WO2009100674A1

WO2009100674A1 - A method of utilizing rdl1 gene to promote seed enlargement and cotton fiber elongation

Info

Publication number: WO2009100674A1
Application number: PCT/CN2009/070355
Authority: WO
Inventors: Xiaoya Chen; Bing Xu; Jinying Gou; Xiaoxia Shangguan; Yingbo Mao; Zhiping Lin; Lingjian Wang
Original assignee: Shanghai Institutes For Biological Sciences, Cas
Priority date: 2008-02-04
Filing date: 2009-02-04
Publication date: 2009-08-20
Also published as: BRPI0907472B1; CN101503690A; BRPI0907472A2; CN101503690B; BRPI0907472A8

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 RDL1gene 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 RDL1gene, 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.

Description

Method for promoting plant seed enlargement and cotton fiber growth by using RDL1 gene

The invention belongs to the field of plant bioengineering and plant improvement genetic engineering. Specifically, the present invention relates to isolation of a cotton fiber-specific expression gene RDL1 cDNA and construction of an overexpression vector, and a method for promoting superior traits such as seed enlargement and fiber growth of a transgenic crop by transferring the RDL1 gene. Background technique

Crop seeds are important raw materials in agriculture and industry such as grain, cotton and oil. Their traits are directly related to the quality of seeds and the quality of their processed products. These traits primarily include the size of the seed volume, the weight of the seed, the length of the seed fiber (for the use of seed fibers;) and/or the strength of the seed fiber.

Cotton is an important economic crop, and cotton fiber is an important raw material for the textile industry. In 2006, the world produced a total of 25.22 million tons of cotton, of which China's output was 6.73 million tons. The cotton textile industry is increasingly demanding the quality of cotton fibres, such as longer fibres, stronger hardness, finer fibres and more tidy fibres, so increasing the quality and yield of cotton is essential. Cotton fiber development is a highly programmed process, and improving the quality of cotton fiber is a major goal of cotton breeding research.

Cotton fiber is a single-cell fiber formed by the differentiation and development of ovule epidermal cells. Its development process can be divided into four stages: the initial stage of fiber development, the elongation stage, the secondary wall thickening stage and the mature stage, in which the elongation period and The secondary wall thickening period has overlapping time domains. In these four periods, the morphological structure of fibroblasts is accompanied by important physiological and biochemical processes, and a large number of genes are involved in the regulation of fiber development. It is important to study the expression and regulation of these genes.

A gene with high homology to Arabidopsis thaliana RD22 has been found in various cotton varieties. For example, GhRDL1 (Gossypium hirsutum RD22-likel) is a gene that is specifically expressed in cotton fiber elongation, encoding one containing 335 genes. A protein of an amino acid residue that is highly homologous to the RD22 protein of Arabidopsis thaliana. The GhRDL1 protein contains a plant-specific BURP domain protein at the C-terminus, and this function has been studied less.

There is an urgent need in the art to find effective means to improve the traits of crop seeds to improve the yield and quality of crops such as grains, cotton and oil. Summary of the invention

The object of the present invention is to use the plant RDL1 gene, especially the cotton RDL1 gene, to improve the traits of crop seeds, thereby improving the quality of crop seeds.

In a first aspect of the invention there is provided the use of a plant RDL1 gene or an encoded RDL1 protein thereof for improving the seed trait of a crop.

In one embodiment, the plant RDL1 gene is a cotton RDL1 gene. In one embodiment, the sequence of the plant RDL1 gene is selected from the group consisting of:

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

(b) a molecule that hybridizes under stringent conditions to (a) a defined sequence and has activity to improve crop seed traits. In another embodiment, the sequence of the RDL1 protein is selected from the group consisting of:

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

(b) A protein derived from (a) which has been substituted, deleted or added with one or several amino acids in (a) a defined amino acid sequence and which has activity to improve crop seed traits.

In another embodiment, the improvement of the crop seed trait comprises: increased seed volume, increased seed weight, seed fiber growth, and/or increased seed fiber strength.

