text	A METHOD OF UTILIZING RDL1 GENE TO PROMOTE SEED ENLARGEMENT AND COTTON FIBER ELONGATION BACKGROUND OF INVENTION Field of the Invention The invention relates to plant bioengineering and plant gene improvement engineering. Especially, the invention relates to isolation of a specific cotton fiber gene RDL1 cDNA and construction of over-expression vectors, and methods for increasing the sizes of the seeds and the length of fibers of transgenic plant by RDL1 gene transfection. Background Art Seeds are important materials in agriculture and industries, relating to oil, cotton and food. The characteristics of seed directly determine the quality of seeds and products made from seeds. These characteristics mainly include seed size, seed weight, seed fiber length (for utility of seed fibers) and/or seed fiber strength. Cotton is an important crop in economy. Cotton fibers are important raw materials in the textile industry. In 2006, the cotton output in the world was 25,220,000 tons, among which China produced 6,730,000 tons. The textile industry increasingly demands higher quality cotton fibers. For example, the cotton fibers are desired to be longer, stronger, thinner and more homogeneous. Therefore, it is important to increase both the quality and quantity of cotton products. Developmental processes of cotton fibers are highly regulated. One of the main objectives of cotton breading research includes finding ways to increase the quality of cotton fibers. Cotton fibers are unicellular fibers differentiated and developed from ovule epidermal cell. The developmental process are divided into four stages: fiber dvelopment initiation, elongation, secondary wall biosynthesis, and maturation, in which the elongation stage and the secondary wall biosynthesis stage partially overlap. During these 4 stages, the morphology and structure changes of fiber cells changes are accompanied by important physiological and biochemical processes. During these processes, many genes participate in the regulation of fiber development. Therefore, it is important to study the expression and regulation of these genes. It has been found that many cotton strains have genes that are highly homologous to Arabidopsis RD22, such as GhRDL1 (Grossypium hirsutum RD22-like). GhRDL1 is specifically and highly expressed during the fiber elongation stage. The gene encodes a protein having 335 amino acids. This protein is highly homologous with Arabidopsis RD22 protein. The C-terminal region of GhRDL1 protein contains a plant-specific BURP kringle domain. However, the function of BURP domain is not well studied. Therefore, there is a urgent need to develop methods that could effectively improve the characteristics of plant seeds, thereby enhancing the quality and quantity of the seeds of food, cotton and oil crops. SUMMARY OF INVENTION The objects of the invention include the use of plant RDL1 genes, particularly cotton RDL1 gene, to improve the characteristics of plant seeds, thereby enhancing the quality of the seeds of the crops. The first aspect of the present invention includes a use of plant RDL1 genes or the encoded RDL1 proteins to improve the plant seed characteristics. In accordance with one embodiment of the invention, the plant RDL1 genes is cotton RDL1 genes. In accordance with another embodiment of the invention, the sequence of plant RDL1 gene is selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (b) a molecule that can hybridize, under stringent conditions, with the sequences specified in (a), wherein the molecule can improve plant seed characteristics. In accordance with yet another embodiment of the invention, the sequence of RDL1 protein is selected from: (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) a protein derived from (a) and having substitution, deletion, or insertion of one or more amino acids of the sequences specified in (a), wherein the protein has an activity for improving the plant seed characteristics. In accordance with yet another embodiment of the invention, improvement of plant seed characteristics may include increased seed size, increased seed weight, increased seed fiber length, and/or increased fiber strength. In accordance with yet another embodiment of the invention, the plants may be dicotyledons or monocotyledons. In accordance with a preferred embodiment, the crops or plants may be selected from marram, hibiscusmutabilis, and crucifer; more preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare (kaoliang); even more preferably, cotton or cole; and most preferably, cotton. The second aspect of the present invention includes vectors, wherein the vectors contain plant RDL1 genes. In accordance with one preferred embodiment, the vectors contain cotton RDL1 genes. In accordance with one preferred embodiment, the sequences of the DL1 genes are selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (b) DNA molecules that can hybridize, under stringent conditions, with the DNA sequences specified in (a) and have activities for improving characteristics of crop plant seeds, wherein the DNA molecules include molecules or fragments that have sequences highly homologous to the sequences of cotton RDL1 gene or gene fragments or Arabidopsis RD22 gene or gene fragments. In accordance with a preferred embodiment, the vectors are selected from bacterial plasmids, bacterial phages, yeast plasmids, plant viruses, or mammal viruses; and preferably,EGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121. The third aspect of the invention includes genetically engineered host cells, wherein the host cells harbor vectors in accordance with embodiments of the invention. In accordance with a preferred embodiment, host cells may be selected from prokaryotes, lower eukaryotes, or higher eukaryotes; preferably, bacterial cells, yeast cells, or plant cells; more preferably, Escherichia coli, streptomyces, agrobacteria, and yeasts; and most preferably, agrobacteria. The forth aspect of the present invention includes methods for making transgenic plants. The methods may include: (1) providing vectors containing RDL1 genes; (2) providing host cells that harbor the vectors generated in step (1); (3) contacting plant cells or tissues with the host cells of step (2), the vectors of step (1), or the RDL1 gene, thereby allowing the RDL1 gene to be transfected into the plant cells and integrated into the chromosomes of the plant cells; (4) selecting for plant cells, tissues, or organs having the RDL1 transfected therein; and (5) regenerating/growing the RDL1 transgenic plant cells, tissues, or organs obtained in step (4), wherein the seeds of the transgenic plants may have improved characteristics. In accordance with one preferred embodiment, the RDL1 genes are cotton RDL1 genes. In accordance with one preferred embodiment, the host cells are agrobacteria. In accordance with yet another preferred embodiment, the crop plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare; more preferably, cotton or cole; and most preferably cotton. The fifth aspect of the invention includes uses of transgenic crop plants generated by the methods of the invention. The transgenic plants may be used to produce crop plant seeds having improved characteristics. In accordance with one preferred embodiment, the improved characteristics may include increased seed sizes, increased seed weights, increased seed fiber lengths, and/or increased fiber strengths. In accordance with another preferred embodiment, the plant may be cotton. The sixth aspect of the present invention includes methods for producing crop plant seeds having improved characteristics, wherein the methods may include increasing the expression levels of RDL1 gene in such crop plants. In accordance with one preferred embodiment, RDL1 gene may be transfected into the crop plants to increase the expression levels of RDL1 gene. In accordance with another preferred embodiment, the methods may include the following steps: (i) transfecting, using transgenic methods, RDL1 gene into plants and allowing the transfected gene to integrate into plant chromosomes; (ii) selecting for RDL1 transgenic plant cells, tissues or organs; and (iii) regenerating/growing the RDL1 transgenic plant cells, tissues or organs obtained from step (ii) to produce plants seeds with improved characteristics. In accordance with another preferred embodiment, the improved characteristics may include increased seed sizes, increased seed weights, increased fiber lengths, and/or increased fiber strengths. In accordance with yet another preferred embodiment, the plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare, more preferably, cotton or cole, and most preferably, cotton. In accordance with one embodiment, the methods include producing transgenic plants having improved seed characteristics and obtaining seeds therefrom, by using methods of the invention. In accordance with another embodiment, the methods include producing transgenic plants having improved seed characteristics by using methods of the invention and crossing such transgenic plants with non-transgenic plants or other transgenic plants to produce hybrid plants. Then, select for the hybrid plants having improved seed characteristics and obtain seeds from such hybrid planta. The seventh aspect of the invention includes transgenic plants that harbor plant RDL1 genes. In accordance with one embodiment, the sequence of RDL1 gene is selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (b) molecules that can hybridize, under stringent conditions, with the sequences specified in (a), and have activities for improving the characteristics of seeds, wherein the DNA molecules include molecules or their fragments that have sequences highly homologous to those of cotton RDL1 gene or gene fragments or Arabidopsis RD22 gene or gene fragments. In accordance with another embodiment, the transgenic plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, hibiscusmutabilis or crucifer; more preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare. The eighth aspect of the invention includes methods for generating plants. The methods may include crossing the transgenic plants, obtained by using methods of the invention, with non-transgenic plants or other transgenic plants to produce hybrid plants containing plant RDLlgene. In accordance with one embodiment, the transgenic plants and the non-transgenic plants, or other transgenic plants, used in the crossing may belong to the same or different families, preferably the same plant family. The said plants are preferably selected from marram, hibiscusmutabilis or crucifer; more preferably, hibiscusmutabilis or crucifer; most preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare. In accordance with another embodiment, the hybrid offsprings have stable genetic characteristics. Preferred embodiments of the invention use cotton RDL1 gene or its encoded RDL1 protein. Other aspects of the present invention would be apparent to one skilled in the art, based on this description. Brief Description of Drawings: FIG 1 shows the construction of GFP-GhRDL1 vector. FIG 2 shows molecular characterization of cotton transfected with GFP-GhRDL1. FIG 3 shows subcellular localization of GhRDL1 using GFP-GhRDL1 fusion protein, wherein A, 35S::GFP/GhRDLl, 2 DPA fibers; B, R 15, 2 DPA fiber; C, 35S::GFP-GhRDLl, leaftrichomes. FIG 4 shows weight (100 seeds with fibers) analysis of GFP/GhRDL1 transgenic cotton seeds (T2 generation), wherein A, morphological comparison of seeds; B, weight comparison of seeds. FIG 5 shows size comparison of Arabidopsis seeds transfected with GhRDL1. FIG 5A is a morphological comparison of seeds; FIG 5B is comparison of seed lengths and seed widths (Bar = 500 urn). DETAILED DESCRIPTION After long-term in-depth study of the structure, function, and location of RDL1 gene, the inventors constructed transgenic vectors containing RDL1 gene. By using plant transgenic technology, the inventors obtained new plants with improved seed characteristics, such as transgenic cotton having increased seed weight and increased seed fiber length. The methods also produce transgenic Arabidopsis with increased seed sizes. The above observations indicate that RDL1 gene affects seed characteristics. The seeds, obtained from the transgenic plants expressing high levels of RDL1 gene, exhibit improvements, such as increased seed sizes, increased weights, increased fiber lengths, increased fiber strengths, and so on. Based on these, the inventors complete the present invention. For example, the inventors use the subcellular localization of RDL1-GFP fusion protein to show that GhRDL1 protein may be localized at certain areas of cell wall with polarized distributions. The results show that cell wall edges, marked by green fluorescence signals, are filled with the pectin-rich polysaccharide. In view of the main components of the primary cell wall also contain pectin during the rapid cotton fiber elongation stage, these data suggest that the specific localization of GhRDL1 may be linked to the pectin in cell wall. In addition, BURP and GFP fusion proteins exhibit similar localization characteristics. The inventors then use yeast two-hybrid methods to identify that GhEXPA1 protein (Gossypium hirsutum a-expansin 1) is a GhRDL1-interacting protein. By co- transfecting GFP-GhRDL1 and RFP-GhEXPA1 fusion proteins into Arabidopsis, followed by examining the root cells of Arabidopsis using confocal microscopy. The results confirm this protein-protein interaction by showing co-localization of fluorescent signals focused on the cell wall. Subsequent Co-IP experiments also confirm the interactions between these two proteins. To study the functions of GhRDL1, the inventors use agrobacteria-mediated methods to transfect 35S::GFP-GhRDLl into cotton. The results show the seed sizes of the transgenic cotton are significantly larger than the control. A hypocotyl from a 6d old cotton seedling of T3 generation was used to analyze mechanical characteristics of tissues before and after gene transfer. The results show the average force needed for breaking the hypocotyl is significantly greater in transgenic plants than that in the control. The statistical results obtained from T3-generation plants growing in the fields show average flower buds of most transgenic plants are heavier than the control. In addition, transgenic plants have increased weights (100 seeds) and increased fiber lengths as compared with the control. The above results show that over-expression of GFP-GhRDL1 fusion protein in cotton may improve the development of cotton fibers and ovules, and thus, it is valuable for cotton improvement. RDL1 gene and its encoded protein In this description, the term "plant RDL1 gene" or "RDL1 gene" is used interchangeably and means a gene that encodes a protein that has a sequence highly homologous to that of the cotton RDL1 protein, or molecules that can hybridize, under stringent conditions, with the above-noted gene sequences, or gene family molecules that are highly homologous to the above-noted molecules. The expression of the above said genes can improve seed characteristics, such as increased seed sizes, increased seed weights, increased seed fiber lengths, and/or increased seed fiber strengths. This definition also includes molecules that can hybridize, under stringent conditions, with the sequences of cotton RDL1 gene or other highly homologous gene family molecules. The term "cotton RDL1 gene" means a gene encoding a protein having a sequence j highly homologous to the sequence of Arabidopsis RD 22 protein. This definition also includes molecules that can hybridize, under stringent conditions, with the sequence of cotton RDL1 gene, or other highly homogenous gene family molecules. The above genes are the ones specifically over-expressed in the cotton fiber elongation stage. For example, land cotton (Grossypium hirsutum) RDL1 gene (GhRDL1) encodes a protein having 355 amino acids, which contains a plant-specific BURP domain at the C-teraiinus. NCBI discloses the sequences of RDL1 gene and its sequence homologs, such as AY072821 [(Li C-H, Grossypium Tursutum dehydration-induced protein RD22-like protein (RDL) mRNA, complete cds; AY641990 [Wang S, Grossypium arboreum dehydration-induced protein RD22-like protein 1(RDL1) mRNA, RDL1-1 allele, complete cds; and AY641991 [Wang S, Grossypium arboreum dehydration-induced protein RD22-like protein 2(RDL2) mRNA, RDL2-2 allele, complete cds. These genes mentioned above are included in the present invention. The RDL1 gene in accordance with embodiments of the present invention may be selected from (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ED NO: 5 (each corresponding to AY072821, AY641990, and AY641991, respectively); or (b) functionally active molecules that can hybridize, under stringent conditions, with the sequences specified in (a) and can improve the characteristics of plant seeds. The term "under stringent conditions" means (1) hybridization and wash under conditions of low ionic strengths and high temperatures, such as 0.2X SSC, 0.1% SDS, 60°C; or (2) hybridization in the presence of denaturants, such as 50% (v/v) formamide, 0.1 % calf serum/0.1% Ficoll at 42^, and so on; or (3) the sequence identity between two sequences may be at least 50%; preferably, > 55% > 60%, > 65%, > 70%, > 75%, > 80%, > 85%, or 90%; more preferably, > 95%, when hybridization takes place. For example, the above sequences may be complementary to the sequences specified in (a). The full-length nucleotide sequence of RDL1 gene or its fragments may be readily obtained using PCR amplification techniques, recombinant DNA techniques, or artificial synthesis. With regard to using PCR amplification to obtain related sequences, the appropriate primers may be designed based on the sequences disclosed in the present invention, especially the open reading frame sequences, and the templates may be a i commercial cDNA library or a cDNA library prepared using conventional methods known to one skilled in the art. When the sequences are long, two or more PCR amplification reactions may be performed, followed by ligation of these amplified fragments in proper order. The RDL1 gene in accordance with embodiments of the present invention may be selected from cotton or other plants which may contain genes having high sequence homology with cotton RDL1 gene (for example, at least 50%; or preferably, > 55%, > 60%, > 65%, > 70%, > 75%, > 80%; more preferably, > 85%, > 90%, > 95%; or even 98% in sequence identity). These genes are also considered within the scope of the present invention. The tools and methods for sequence identity or homology comparison are well known in the art, such as BLAST. In this description, the term "RDL1 protein" refers to a polypeptide encoded by the RDL1 gene. This definition includes mutant polypeptides that can also improve seed characteristics. The proteins of the invention may be purified from natural sources, or obtained from chemical synthesis, or obtained from prokaryotic or eukaryotic host cells (such as bacteria, yeasts, plants, insects, or mammals) using recombinant DNA techniques. The proteins of the invention may be encoded by genes selected from cotton RDL1 gene or other homologous genes or gene families. The protein sequences of RDL1 may be selected from (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) protein sequences derived from the amino acid sequences specified in (a) with substitution, deletion, or insertion of one or more amino acids, which have activities for improving seed characteristics. The said mutations include (but not limited to): one or more (usually, 1-50; good, 1-30; better, 1-20; best, 1-10; such as, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletion, insertion, and/or substitution; and insertion at the C-terminus and/or at N-terminus of one or more (usually, To obtain the above (b) proteins, radiation or mutagens may be used to induce random mutations. Alternatively, the mutant proteins may be obtained by performing site-directed mutagenesis or other known molecular biology techniques. The protein sequences can be used to construct the transgenic plants. Then, monitoring whether seed characteristics have been improved to select and identify the proteins (for example, referring to the methods described in the present invention). Depending on the host cells used in the recombinant DNA production process, the present proteins may be glycosylated or non-glycosylated. This term may also include the active fragments or functionally active derivatives of the RDL1 proteins. The mutant proteins include those having: homologous sequences, conservative mutations, allelic variants, natural mutants, induced mutants, sequences capable of hybridizing, under high or low stringent conditions, with the sequences encoding RDL1 proteins, the proteins or polypeptides obtained by using anti-serum against RDL1 proteins. In addition, other polypeptides, such as fusion proteins containing RDL1 protein or other RDL1 protein fragments may be used as well. In addition to the near full-length polypeptides, embodiments of the present invention may include soluble fragments of RDL1 proteins. In general, the protein fragments usually contain at least 10 continuous amino acids of the RDL1 protein sequences; usually, at least 30 continuous amino acids; preferably, at least 50 continuous amino acids; more preferably, at least 80 continuous amino acids; most preferably, at least 100 continuous amino acids. Plant seeds and their characteristics: The term "plant" refers to the plants which have economic values in the food, cotton and oil industries. The economic values are mostly based on their seeds. The plants include, but not limited to, dicotyledons or monocotyledons. The preferred monocotyledon is marram; more preferably, rice, wheat, barley, corn, and sorghum vulgare. The preferred dicotyledons include, but not limited to, malvaceae gossypium plants and brassicaceae brassica, etc.; more preferably, cotton or cole, etc.; most preferably, cotton. The seed characteristics in accordance with embodiments of the present invention include, but not limited to, seed sizes, seed weights, seed fiber lengths, and/or seed fiber strengths. Improvements of seeds characteristics refer to improvements having increased sizes, increased weights, increased fiber lengths, and/or increased fiber strengths, as compared with the seeds prior to improvements In this description, the terms "seed index" and "100 seeds weight" are interchangeable, referring to the weight per 100 seeds. These data indicate the grain sizes and the degree of fullness of seeds. Embodiments of the present invention also provide methods for producing plant seeds having improved