In another embodiment, the crop is a dicot or a monocot.

In a preferred embodiment, the crop is selected from the group consisting of: a gramineous crop, a Malvaceae cotton crop, a Brassica Brassica crop, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably cotton. Or rapeseed, most preferably cotton. In a second aspect of the invention, there is provided a vector comprising a plant RDL1 gene.

In a preferred embodiment, the vector contains the cotton RDL1 gene.

In a preferred embodiment, the sequence of the RDL1 gene is selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or a DNA sequence defined under (a) under stringent conditions A DNA molecule that hybridizes and has activity to improve crop seed traits. Such DNA molecules include molecules or fragments thereof that are highly homologous to the cotton RDL1 or Arabidopsis RD22 gene or fragments thereof.

In a preferred embodiment, the vector is selected from the group consisting of: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a mammalian cell virus, preferably pEGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121 o. In a third aspect of the invention, A genetically engineered host cell is provided, the host cell comprising a vector of the invention.

In a preferred embodiment, the host cell is selected from the group consisting of a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell, preferably a bacterial cell, a yeast cell or a plant cell, more preferably Escherichia coli, Streptomyces, Agrobacterium, yeast, Agrobacterium is most preferred. In a fourth aspect of the invention, a method of preparing a transgenic crop is provided, the method comprising:

(1) providing a vector containing the RDL1 gene;

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

(3) contacting the plant cell or tissue with the host cell in the step (2) or directly contacting the vector in the step (1) or the RDL1 gene therein, thereby transferring the RDL1 gene into the plant cell, and integrating into the plant cell. On the chromosome;

(4) selecting a plant cell, tissue or organ that is transferred to the RDL1 gene;

(5) regenerating the plant cells, tissues or organs in step (4),

The seed of the transgenic crop has an improved trait.

In a preferred embodiment, the RDL1 gene is a cotton RDL1 gene.

In a preferred embodiment, the host cell is Agrobacterium.

In another preferred embodiment, the crop is selected from the group consisting of: gramineous crops, Malvaceae, Brassica, Brassica, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably Cotton or canola, most preferably cotton. In a fifth aspect of the invention, there is provided the use of a transgenic crop prepared by the method of the invention for producing a crop seed having an improved trait.

In a preferred embodiment, the improved trait comprises: increased seed volume, increased seed weight, increased seed fiber, and/or increased seed fiber strength.

In another preferred embodiment, the crop is cotton. In a sixth aspect of the invention, there is provided a method of producing a crop seed having an improved trait, the method comprising: increasing the expression level of the RDL1 gene in the crop.

In a preferred embodiment, the expression level of the RDL1 gene in the crop is increased by transferring the RDL1 gene into the crop and expressing the gene.

In another preferred embodiment, the method includes the steps of:

(i) transferring the RDL1 gene into the crop by transgenic methods and integrating the gene into the chromosome of the crop cell;

(ii) selecting a plant cell, tissue or organ that has been transferred to the RDL1 gene;

(iii) regenerating the plant cell, tissue or organ in step (ii), and using the plant to produce a crop seed having a modified trait.

In another preferred embodiment, the improved trait comprises: increasing seed volume, increasing seed weight, increasing seed fibers, and/or increasing seed fiber strength.

In another preferred embodiment, the crop is selected from the group consisting of: a gramineous crop, a Malvaceae cotton crop, a Brassicaceae Brassica crop, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably Cotton or canola, most preferably cotton.

In one embodiment, the method comprises preparing a seed trait modified transgenic product using the method of the invention and obtaining a seed thereof.

In another embodiment, the method comprises preparing a transgenic crop by the method of the present invention, and hybridizing the transgenic crop with a non-transgenic crop or other transgenic crop to produce a hybrid progeny, the seed being improved The hybrid progeny are obtained and their seeds are obtained. In a seventh aspect of the invention, a transgenic plant comprising a plant RDL1 gene is provided.