characteristics. The methods may include increasing the expression levels of the said RDL1 gene (preferably, cotton RDL1 gene). The improvements may be achieved by increasing the expression levels of plant RDL1 gene or increasing the quantity of the RDL1 proteins. One skilled in the art may, based on particular purposes, select appropriate improvement methods, such as transfection methods. These methods usually include steps of constructing transfection vectors containing RDL1 gene and transfecting them into plants and plant clones, etc. Vectors, host cells, and transgenic plants The present invention also relates to vectors containing RDL1 gene and host cells harboring said vectors produced by genetic engineering, and RDL1 over-expressing transgenic plants obtained using transgenic technology. Using the conventional recombinant DNA techniques (Science, 1984; 224: 1431) and the sequences disclosed in the present invention, the recombinant RDL1 proteins can be obtained. The method may generally include the following steps: (1) transforming or transfecting appropriate host cells with the polynucleotides (or variants) encoding RDL1 proteins in accordance with embodiments of the present invention or with the recombinant expression vectors containing said polynucleotides; (2) incubating host cells in suitable culture media; and (3) isolating and purifying the proteins from the media or the host cells. In this description, the terms "vector' and "recombinant expression vector" may be used interchangeably, referring to bacterial plasmids, bacterial phages, yeast plasmids, plant cell viruses, mammal cell viruses or other vectors well known in the art. In short, any plasmids or vectors may be used as long as they can replicate and are stable in the host cells. Important features of expression vectors include the presence of replication origins, promoters, selection marker genes, and translational control elements. One skilled in the art would know how to construct expression vectors that contain RDL1 gene sequence and suitable transcriptional/translational control elements. These methods include in vitro recombination DNA technology, DNA synthesis techniques, and in vivo recombination technology. The said DNA sequences may be efficiently linked to a promoter on the expression vector to direct the synthesis of mRNA. The vector may also include elements containing ribosomal binding sites and transcription terminators. The preferred vectors for use in the present invention may include pEGFP-1, pCAMBIA 1300, pCAMBIA 2301, or pBI121, etc. In addition, the expression vectors may preferably contain one or more selection marker genes to permit selection of the transformed host cell phenotype, such as dihyrofolate reductase used in culturing eukaryotic cells, neomycin resistance, and green fluorescence protein (GFP), or tetracycline- or ampicillin-resistance used in E. coli. The vectors contain the said DNA sequences and a suitable promoter or control elements may be used to selectively transform host cells to express proteins. Host cells may be prokaryotes, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include E. coli, streptomyces, agrobacteria; fungal cells, such as yeasts, plant cells, etc. The preferred host cells for use in the embodiments of the present invention may be agrobacteria. In the case that the polynucleotides expressed in the higher eukaryotic cells, enhancer sequences may be inserted into the vector to increase transcription. Enhancer is a cw-acting DNA element, which usually have 10-300 base pairs acting on the promoters to enhance gene transcription. One skilled in the art would know how to select suitable vectors, promoters, enhancers, and host cells. In this description, the terms "transgenic plants," "transfectants," and "transfectant plants" are used interchangeably, referring to cells, organs, or plants, which contain RDL1 genes and stably express RDL1 proteins, obtained by using conventional gene transfer techniques. Transgenic plants may be obtained by using agrobacteria transfection or gene gun bombardment methods, such as, leaf disc method. The transgenic plants, tissues, or organs may be re-cultivated (re-grown) using conventional methods to obtain plants having high disease resistance. The transfectants may be cultivated using conventional methods to express polypeptides in accordance with the embodiments of the present invention. Depending on the host cells used, various culture media selected from the conventional media may be used to incubate the cells under the conditions suitable for growth. When the growth of host cells reaches appropriate cell density, the promoters can be selectively activated by suitable methods (for example, temperature changes or chemical induction), followed by incubating cells for a period of time. The above recombinant polypeptides may be expressed within the cells, or on the cell membrane, or secreted out of the cells. If necessary, based on the physical, chemical, and other specific features, the recombinant proteins may be isolated and purified using different separation methods. These separation methods are well known to one skilled in the art. These methods may include, but not limited to,: conventional renaturation treatment, protein precipitation treatment (salting-out), centrifugation, osmotic lysis of bacteria, super process, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chromatography (HPLC), other liquid chromatography, and the combination thereof. Advantages of the invention (1) providing new use of RDL1 gene and its proteins to effectively increase the seed quality; (2) providing transgenic plants with improved characteristics, which include improved seed sizes, weights, fiber lengths, fiber strengths, etc., thus, providing excellent raw materials for the food, cotton, and oil industries; and (3) providing novel methods to improve seeds characteristics, which may have wide applications. EXAMPLES The following examples are provided to further illustrate the present invention. It is understood that these examples are only used to illustrate the present invention and not to be construed as limiting the scope of this invention. Experimental conditions not noted in the following examples may be performed according to conventional conditions described in "Molecular Cloning Laboratory Manuel," by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions suggested by the manufacturers. Unless otherwise indicated, percentages and fractions are based on weights. Unless otherwise defined, all special or scientific terms used in the present description have the same meanings as known to one skilled in the art. In addition, any methods or materials, which are similar or equivalent to that shown in the present description, can be used to practice the present invention. The preferred methods and materials shown here are for illustration only. Example 1: Isolation of RDL1 cDNA Preparation of cotton RNA using phenol extraction: Grind 2 g of ovular surface fibers (obtained 9 days after land cotton blossom) into powders in liquid nitrogen. Transfer the powders to a 50 ml centrifugation tube. Add 8 ml of extraction buffer (1 M Tris-HCl, 50 mM EDTA, 1 % SDS, pH 9.0) and equal volumes of water-saturated phenol:chloroform:isoamyl alcohol (25:24:1). Mix and place in ice for 1 hour (mix every ten minutes). Centrifuge the mixture (13000g) at 4°C for 20 minutes. Repeat the phenol:chloroform:isoamyl alcohol extraction step 2 ~ 4 times, followed by chloroform:isoamyl alcohol (24:1) extraction once. Collect the supernatant. Add 1/2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol to the supernatant, mix, and place it in -70°C for 1 hour. Centrifuge the mixture (13000g) at 4C for 20 minutes. Discard the supernatant and dissolve the precipitates in 1 ml DEPC treated-water. Centrifuge the solution (13000g) at 4"C for 10 minutes. Transfer the supernatant to a 1.5 ml Enppendorf tube. Add 1/3 volume of 8 M LiCl and equal volume of NaAC and place it at -20"C overnight. Centrifuge the solution (13000g) at 4°C for 20 minutes. Discard the supernatant and wash the precipitates twice with 1 ml 70 % ethanol. Air-dry the precipitates at room temperature. Dissolve the precipitates in 100~200 uL DEPC treated-water. Based on AY641990 sequence to synthesize a pair of primers (which may contain restriction enzyme cutting sites and protective amines) and use the primers to perform PCR reactions using RDL1 cDNA as templates: RDLl-S-BamHI: S'-CGGGATCCATGAAGGTTCTCTCCCCAATTCTTGCT-S' (SEQ ID NO: 7); RDLl-A-SacI: S'-CCGGAGCTCTTACTTAGGGACCCAAACAATGTG - 3' (SEQ ID NO: 8). Conditions for PCR reactions: pre-denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 30 seconds, renaturation at 56"C for 30 seconds, and extension at 72°C for 1 minute, for a total 35 circles, followed by a final extension at 72 °C for 10 minutes. DNAs of the subclones are then sequenced to determine whether the subclones are correct. The results show that the sequences obtained are identical to AY641990 sequence. Example 2: Construction of RDL1 and GFP fusion vectors and transformation of agrobacteria. The GFP gene may be obtained from pEGFP-1 (Clontech, Palo Alto, USA). Inserting appropriate restriction enzyme sites using suitable PCR methods. Remove stop codons from GFP gene sequence and insert a fragment having a non-overlapping sequence of 6 amine acids (GPGGGG). Use GS1: 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG - 3'(SEQ ID NO:9) and GA1: 5'-GTCCCCCGGGCGTCCTCCTCCTCCTGGTCCCTGGTACAGC TCGTCCATGCC-3(SEQ ID NO: 10) as primers, pEGFP-1 vector as a template, and Pyrobest DNA polymerase (purchased from TAKARA company) to amplify the coding region of GFP gene. Ligate the amplified GFP fragments into pET32c between Eco RV and Nco I sites to generate an intermediate vector, GFP-32c. Use Pyrobest DNA polymerase to amplify the target sequences containing appropriate restriction enzyme sites, e.g., Bam HI and Sac I (see Example 1). After confirmation of the reading frames, use double-enzyme digestion and ligate the fragments to the corresponding cutting sites in GFP-32c intermediate vector. Use Pyrobest DNA polymerase to amplify the fusion fragments containing Xba I and Kpn I enzyme sites. After double-enzyme digestion, the fragments are ligated downstream from the 35S promoter of pCAMBIA2301 to generate 35S:GFP-RDL1 transgenic vector (abbreviation: RG, for the vector map see FIG. 1). Correct sequence is confirmed. Transform agrobacteria using freeze-thaw method. Select a single colony LBA4404 or GV3101 (Invitrogen) into 3 ml LB culture medium (25 fag/ml rifamycin and 50 ug/ml kanamycin (Kan) or gentamycin (Gen), and incubate at 28 °C, 220 rpm overnight. Add 2 ml bacteria liquid to a 50 ml LB culture medium (25 ug/ml Rif and 50 ug/ml Gen), incubate at 28°C, 220 rpm to reach OD6oo = 0.5 (about 6 hours). Place the culture on ice for 30 minutes, and then centrifuge it (5000g) for at 4"C 5 minutes. Re-suspend the bacterial pellet inl0mlof0.15M NaCl and centrifuge (5000g) at 4 °C for 5 minutes. Suspend the bacterial pellet in 1 ml of 20 mM CaCh. Aliquot the bacterial mixture into 50 ul tubes, quick-freeze in liquid nitrogen and keep the competent cells at -70 °C. Mix the vectors containing target genes with 50 ul/tube competent cells and place them on ice for 30 minutes, followed by quick-freeze in liquid nitrogen for 1 minute. Dissolve the bacteria in water bath at 37 °C for 5 minutes. Add 1 ml LB culture medium and incubate at 4"C, 220 rpm, for 2-4 hours. Use 50~ 100 ul of bacterial solution to plate on LB culture medium plates (which contain 25 fig/ml Rif, 50 ng/ml Gen, and 50 u.g/ml Kan or hygromycin (Hyg)). After two days, select a single colony for PCR identification. Example 3. Plant transfection and screening for transgenic progeny a. Transeenes in cotton Culture agrobacteria harboring the vector plasmid in YEB bacteria culture plates containing 50 mg/L kanamycin, 100 mg/L rifampicin, and 300 mg/L streptomycin for 2 to 3 days. Select a single colony and inoculate it in YEB liquid nutrient medium containing the same antibiotics, incubate in suspension culture on a table shaker at 28 °C, 200 rpm/min overnight. Centrifuge the bacteria at 4000 rpm/min for 10 minute. Re-suspend the bacteria pellet in the 1/2MS liquid nutrient medium (containing 30 g/L glucose and 100 umol/L acetosyringone) and allow them to grow until OD600 reaches 0.4 ~ 0.6, which may then be used as inoculants Disinfect, using traditional methods, cotton R15 seeds (parent plants, a wild type of tetraploid land cotton). Place the seeds in 1/2MS0 culture plates [1/2MS salt (purchased from DUCHEFA M0221) + 5 g/L glucose + 7 g/L agarose powder, pH 6.0] in darkness. After 5 ~ 7 days, slice the hypocotyledonary axis of aseptic sprout into 1 cm segments about 1 cm (explants) for subsequent transfection. Submerge the explants in the agrobacteria liquid for 15 ~ 20 minutes, and then transfer them to co-culture plates MSB1 (MS salt + B5 organic compounds (one contains inositol, nicotinic acid, VBI and VB6) + 30 g/L glucose + 0.1 mg/L KT (Cell kinetin) + 0.1 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) + 2.2 g/L Gelrite (a solidifying agent), pH 6.0). Culture the explants in darkness at 22 V for 2 days. Transfer the explants to culture plates MSB2 (MSB1 + 500 mg/L cephalosporin + 80 mg/L kanamycin) to induce callus. The explants regenerate test-tube plantlets through resistance to callus induction, callus proliferation, embryonic callus induction (culture medium MSB3: MS salt + B5 organic compounds + 30 g/L glucose + 2.5 g/L Gelrite, pH 6.0), and embryonic cell development (culture medium MSB4: MS salt + B5 organic compounds + 30 g/L glucose + 1.0 g/L asparagines + 2.0 g/L glutamine + 3.0 g/L Gelrite, pH 6.0; double amount of KNO3 in the MS salt and removal of NH4NO3).' Wait until the test-tube plantlets develop 3 to 4 leaves, transplant them to a pot and grow in a climate control room. b. Transgenes in Arabidopsis Transfect Arabidopsis using floral dip methods (Clough and Bent, 1998, Plant J. 16:735-743). The methods of culturing Agrobacteria are similar to that mentioned above. Centrifuge (4000 rpm/min) bacteria solution for 10 minutes. Re-suspend the bacteria pellet in the 500 ml of 5% sucrose solution containing 0.02% Silwet L-77. Submerge the aboveground parts of the plants in the bacteria liquid for 5 seconds. Lay them flat on a plastic container, keep moisten, and avoid light for 16 ~ 24 hours. Vernalize To-generation seeds at 4C for 2 ~ 4 days. Treat them with 20% bleach water for 15 minutes. Wash them with sterile water 3 ~ 4 times. Suspend them in 0.5% agarose (55 C), lay them in LB culture plates with 0.6% agar (50 ug/ml Kan or Hyg), at 22'C, under continuous light for one week. Transplant the antibiotic-resistant green sprout to a nutritious soil (peat:vermiculite:perlite = 1:1:1) for growth. Example 4. Analysis of transgenic plants using molecular biology techniques a. PCR DNA isolation by cold phenol methods. Take 2 g of young leaves obtained from transgenic cotton (transgenic cotton that uses R15 as receptors). Grind the leaves into powders in liquid nitrogen. Transfer the powders into a 50 ml centrifugation tube. Add 8 ml of extraction buffer (1M Tris-HCl, 50 mM EDTA, 1% SDS, pH 9.0) and equal volumes of water-saturated phenol:chloroform:isoamylol (25:24:1) into the tube. Then mix the liquid and place it on ice for 1 hour. Mix every 10 minutes and centrifuge 13000 g at A°C for 20 minutes. Repeat the extraction step with phenol:chloroform:isoamylol for 2 ~ 4 times, followed by a final extraction with chlorofonndsoamyl alcohol (24:1). Collect the supernatant. Add V2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and V2 volume isoamyl alcohol, mix, and place it at -70°C for one hour. Centrifuge 13000 g at 4°C for 20 minutes and discard the supernatant. Wash the precipitates two times with 1 ml of 70% ethanol, dry the precipitates at room temperature, and dissolve them in 1 ml of sterile water. Centrifuge 13000 g at 4°C for 10 minutes. Collect the supernatant and add 5~ 10 ul RNase (10 mg/ml) followed by incubation at 37 V for 30 minutes. PCR reactions may be carried out using GFP primers (which may be the same ones shown in Example 2). The templates may be pEGFP-1 plasmids. The reaction conditions of PCR may be: pre-denaturization at 94C for 5 minutes; denaturization at 94°C for 30 seconds; renaruration at 56 °C for 30 seconds; extension at 72 °C for 1 minute; and 35 total circles; followed by a final extension at 72 °C for 10 minutes. b. GUS staining analysis Submerge the plant materials in GUS dye solution (100 mM pH 7.0 phosphate buffer, 50 mM K3[Fe(CN)6], 50 mM K4[Fe(CN)6], 10 mM EDTA, 1 mM X-gluc, and 0.1% Triton X-100) at 37 °C for 12 ~ 24 hours. Destain samples in 70% ethanol. Preserve samples in 70% ethanol. Example 5. Analyzing the characteristics of transgenic plants a. Analyzing the characteristics of transgenic cotton Plant RDL1 transgenic cotton and the parental R15 at different locations: Plant clone No. 105 and plant clone No. 117 are planted in Shanghai. Plant clone No.l 15 and plant clone No. 119 are planted in Hainan. Collect ripe cotton balls from each T2-genetation transgenic plants. The positions from which the cotton balls are collected are kept constant as much as possible. 100 seeds are randomly collected from each ripe cotton balls from individual plants. More than 10 seeds are obtained and their fibers are combed down to measure the length of each fiber. Weigh the 100 seeds (which may or may not contain fibers). The results are summarized in a chart. The results of T3-generation of plant clone No.1 15 and plant clone No. 119 in Hainan are obtained from 5 plants. As shown in FIG. 4, plant clone No. 115 and plant clone No. 117 of T2-generation in Hainan have a weight (100 seeds) (containing fibers) of 19 g and the weight (100 seeds) of the parental R15 is 16 g. The difference in weight between them is statistically significant (p Table 1. Analysis of fiber length and seed index of GFP-GhRDL1 transgenic cotton b. Analyzing the characteristics of transgenic Arabidopsis Transfect GhRDL1 vectors (without GUS genes) into Arabidopsis using Agrobacteria as described above. Obtain transgene-positive plant clones through antibiotic-resistance screening and RT-PCR. Collect seeds from the next generation and the WT seeds. Measure the length and the width of seeds (n > 50) under a dissecting microscope with 20X magnifications. As shown in FIG. 5, there is a statistically difference (p All documents mentioned in this invention are used as references in a manner mat every reference is used as a stand-alone reference. It is also understood that one skilled in the art having read the disclosure of the present invention could modify and change the present invention. These modification and changes may fall within the scope of the present invention, which is limited only by the attached claims. Claims 1. Use of a plant RDL1 gene or its encoded RDL1 protein for improving plant seed characteristics. 2. The use of claim 1, characterized in that the sequence of the plant RDL1 gene is selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (b) a molecule that hybridizes, under a stringent condition, with the sequence specified in (a), wherein the molecule has an activity for improving the plant seed characteristics. 3. The use of claim 1, characterized in that the sequence of the RDL1 protein is selected from: (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) a protein derived from (a) having substitution, deletion, or insertion of one or more amino acids of the sequence specified in (a), and having an activity for improving the plant seed characteristics. 4. The use of claim 1, characterized in that the improved seed characteristics comprise an increased seed size, an increased seed weight, an increased seed fiber length, and/or an increased seed fiber strength. 5. The use of claim 1, characterized in that the plant is a dicotyledon or a monocotyledon. 6. A vector, characterized in that the vector comprises a plant RDL1 gene. 7. A genetically engineered host cell, characterized in that the host cell harbors the vector of claim 6. 8. A method for producing a transgenic plant, characterized in that the method comprises: (1) providing a vector containing an RDL1 gene; (2) providing a host cell harboring the vector described in step (1); (3) contacting a plant cell or a tissue with the host cell from step (2) or with the vector from step (1) or the RDL1 gene contained therein, causing the RDL1 gene to be transferred into the plant cell and allowing the RDL1 gene to integrate into chromosomes of the plant cell; (4) selecting for a RDL1 transgenic plant cell, tissue, or organ; and (5) regenerating or growing a transgenic plant from the plant cell, tissue, or organ obtained in step (4), wherein seeds produced by the transgenic plant have improved characteristics. 9. Use of the transgenic plant produced by the method of claim 8, characterized in that the transgenic plant is used to produce plant seeds having improved characteristics. 10. A method for producing plant seeds having improved characteristics, comprising increasing expression level of RDL1 gene in a plant. 11. The method of claim 10, comprising: producing a transgenic plant using the method of claim 8; and obtaining seeds from the transgenic plant. 12. The method of claim 10, comprising: generating a transgenic plant using the method of claim 8; crossing the transgenic plant with a non-transgenic plant or another transgenic plant to obtain hybrid offsprings; selecting a hybrid offspring having improved seed characteristics; and obtaining seeds from the selected hybrid offspring. 13. A transgenic plant, characterized in that a gene transfected therein comprises a plant RDL1 gene. 14. The transgenic plant of claim 13, characterized in that the sequence of the RDL1 gene is selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (b) a molecule that hybridizes, under a stringent condition, with the sequence specified in (a), and having an activity for improving plant seed characteristics. 15. The transgenic plant of claim 13, characterized in that the plant is marram, hibiscusmutabilis or crucifer. 16. The transgenic plant of claim 13, characterized in that the plant is a hibiscusmutabilis crop plant or a crucifer crop plant. 17. The transgenic plant of claim 13, characterized in that the plant is selected from the group consisting of cotton, cole, rice, wheat, barley, corn, sorghum vulgare, and arabidopsis. 18. A method for producing a plant, comprising crossing the transgenic plant of claim 13 with a non-transgenic plant or another transgenic plant to produce a hybrid plant harboring the RDL1 gene.