In one embodiment, the sequence of the RDL1 gene is selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (; b) under stringent conditions and (a) A defined sequence hybridizes and has a molecule that improves the activity of the crop seed trait. Such DNA molecules include molecules or fragments thereof which are highly homologous to the cotton RDL 1 or Arabidopsis RD22 gene or a fragment thereof.

In another embodiment, the transgenic plant is a gramineous plant, a malvaceae plant or a cruciferous plant, preferably a Malvaceae cotton plant or a Brassicaceae Brassica crop, more preferably the plant is selected from the group consisting of: Cotton, canola, rice, wheat, barley, corn or sorghum. In an eighth aspect of the invention, a method of producing a plant comprising crossing a transgenic plant of the invention with a non-transgenic plant or other transgenic plant to obtain a hybrid progeny comprising the plant RDL1 gene is provided.

In one embodiment, the transgenic plant for hybridization and the non-transgenic plant or other transgenic plant are classified into the same family or different families of plants, preferably the same family of plants. The plant is preferably a gramineous plant, a malvaceae plant or a cruciferous plant, preferably a Malvaceae cotton plant or a Brassicaceae Brassica crop, more preferably the plant is selected from the group consisting of: cotton, rapeseed, rice, wheat , barley, corn or sorghum.

In another embodiment, the hybrid progeny have a stable genetic trait. In a preferred embodiment of the invention, the cotton RDL1 gene or the RDL1 protein encoded thereby is employed. Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

Figure 1: Construction of the GFP-GhRDL1 transgenic vector.

Figure 2: Molecular identification of GFP-GhRDL1 transgenic cotton.

Figure 3: GhRDL1 subcellular localization by the GFP-GhRDL1 fusion protein. Where A is

35S: : GFP/GhRDL l , 2 DPA fiber; B is R15, 2 DPA fiber; C is 35S : : GFP/GhRDL l , leaf epidermis.

Figure 4: Analysis of 100-grain weight (fiber-containing) of GFP-GhRDL1 transgenic cotton seeds (; T ₂ generation;). Among them, A is the comparison of the morphology of the seeds; B is the comparison of the 100-grain weight of the seeds.

Figure 5: Seed size analysis of GhRDL l transgenic Arabidopsis thaliana. Figure 5A is a comparison of the morphology of the seeds; Figure 5B is a comparison of the length and width of the seeds (Bar = 500 μηι). detailed description

The present inventors conducted a long-term and in-depth study on the structure, localization and function of the RDL1 gene, and constructed a transgenic vector containing the RDL1 gene, and obtained plants having improved seed traits by plant transgenic technology, for example, an increase in 100-grain weight and Fiber-grown transgenic cotton, and transgenic Arabidopsis with increased seed volume. This proves that the RDL1 gene is involved in the traits of crop seeds, and the traits of the seeds of plants expressing the gene can be improved, such as increased volume, increased weight, fiber growth, and increased fiber strength. On the basis of this, the inventors completed the present invention.

Specifically, the present inventors confirmed that the GhRDL1 protein is localized in certain regions of the cell wall by a subcellular localization of the RDL1-GFP fusion protein, and has a certain polarity distribution. The green fluorescent signal is located at the corners of the cell wall filled with pectin-rich polysaccharides, and the main component of the primary cell wall in the rapid elongation stage of cotton fibers is also pectin. This result suggests that the localization of GhRDL1 may be related to the pectin of the cell wall; The fusion protein of BURP and GFP showed similar localization characteristics.

Then, the inventors used the yeast two-hybrid method to screen the GhEXPAl (Gossypium hirsutum α-expansinl) protein to bind to the GhRDL1 protein. By GFP-GhRDLl and

The RFP-GhEXPAl fusion protein was co-transformed into Arabidopsis thaliana, and Arabidopsis root cells were observed by confocal microscopy, and fluorescence signals were found to be concentrated on the cell wall and co-localized. Further interactions between the two proteins were verified by Co-IP experiments.