Full Text

A METHOD OF UTILIZING RDL1 GENE TO PROMOTE SEED ENLARGEMENT AND COTTON FIBER
ELONGATION

BACKGROUND OF INVENTION
Field of the Invention
The invention relates to plant bioengineering and plant gene improvement engineering. Especially, the invention relates to isolation of a specific cotton fiber gene RDL1 cDNA and construction of over-expression vectors, and methods for increasing the sizes of the seeds and the length of fibers of transgenic plant by RDL1 gene transfection.

Background Art
Seeds are important materials in agriculture and industries, relating to oil, cotton and food. The characteristics of seed directly determine the quality of seeds and products made from seeds. These characteristics mainly include seed size, seed weight, seed fiber length (for utility of seed fibers) and/or seed fiber strength.

Cotton is an important crop in economy. Cotton fibers are important raw materials in the textile industry. In 2006, the cotton output in the world was 25,220,000 tons, among which China produced 6,730,000 tons. The textile industry increasingly demands higher quality cotton fibers. For example, the cotton fibers are desired to be longer, stronger, thinner and more homogeneous. Therefore, it is important to increase both the quality and quantity of cotton products. Developmental processes of cotton fibers are highly regulated. One of the main objectives of cotton breading research includes finding ways to increase the quality of cotton fibers.

Cotton fibers are unicellular fibers differentiated and developed from ovule epidermal cell. The developmental process are divided into four stages: fiber dvelopment initiation, elongation, secondary wall biosynthesis, and maturation, in which the elongation stage and the secondary wall biosynthesis stage partially overlap. During these 4 stages, the morphology and structure changes of fiber cells changes are accompanied by important physiological and biochemical processes. During these processes, many genes participate in the regulation of fiber development. Therefore, it is important to study the expression and regulation of these genes.

It has been found that many cotton strains have genes that are highly homologous to Arabidopsis RD22, such as GhRDL1 (Grossypium hirsutum RD22-like). GhRDL1 is specifically and highly expressed during the fiber elongation stage. The gene encodes a protein having 335 amino acids. This protein is highly homologous with Arabidopsis RD22 protein. The C-terminal region of GhRDL1 protein contains a plant-specific BURP kringle domain. However, the function of BURP domain is not well studied.

Therefore, there is a urgent need to develop methods that could effectively improve the characteristics of plant seeds, thereby enhancing the quality and quantity of the seeds of food, cotton and oil crops.

SUMMARY OF INVENTION
The objects of the invention include the use of plant RDL1 genes, particularly cotton RDL1 gene, to improve the characteristics of plant seeds, thereby enhancing the quality of the seeds of the crops.
The first aspect of the present invention includes a use of plant RDL1 genes or the encoded RDL1 proteins to improve the plant seed characteristics.
In accordance with one embodiment of the invention, the plant RDL1 genes is cotton RDL1 genes.
In accordance with another embodiment of the invention, the sequence of plant RDL1 gene is selected from:
(a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or
(b) a molecule that can hybridize, under stringent conditions, with the sequences specified in (a), wherein the molecule can improve plant seed characteristics.
In accordance with yet another embodiment of the invention, the sequence of RDL1 protein is selected from:
(a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or
(b) a protein derived from (a) and having substitution, deletion, or insertion of one or more amino acids of the sequences specified in (a), wherein the protein has an activity for
improving the plant seed characteristics.

In accordance with yet another embodiment of the invention, improvement of plant seed characteristics may include increased seed size, increased seed weight, increased seed fiber length, and/or increased fiber strength.

In accordance with yet another embodiment of the invention, the plants may be dicotyledons or monocotyledons.

In accordance with a preferred embodiment, the crops or plants may be selected from marram, hibiscusmutabilis, and crucifer; more preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare (kaoliang); even more preferably, cotton or cole; and most preferably, cotton.

The second aspect of the present invention includes vectors, wherein the vectors contain plant RDL1 genes.

In accordance with one preferred embodiment, the vectors contain cotton RDL1 genes.

In accordance with one preferred embodiment, the sequences of the DL1 genes are selected from:
(a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or
(b) DNA molecules that can hybridize, under stringent conditions, with the DNA sequences specified in (a) and have activities for improving characteristics of crop plant seeds, wherein the DNA molecules include molecules or fragments that have sequences highly homologous to the sequences of cotton RDL1 gene or gene fragments or
Arabidopsis RD22 gene or gene fragments.

In accordance with a preferred embodiment, the vectors are selected from bacterial
plasmids, bacterial phages, yeast plasmids, plant viruses, or mammal viruses; and preferably,EGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121.

The third aspect of the invention includes genetically engineered host cells, wherein the host cells harbor vectors in accordance with embodiments of the invention.

In accordance with a preferred embodiment, host cells may be selected from prokaryotes, lower eukaryotes, or higher eukaryotes; preferably, bacterial cells, yeast cells, or plant cells; more preferably, Escherichia coli, streptomyces, agrobacteria, and yeasts; and most preferably, agrobacteria.

The forth aspect of the present invention includes methods for making transgenic plants. The methods may include:
(1) providing vectors containing RDL1 genes;
(2) providing host cells that harbor the vectors generated in step (1);
(3) contacting plant cells or tissues with the host cells of step (2), the vectors of step (1), or the RDL1 gene, thereby allowing the RDL1 gene to be transfected into the plant cells and integrated into the chromosomes of the plant cells;
(4) selecting for plant cells, tissues, or organs having the RDL1 transfected therein; and
(5) regenerating/growing the RDL1 transgenic plant cells, tissues, or organs obtained in step (4), wherein the seeds of the transgenic plants may have improved characteristics.

In accordance with one preferred embodiment, the RDL1 genes are cotton RDL1 genes.

In accordance with one preferred embodiment, the host cells are agrobacteria.

In accordance with yet another preferred embodiment, the crop plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare; more preferably, cotton or cole; and most preferably cotton.

The fifth aspect of the invention includes uses of transgenic crop plants generated by the methods of the invention. The transgenic plants may be used to produce crop plant seeds having improved characteristics.

In accordance with one preferred embodiment, the improved characteristics may include increased seed sizes, increased seed weights, increased seed fiber lengths, and/or increased fiber strengths.

In accordance with another preferred embodiment, the plant may be cotton.

The sixth aspect of the present invention includes methods for producing crop plant seeds having improved characteristics, wherein the methods may include increasing the expression levels of RDL1 gene in such crop plants.

In accordance with one preferred embodiment, RDL1 gene may be transfected into the crop plants to increase the expression levels of RDL1 gene.

In accordance with another preferred embodiment, the methods may include the following steps:

(i) transfecting, using transgenic methods, RDL1 gene into plants and allowing the transfected gene to integrate into plant chromosomes;
(ii) selecting for RDL1 transgenic plant cells, tissues or organs; and
(iii) regenerating/growing the RDL1 transgenic plant cells, tissues or organs obtained from step (ii) to produce plants seeds with improved characteristics.

In accordance with another preferred embodiment, the improved characteristics may include increased seed sizes, increased seed weights, increased fiber lengths, and/or increased fiber strengths.

In accordance with yet another preferred embodiment, the plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare, more preferably, cotton or cole, and most preferably, cotton.

In accordance with one embodiment, the methods include producing transgenic plants having improved seed characteristics and obtaining seeds therefrom, by using methods of the invention.

In accordance with another embodiment, the methods include producing transgenic plants having improved seed characteristics by using methods of the invention and crossing such transgenic plants with non-transgenic plants or other transgenic plants to produce hybrid plants. Then, select for the hybrid plants having improved seed characteristics and obtain seeds from such hybrid planta.

The seventh aspect of the invention includes transgenic plants that harbor plant RDL1
genes.
In accordance with one embodiment, the sequence of RDL1 gene is selected from:
(a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or
(b) molecules that can hybridize, under stringent conditions, with the sequences
specified in (a), and have activities for improving the characteristics of seeds, wherein the
DNA molecules include molecules or their fragments that have sequences highly homologous to those of cotton RDL1 gene or gene fragments or Arabidopsis RD22 gene or gene fragments.

In accordance with another embodiment, the transgenic plants may be selected from marram, hibiscusmutabilis or crucifer; preferably, hibiscusmutabilis or crucifer; more preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare.
The eighth aspect of the invention includes methods for generating plants. The methods may include crossing the transgenic plants, obtained by using methods of the invention, with non-transgenic plants or other transgenic plants to produce hybrid plants containing plant RDLlgene.

In accordance with one embodiment, the transgenic plants and the non-transgenic plants, or other transgenic plants, used in the crossing may belong to the same or different families, preferably the same plant family. The said plants are preferably selected from marram, hibiscusmutabilis or crucifer; more preferably, hibiscusmutabilis or crucifer; most preferably, cotton, cole, rice, wheat, barley, corn, or sorghum vulgare.

In accordance with another embodiment, the hybrid offsprings have stable genetic characteristics.

Preferred embodiments of the invention use cotton RDL1 gene or its encoded RDL1 protein.

Other aspects of the present invention would be apparent to one skilled in the art, based on this description.

Brief Description of Drawings:

FIG 1 shows the construction of GFP-GhRDL1 vector.
FIG 2 shows molecular characterization of cotton transfected with GFP-GhRDL1.
FIG 3 shows subcellular localization of GhRDL1 using GFP-GhRDL1 fusion protein, wherein A, 35S::GFP/GhRDLl, 2 DPA fibers; B, R 15, 2 DPA fiber; C, 35S::GFP-GhRDLl, leaftrichomes.
FIG 4 shows weight (100 seeds with fibers) analysis of GFP/GhRDL1 transgenic cotton seeds (T2 generation), wherein A, morphological comparison of seeds; B, weight comparison of seeds.
FIG 5 shows size comparison of Arabidopsis seeds transfected with GhRDL1. FIG 5A is a morphological comparison of seeds; FIG 5B is comparison of seed lengths and seed widths (Bar = 500 urn).

DETAILED DESCRIPTION
After long-term in-depth study of the structure, function, and location of RDL1 gene, the inventors constructed transgenic vectors containing RDL1 gene. By using plant transgenic technology, the inventors obtained new plants with improved seed characteristics, such as transgenic cotton having increased seed weight and increased seed fiber length. The methods also produce transgenic Arabidopsis with increased seed sizes.
The above observations indicate that RDL1 gene affects seed characteristics. The seeds, obtained from the transgenic plants expressing high levels of RDL1 gene, exhibit improvements, such as increased seed sizes, increased weights, increased fiber lengths, increased fiber strengths, and so on. Based on these, the inventors complete the present invention.

For example, the inventors use the subcellular localization of RDL1-GFP fusion protein to show that GhRDL1 protein may be localized at certain areas of cell wall with polarized distributions. The results show that cell wall edges, marked by green fluorescence signals, are filled with the pectin-rich polysaccharide. In view of the main components of the primary cell wall also contain pectin during the rapid cotton fiber elongation stage, these data suggest that the specific localization of GhRDL1 may be linked to the pectin in cell wall. In addition, BURP and GFP fusion proteins exhibit similar localization characteristics.

The inventors then use yeast two-hybrid methods to identify that GhEXPA1 protein (Gossypium hirsutum a-expansin 1) is a GhRDL1-interacting protein. By co-
transfecting GFP-GhRDL1 and RFP-GhEXPA1 fusion proteins into Arabidopsis, followed by examining the root cells of Arabidopsis using confocal microscopy. The results confirm this protein-protein interaction by showing co-localization of fluorescent signals focused on the cell wall. Subsequent Co-IP experiments also confirm the interactions between these two proteins.

To study the functions of GhRDL1, the inventors use agrobacteria-mediated methods to transfect 35S::GFP-GhRDLl into cotton. The results show the seed sizes of the transgenic cotton are significantly larger than the control. A hypocotyl from a 6d old cotton seedling of T3 generation was used to analyze mechanical characteristics of tissues before and after gene transfer. The results show the average force needed for breaking the hypocotyl is significantly greater in transgenic plants than that in the control. The statistical results obtained from T3-generation plants growing in the fields show average flower buds of most transgenic plants are heavier than the control. In addition, transgenic plants have increased weights (100 seeds) and increased fiber lengths as compared with the control. The above results show that over-expression of GFP-GhRDL1 fusion protein in cotton may improve the development of cotton fibers and ovules, and thus, it is valuable for cotton improvement.

RDL1 gene and its encoded protein
In this description, the term "plant RDL1 gene" or "RDL1 gene" is used interchangeably and means a gene that encodes a protein that has a sequence highly homologous to that of the cotton RDL1 protein, or molecules that can hybridize, under stringent conditions, with the above-noted gene sequences, or gene family molecules that are highly homologous to the above-noted molecules. The expression of the above said genes can improve seed characteristics, such as increased seed sizes, increased seed weights, increased seed fiber lengths, and/or increased seed fiber strengths. This definition also includes molecules that can hybridize, under stringent conditions, with the sequences of cotton RDL1 gene or other highly homologous gene family molecules.
The term "cotton RDL1 gene" means a gene encoding a protein having a sequence j
highly homologous to the sequence of Arabidopsis RD 22 protein. This definition also includes molecules that can hybridize, under stringent conditions, with the sequence of cotton RDL1 gene, or other highly homogenous gene family molecules. The above genes are the ones specifically over-expressed in the cotton fiber elongation stage. For example, land cotton (Grossypium hirsutum) RDL1 gene (GhRDL1) encodes a protein having 355 amino acids, which contains a plant-specific BURP domain at the C-teraiinus.
NCBI discloses the sequences of RDL1 gene and its sequence homologs, such as AY072821 [(Li C-H, Grossypium Tursutum dehydration-induced protein RD22-like protein (RDL) mRNA, complete cds; AY641990 [Wang S, Grossypium arboreum dehydration-induced protein RD22-like protein 1(RDL1) mRNA, RDL1-1 allele, complete cds; and AY641991 [Wang S, Grossypium arboreum dehydration-induced protein RD22-like protein 2(RDL2) mRNA, RDL2-2 allele, complete cds. These genes mentioned above are included in the present invention.

The RDL1 gene in accordance with embodiments of the present invention may be selected from (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ED NO: 5 (each corresponding to AY072821, AY641990, and AY641991, respectively); or (b) functionally active molecules that can hybridize, under stringent conditions, with the sequences specified in (a) and can improve the characteristics of plant seeds.

The term "under stringent conditions" means (1) hybridization and wash under conditions of low ionic strengths and high temperatures, such as 0.2X SSC, 0.1% SDS, 60°C; or (2) hybridization in the presence of denaturants, such as 50% (v/v) formamide, 0.1 % calf serum/0.1% Ficoll at 42^, and so on; or (3) the sequence identity between two sequences may be at least 50%; preferably, > 55% > 60%, > 65%, > 70%, > 75%, > 80%, > 85%, or 90%; more preferably, > 95%, when hybridization takes place. For example, the above sequences may be complementary to the sequences specified in (a).

The full-length nucleotide sequence of RDL1 gene or its fragments may be readily
obtained using PCR amplification techniques, recombinant DNA techniques, or artificial
synthesis. With regard to using PCR amplification to obtain related sequences, the
appropriate primers may be designed based on the sequences disclosed in the present
invention, especially the open reading frame sequences, and the templates may be a
i commercial cDNA library or a cDNA library prepared using conventional methods known to one skilled in the art. When the sequences are long, two or more PCR amplification reactions may be performed, followed by ligation of these amplified fragments in proper order.

The RDL1 gene in accordance with embodiments of the present invention may be selected from cotton or other plants which may contain genes having high sequence

homology with cotton RDL1 gene (for example, at least 50%; or preferably, > 55%, > 60%, > 65%, > 70%, > 75%, > 80%; more preferably, > 85%, > 90%, > 95%; or even 98% in sequence identity). These genes are also considered within the scope of the present invention. The tools and methods for sequence identity or homology comparison are well known in the art, such as BLAST.

In this description, the term "RDL1 protein" refers to a polypeptide encoded by the RDL1 gene. This definition includes mutant polypeptides that can also improve seed characteristics. The proteins of the invention may be purified from natural sources, or obtained from chemical synthesis, or obtained from prokaryotic or eukaryotic host cells (such as bacteria, yeasts, plants, insects, or mammals) using recombinant DNA techniques. The proteins of the invention may be encoded by genes selected from cotton RDL1 gene or other homologous genes or gene families. The protein sequences of RDL1 may be selected from (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) protein sequences derived from the amino acid sequences specified in (a) with substitution, deletion, or insertion of one or more amino acids, which have activities for improving seed characteristics.

The said mutations include (but not limited to): one or more (usually, 1-50; good, 1-30; better, 1-20; best, 1-10; such as, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletion, insertion, and/or substitution; and insertion at the C-terminus and/or at N-terminus of one or more (usually,
To obtain the above (b) proteins, radiation or mutagens may be used to induce random mutations. Alternatively, the mutant proteins may be obtained by performing site-directed mutagenesis or other known molecular biology techniques. The protein sequences can be used to construct the transgenic plants. Then, monitoring whether seed characteristics have been improved to select and identify the proteins (for example, referring to the methods described in the present invention).

Depending on the host cells used in the recombinant DNA production process, the present proteins may be glycosylated or non-glycosylated. This term may also include the active fragments or functionally active derivatives of the RDL1 proteins.
The mutant proteins include those having: homologous sequences, conservative mutations, allelic variants, natural mutants, induced mutants, sequences capable of hybridizing, under high or low stringent conditions, with the sequences encoding RDL1 proteins, the proteins or polypeptides obtained by using anti-serum against RDL1 proteins. In addition, other polypeptides, such as fusion proteins containing RDL1 protein or other RDL1 protein fragments may be used as well. In addition to the near full-length polypeptides, embodiments of the present invention may include soluble fragments of RDL1 proteins. In general, the protein fragments usually contain at least 10 continuous amino acids of the RDL1 protein sequences; usually, at least 30 continuous amino acids; preferably, at least 50 continuous amino acids; more preferably, at least 80 continuous amino acids; most preferably, at least 100 continuous amino acids.
Plant seeds and their characteristics:

The term "plant" refers to the plants which have economic values in the food, cotton and oil industries. The economic values are mostly based on their seeds. The plants include, but not limited to, dicotyledons or monocotyledons. The preferred monocotyledon is marram; more preferably, rice, wheat, barley, corn, and sorghum vulgare. The preferred dicotyledons include, but not limited to, malvaceae gossypium plants and brassicaceae brassica, etc.; more preferably, cotton or cole, etc.; most preferably, cotton.
The seed characteristics in accordance with embodiments of the present invention include, but not limited to, seed sizes, seed weights, seed fiber lengths, and/or seed fiber strengths. Improvements of seeds characteristics refer to improvements having increased sizes, increased weights, increased fiber lengths, and/or increased fiber strengths, as compared with the seeds prior to improvements In this description, the terms "seed index" and "100 seeds weight" are interchangeable, referring to the weight per 100 seeds. These data indicate the grain sizes and the degree of fullness of seeds.
Embodiments of the present invention also provide methods for producing plant seeds having improved characteristics. The methods may include increasing the expression levels of the said RDL1 gene (preferably, cotton RDL1 gene). The improvements may be achieved by increasing the expression levels of plant RDL1 gene or increasing the quantity of the RDL1 proteins. One skilled in the art may, based on particular purposes, select appropriate improvement methods, such as transfection methods. These methods usually include steps of constructing transfection vectors containing RDL1 gene and transfecting them into plants and plant clones, etc.
Vectors, host cells, and transgenic plants

The present invention also relates to vectors containing RDL1 gene and host cells harboring said vectors produced by genetic engineering, and RDL1 over-expressing transgenic plants obtained using transgenic technology.

Using the conventional recombinant DNA techniques (Science, 1984; 224: 1431) and the sequences disclosed in the present invention, the recombinant RDL1 proteins can be obtained. The method may generally include the following steps:
(1) transforming or transfecting appropriate host cells with the polynucleotides (or variants) encoding RDL1 proteins in accordance with embodiments of the present invention or with the recombinant expression vectors containing said polynucleotides;
(2) incubating host cells in suitable culture media; and
(3) isolating and purifying the proteins from the media or the host cells.

In this description, the terms "vector' and "recombinant expression vector" may be used interchangeably, referring to bacterial plasmids, bacterial phages, yeast plasmids, plant cell viruses, mammal cell viruses or other vectors well known in the art. In short, any plasmids or vectors may be used as long as they can replicate and are stable in the host cells. Important features of expression vectors include the presence of replication origins, promoters, selection marker genes, and translational control elements.
One skilled in the art would know how to construct expression vectors that contain RDL1 gene sequence and suitable transcriptional/translational control elements. These methods include in vitro recombination DNA technology, DNA synthesis techniques, and in vivo recombination technology. The said DNA sequences may be efficiently linked to a promoter on the expression vector to direct the synthesis of mRNA. The vector may also include elements containing ribosomal binding sites and transcription terminators. The preferred vectors for use in the present invention may include pEGFP-1, pCAMBIA 1300, pCAMBIA 2301, or pBI121, etc.

In addition, the expression vectors may preferably contain one or more selection marker genes to permit selection of the transformed host cell phenotype, such as dihyrofolate reductase used in culturing eukaryotic cells, neomycin resistance, and green fluorescence protein (GFP), or tetracycline- or ampicillin-resistance used in E. coli.
The vectors contain the said DNA sequences and a suitable promoter or control elements may be used to selectively transform host cells to express proteins. Host cells may be prokaryotes, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include E. coli, streptomyces, agrobacteria; fungal cells, such as yeasts, plant cells, etc. The preferred host cells for use in the embodiments of the present invention may be agrobacteria.
In the case that the polynucleotides expressed in the higher eukaryotic cells, enhancer sequences may be inserted into the vector to increase transcription. Enhancer is a cw-acting DNA element, which usually have 10-300 base pairs acting on the promoters to enhance gene transcription. One skilled in the art would know how to select suitable vectors, promoters, enhancers, and host cells.

In this description, the terms "transgenic plants," "transfectants," and "transfectant plants" are used interchangeably, referring to cells, organs, or plants, which contain RDL1 genes and stably express RDL1 proteins, obtained by using conventional gene transfer techniques.

Transgenic plants may be obtained by using agrobacteria transfection or gene gun bombardment methods, such as, leaf disc method. The transgenic plants, tissues, or organs may be re-cultivated (re-grown) using conventional methods to obtain plants having high disease resistance. The transfectants may be cultivated using conventional methods to express polypeptides in accordance with the embodiments of the present invention. Depending on the host cells used, various culture media selected from the conventional media may be used to incubate the cells under the conditions suitable for growth. When the growth of host cells reaches appropriate cell density, the promoters can be selectively activated by suitable methods (for example, temperature changes or chemical induction), followed by incubating cells for a period of time.
The above recombinant polypeptides may be expressed within the cells, or on the cell membrane, or secreted out of the cells. If necessary, based on the physical, chemical, and other specific features, the recombinant proteins may be isolated and purified using different separation methods. These separation methods are well known to one skilled in the art. These methods may include, but not limited to,: conventional renaturation treatment, protein precipitation treatment (salting-out), centrifugation, osmotic lysis of bacteria, super process, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chromatography (HPLC), other liquid chromatography, and the combination thereof.

Advantages of the invention
(1) providing new use of RDL1 gene and its proteins to effectively increase the seed quality;
(2) providing transgenic plants with improved characteristics, which include improved seed sizes, weights, fiber lengths, fiber strengths, etc., thus, providing excellent raw materials for the food, cotton, and oil industries; and
(3) providing novel methods to improve seeds characteristics, which may have wide applications.

EXAMPLES
The following examples are provided to further illustrate the present invention. It is understood that these examples are only used to illustrate the present invention and not to be construed as limiting the scope of this invention. Experimental conditions not noted in the following examples may be performed according to conventional conditions described in "Molecular Cloning Laboratory Manuel," by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions suggested by the manufacturers. Unless otherwise indicated, percentages and fractions are based on weights.

Unless otherwise defined, all special or scientific terms used in the present description have the same meanings as known to one skilled in the art. In addition, any methods or materials, which are similar or equivalent to that shown in the present description, can be used to practice the present invention. The preferred methods and materials shown here are for illustration only.

Example 1: Isolation of RDL1 cDNA
Preparation of cotton RNA using phenol extraction:
Grind 2 g of ovular surface fibers (obtained 9 days after land cotton blossom) into powders in liquid nitrogen. Transfer the powders to a 50 ml centrifugation tube. Add 8 ml of extraction buffer (1 M Tris-HCl, 50 mM EDTA, 1 % SDS, pH 9.0) and equal volumes of water-saturated phenol:chloroform:isoamyl alcohol (25:24:1). Mix and place in ice for 1 hour (mix every ten minutes). Centrifuge the mixture (13000g) at 4°C for 20 minutes. Repeat the phenol:chloroform:isoamyl alcohol extraction step 2 ~ 4 times, followed by chloroform:isoamyl alcohol (24:1) extraction once. Collect the supernatant. Add 1/2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol to the supernatant, mix, and place it in -70°C for 1 hour. Centrifuge the mixture (13000g) at 4*C for 20 minutes. Discard the supernatant and dissolve the precipitates in 1 ml DEPC treated-water. Centrifuge the solution (13000g) at 4"C for 10 minutes. Transfer the supernatant to a 1.5 ml Enppendorf tube. Add 1/3 volume of 8 M LiCl and equal volume of NaAC and place it at -20"C overnight. Centrifuge the solution (13000g) at 4°C for 20 minutes. Discard the supernatant and wash the precipitates twice with 1 ml 70 % ethanol. Air-dry the precipitates at room temperature. Dissolve the precipitates in 100~200 uL DEPC treated-water.
Based on AY641990 sequence to synthesize a pair of primers (which may contain restriction enzyme cutting sites and protective amines) and use the primers to perform PCR reactions using RDL1 cDNA as templates:
RDLl-S-BamHI:
S'-CGGGATCCATGAAGGTTCTCTCCCCAATTCTTGCT-S' (SEQ ID NO: 7);
RDLl-A-SacI:
S'-CCGGAGCTCTTACTTAGGGACCCAAACAATGTG - 3' (SEQ ID NO: 8).
Conditions for PCR reactions: pre-denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 30 seconds, renaturation at 56"C for 30 seconds, and extension at 72°C for 1 minute, for a total 35 circles, followed by a final extension at 72 °C for 10 minutes. DNAs of the subclones are then sequenced to determine whether the subclones are correct. The results show that the sequences obtained are identical to AY641990 sequence.

Example 2: Construction of RDL1 and GFP fusion vectors and transformation of agrobacteria.

The GFP gene may be obtained from pEGFP-1 (Clontech, Palo Alto, USA). Inserting appropriate restriction enzyme sites using suitable PCR methods. Remove stop codons from GFP gene sequence and insert a fragment having a non-overlapping sequence of 6 amine acids (GPGGGG).

Use GS1: 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG - 3'(SEQ ID NO:9)
and GA1: 5'-GTCCCCCGGGCGTCCTCCTCCTCCTGGTCCCTGGTACAGC
TCGTCCATGCC-3*(SEQ ID NO: 10) as primers, pEGFP-1 vector as a template, and Pyrobest DNA polymerase (purchased from TAKARA company) to amplify the coding region of GFP gene. Ligate the amplified GFP fragments into pET32c between Eco RV and Nco I sites to generate an intermediate vector, GFP-32c.

Use Pyrobest DNA polymerase to amplify the target sequences containing appropriate restriction enzyme sites, e.g., Bam HI and Sac I (see Example 1). After confirmation of the reading frames, use double-enzyme digestion and ligate the fragments to the corresponding cutting sites in GFP-32c intermediate vector. Use Pyrobest DNA polymerase to amplify the fusion fragments containing Xba I and Kpn I enzyme sites. After double-enzyme digestion, the fragments are ligated downstream from the 35S promoter of pCAMBIA2301 to generate 35S:GFP-RDL1 transgenic vector (abbreviation: RG, for the vector map see FIG. 1). Correct sequence is confirmed.

Transform agrobacteria using freeze-thaw method.
Select a single colony LBA4404 or GV3101 (Invitrogen) into 3 ml LB culture medium (25 fag/ml rifamycin and 50 ug/ml kanamycin (Kan) or gentamycin (Gen), and incubate at 28 °C, 220 rpm overnight. Add 2 ml bacteria liquid to a 50 ml LB culture medium (25 ug/ml Rif and 50 ug/ml Gen), incubate at 28°C, 220 rpm to reach OD6oo = 0.5 (about 6 hours). Place the culture on ice for 30 minutes, and then centrifuge it (5000g) for at 4"C 5 minutes. Re-suspend the bacterial pellet inl0mlof0.15M NaCl and centrifuge (5000g) at 4 °C for 5 minutes. Suspend the bacterial pellet in 1 ml of 20 mM CaCh. Aliquot the bacterial mixture into 50 ul tubes, quick-freeze in liquid nitrogen and keep the competent cells at -70 °C. Mix the vectors containing target genes with 50 ul/tube competent cells and place them on ice for 30 minutes, followed by quick-freeze in liquid nitrogen for 1 minute. Dissolve the bacteria in water bath at 37 °C for 5 minutes. Add 1 ml LB culture medium and incubate at 4"C, 220 rpm, for 2-4 hours. Use 50~ 100 ul of bacterial solution to plate on LB culture medium plates (which contain 25 fig/ml Rif, 50 ng/ml Gen, and 50 u.g/ml Kan or hygromycin (Hyg)). After two days, select a single colony for PCR identification.

Example 3. Plant transfection and screening for transgenic progeny
a. Transeenes in cotton
Culture agrobacteria harboring the vector plasmid in YEB bacteria culture plates containing 50 mg/L kanamycin, 100 mg/L rifampicin, and 300 mg/L streptomycin for 2 to 3 days. Select a single colony and inoculate it in YEB liquid nutrient medium containing the same antibiotics, incubate in suspension culture on a table shaker at 28 °C, 200 rpm/min overnight. Centrifuge the bacteria at 4000 rpm/min for 10 minute. Re-suspend the bacteria pellet in the 1/2MS liquid nutrient medium (containing 30 g/L glucose and 100 umol/L acetosyringone) and allow them to grow until OD600 reaches 0.4 ~ 0.6, which may then be used as inoculants

Disinfect, using traditional methods, cotton R15 seeds (parent plants, a wild type of tetraploid land cotton). Place the seeds in 1/2MS0 culture plates [1/2MS salt (purchased from DUCHEFA M0221) + 5 g/L glucose + 7 g/L agarose powder, pH 6.0] in darkness. After 5 ~ 7 days, slice the hypocotyledonary axis of aseptic sprout into 1 cm segments about 1 cm (explants) for subsequent transfection.

Submerge the explants in the agrobacteria liquid for 15 ~ 20 minutes, and then transfer them to co-culture plates MSB1 (MS salt + B5 organic compounds (one contains inositol, nicotinic acid, VBI and VB6) + 30 g/L glucose + 0.1 mg/L KT (Cell kinetin) + 0.1 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) + 2.2 g/L Gelrite (a solidifying agent), pH 6.0). Culture the explants in darkness at 22 V for 2 days. Transfer the explants to culture plates MSB2 (MSB1 + 500 mg/L cephalosporin + 80 mg/L kanamycin) to induce callus. The explants regenerate test-tube plantlets through resistance to callus induction, callus proliferation, embryonic callus induction (culture medium MSB3: MS salt + B5 organic compounds + 30 g/L glucose + 2.5 g/L Gelrite, pH 6.0), and embryonic cell development
(culture medium MSB4: MS salt + B5 organic compounds + 30 g/L glucose + 1.0 g/L asparagines + 2.0 g/L glutamine + 3.0 g/L Gelrite, pH 6.0; double amount of KNO3 in the MS salt and removal of NH4NO3).' Wait until the test-tube plantlets develop 3 to 4 leaves, transplant them to a pot and grow in a climate control room.
b. Transgenes in Arabidopsis

Transfect Arabidopsis using floral dip methods (Clough and Bent, 1998, Plant J. 16:735-743). The methods of culturing Agrobacteria are similar to that mentioned above. Centrifuge (4000 rpm/min) bacteria solution for 10 minutes. Re-suspend the bacteria pellet in the 500 ml of 5% sucrose solution containing 0.02% Silwet L-77. Submerge the aboveground parts of the plants in the bacteria liquid for 5 seconds. Lay them flat on a plastic container, keep moisten, and avoid light for 16 ~ 24 hours. Vernalize To-generation seeds at 4*C for 2 ~ 4 days. Treat them with 20% bleach water for 15 minutes. Wash them with sterile water 3 ~ 4 times. Suspend them in 0.5% agarose (55 *C), lay them in LB culture plates with 0.6% agar (50 ug/ml Kan or Hyg), at 22'C, under continuous light for one week. Transplant the antibiotic-resistant green sprout to a nutritious soil (peat:vermiculite:perlite = 1:1:1) for growth.
Example 4. Analysis of transgenic plants using molecular biology techniques
a. PCR

DNA isolation by cold phenol methods. Take 2 g of young leaves obtained from transgenic cotton (transgenic cotton that uses R15 as receptors). Grind the leaves into powders in liquid nitrogen. Transfer the powders into a 50 ml centrifugation tube. Add 8 ml of extraction buffer (1M Tris-HCl, 50 mM EDTA, 1% SDS, pH 9.0) and equal volumes of water-saturated phenol:chloroform:isoamylol (25:24:1) into the tube. Then mix the liquid and place it on ice for 1 hour. Mix every 10 minutes and centrifuge 13000 g at A°C for 20 minutes. Repeat the extraction step with phenol:chloroform:isoamylol for 2 ~ 4 times, followed by a final extraction with chlorofonndsoamyl alcohol (24:1). Collect the supernatant. Add V2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and V2 volume isoamyl alcohol, mix, and place it at -70°C for one hour. Centrifuge 13000 g at 4°C for 20 minutes and discard the supernatant. Wash the precipitates two times with 1 ml of

70% ethanol, dry the precipitates at room temperature, and dissolve them in 1 ml of sterile water. Centrifuge 13000 g at 4°C for 10 minutes. Collect the supernatant and add 5~ 10 ul RNase (10 mg/ml) followed by incubation at 37 V for 30 minutes.
PCR reactions may be carried out using GFP primers (which may be the same ones shown in Example 2). The templates may be pEGFP-1 plasmids. The reaction conditions of PCR may be: pre-denaturization at 94C for 5 minutes; denaturization at 94°C for 30 seconds; renaruration at 56 °C for 30 seconds; extension at 72 °C for 1 minute; and 35 total circles; followed by a final extension at 72 °C for 10 minutes.
b. GUS staining analysis

Submerge the plant materials in GUS dye solution (100 mM pH 7.0 phosphate buffer, 50 mM K3[Fe(CN)6], 50 mM K4[Fe(CN)6], 10 mM EDTA, 1 mM X-gluc, and 0.1% Triton X-100) at 37 °C for 12 ~ 24 hours. Destain samples in 70% ethanol. Preserve samples in 70% ethanol.

Example 5. Analyzing the characteristics of transgenic plants
a. Analyzing the characteristics of transgenic cotton
Plant RDL1 transgenic cotton and the parental R15 at different locations: Plant clone No. 105 and plant clone No. 117 are planted in Shanghai. Plant clone No.l 15 and plant clone No. 119 are planted in Hainan. Collect ripe cotton balls from each T2-genetation transgenic plants. The positions from which the cotton balls are collected are kept constant as much as possible. 100 seeds are randomly collected from each ripe cotton balls from individual plants. More than 10 seeds are obtained and their fibers are combed down to measure the length of each fiber. Weigh the 100 seeds (which may or may not contain fibers). The results are summarized in a chart.

The results of T3-generation of plant clone No.1 15 and plant clone No. 119 in Hainan are obtained from 5 plants. As shown in FIG. 4, plant clone No. 115 and plant clone No. 117 of T2-generation in Hainan have a weight (100 seeds) (containing fibers) of 19 g and the weight (100 seeds) of the parental R15 is 16 g. The difference in weight between them is statistically significant (p Table 1. Analysis of fiber length and seed index of GFP-GhRDL1 transgenic cotton
b. Analyzing the characteristics of transgenic Arabidopsis

Transfect GhRDL1 vectors (without GUS genes) into Arabidopsis using Agrobacteria as described above. Obtain transgene-positive plant clones through antibiotic-resistance
screening and RT-PCR. Collect seeds from the next generation and the WT seeds. Measure the length and the width of seeds (n > 50) under a dissecting microscope with 20X magnifications. As shown in FIG. 5, there is a statistically difference (p
All documents mentioned in this invention are used as references in a manner mat every reference is used as a stand-alone reference. It is also understood that one skilled in the art having read the disclosure of the present invention could modify and change the present invention. These modification and changes may fall within the scope of the present invention, which is limited only by the attached claims.

Claims

1. Use of a plant RDL1 gene or its encoded RDL1 protein for improving plant seed
characteristics.

2. The use of claim 1, characterized in that the sequence of the plant RDL1 gene is selected from:

(a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or

(b) a molecule that hybridizes, under a stringent condition, with the sequence specified in (a), wherein the molecule has an activity for improving the plant seed
characteristics.

3. The use of claim 1, characterized in that the sequence of the RDL1 protein is selected from:

(a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or

(b) a protein derived from (a) having substitution, deletion, or insertion of one or more amino acids of the sequence specified in (a), and having an activity for improving the plant seed characteristics.

4. The use of claim 1, characterized in that the improved seed characteristics comprise an increased seed size, an increased seed weight, an increased seed fiber length, and/or an increased seed fiber strength.

5. The use of claim 1, characterized in that the plant is a dicotyledon or a monocotyledon.

6. A vector, characterized in that the vector comprises a plant RDL1 gene.

7. A genetically engineered host cell, characterized in that the host cell harbors the vector of claim 6.

8. A method for producing a transgenic plant, characterized in that the method comprises:

(1) providing a vector containing an RDL1 gene;

(2) providing a host cell harboring the vector described in step (1);

(3) contacting a plant cell or a tissue with the host cell from step (2) or with the vector from step (1) or the RDL1 gene contained therein, causing the RDL1 gene to be transferred into the plant cell and allowing the RDL1 gene to integrate into chromosomes of the plant cell;

(4) selecting for a RDL1 transgenic plant cell, tissue, or organ; and

(5) regenerating or growing a transgenic plant from the plant cell, tissue, or organ obtained in step (4), wherein seeds produced by the transgenic plant have improved characteristics.

9. Use of the transgenic plant produced by the method of claim 8, characterized in that the transgenic plant is used to produce plant seeds having improved characteristics.

10. A method for producing plant seeds having improved characteristics, comprising increasing expression level of RDL1 gene in a plant.

11. The method of claim 10, comprising: producing a transgenic plant using the method of claim 8; and obtaining seeds from the transgenic plant.

12. The method of claim 10, comprising:

generating a transgenic plant using the method of claim 8;

crossing the transgenic plant with a non-transgenic plant or another transgenic plant to
obtain hybrid offsprings;
selecting a hybrid offspring having improved seed characteristics; and obtaining seeds from the selected hybrid offspring.

13. A transgenic plant, characterized in that a gene transfected therein comprises a plant RDL1 gene.

14. The transgenic plant of claim 13, characterized in that the sequence of the RDL1 gene is selected from:

(a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or

(b) a molecule that hybridizes, under a stringent condition, with the sequence specified in (a), and having an activity for improving plant seed characteristics.

15. The transgenic plant of claim 13, characterized in that the plant is marram,
hibiscusmutabilis or crucifer.

16. The transgenic plant of claim 13, characterized in that the plant is a hibiscusmutabilis crop plant or a crucifer crop plant.

17. The transgenic plant of claim 13, characterized in that the plant is selected from the group consisting of cotton, cole, rice, wheat, barley, corn, sorghum vulgare, and arabidopsis.

18. A method for producing a plant, comprising crossing the transgenic plant of claim 13 with a non-transgenic plant or another transgenic plant to produce a hybrid plant harboring the RDL1 gene.

Documents:

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Patent Number

272097

Indian Patent Application Number

5503/CHENP/2010

PG Journal Number

12/2016

Publication Date

18-Mar-2016

Grant Date

17-Mar-2016

Date of Filing

02-Sep-2010

Name of Patentee

SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CAS

Applicant Address

320 YUE YANG ROAD, SHANGHAI 200031

Inventors:

#	Inventor's Name	Inventor's Address
1	CHEN, XIAOYA	320 YUE-YANG ROAD, SHANGHAI 200031
2	XU, BING	320 YUE-YANG ROAD, SHANGHAI 200031
3	GOU, JINYING	320 YUE-YANG ROAD, SHANGHAI 200031
4	SHANGGUAN, XIAOXIA	320 YUE-YANG ROAD, SHANGHAI 200031
5	WANG, LINGJIAN	320 YUE-YANG ROAD, SHANGHAI 200031

PCT International Classification Number

C12N15/29, C12N15/82, C12N15/00

PCT International Application Number

PCT/CN09/070355

PCT International Filing date

2009-02-04

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	200810033537.2	2008-02-04	China