In order to study the function of GhRDL1, the inventors used Agrobacterium-mediated methods to transform 35S::GFP-GhRDLl into cotton, and found that the seed volume of transgenic cotton was significantly enlarged compared with the control. T ₃ generations hypocotyls using cotton seedlings under 6d, the mechanical characteristics were analyzed before and after the transgenic tissue, the results of Table Mingla transgenic plant hypocotyl average breaking force required is significantly greater than in controls. The statistical results of T ₃ plants planted in the field showed that the weight of single bolls in most strains was higher than that of the control, the 100-grain weight increased, and the fiber length was higher than the control. The above results indicate that overexpression of GFP-GhRDL1 fusion protein in cotton can promote the development of cotton fiber and ovule, which is of great value for cotton improvement.

RDL1 encoding genes and proteins

As used herein, "plant RDL1 gene" or "RDL1 gene" is used interchangeably to refer to a gene that is highly homologous to a sequence encoding a cotton RDL1 protein, or a molecule that hybridizes to the gene sequence under stringent conditions, or A family gene molecule highly homologous to the above molecule, the expression of which has a certain improvement effect on the seed trait of the crop, for example, increasing the seed volume, increasing the weight, increasing the fiber, and/or increasing the fiber strength. Also included in the definition are molecules that hybridize to the cotton RDL1 gene sequence under stringent conditions, or family gene molecules that are highly homologous to the above molecules.

As used herein, the term "cotton RDL1 gene" refers to a highly homologous sequence encoding an Arabidopsis RD22 protein, which includes a molecule that hybridizes under stringent conditions to the cotton RDL1 gene sequence, or is highly homologous to the above molecule. Family gene molecules, and preferably the genes are specifically highly expressed during elongation of cotton fibers. For example, the Upland cotton RDL1 encoding gene (; GhRDL1) encodes a protein containing 335 amino acid residues and a plant-specific BURP domain at the C-terminus.

NCBI has published sequences of RDL1 and its homologous genes, such as AY072821 [(Li C-H,

Gossypium hirsutum dehydration-induced protein RD22-like protein (RDL) mRNA, complete cds. "Upland cotton dehydration-induced protein RD22-like protein (RDL1)" mRNA, full-length cds]; AY641990 [Wang S, Gossypium arboreum dehydration-induced protein RD22-like protein 1 (RDL1) mRNA, RDL 1-1 allele, complete cds. "Asian cotton dehydration-inducible protein RD22-like protein 1 (RDL1) "mRNA, RDL1-1 allele, full-length cds]; AY641991 [Wang S Gossypium arboreum dehydration-induced protein RD22-like protein 2 (RDL2) mRNA, RDL2- 2 allele, complete cds, "Asian cotton dehydration-inducible protein RD22-like protein

2 (RDL2) "mRNA, RDL1-1 homologous gene, full-length cds] These genes are all included in the present invention.

The RDL1 gene of the present invention may be selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 (which correspond to AY072821, AY641990, and AY641991, respectively); or (b) under stringent conditions A molecule that hybridizes to the defined sequence of (a) and has activity to improve crop seed traits. As used herein, the term "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 50% identity between the two sequences, Hybridization occurs preferably at 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more. For example, the sequence can be the complement of a sequence defined in ( ).

The full-length nucleotide sequence of the RDL1 gene of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

It is to be understood that the RDL1 gene of the present invention is preferably obtained from cotton, and is highly homologous to the cotton RDL1 gene obtained from other plants (e.g., having 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75). Other genes above %, above 80%, more preferably above 85%, such as 85%, 90%, 95%, or even 98% sequence identity, are also within the equivalent scope of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

In the present invention, the term "RDL1 protein" refers to a polypeptide having a gene encoded by the RDL1 gene of the present invention, and the definition also includes a variant form of the above polypeptide having a function of improving plant seed traits. The proteins of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells;). The RDL1 protein of the present invention is preferably encoded by the cotton RDL1 gene or a homologous gene or family gene thereof. The sequence of the RDL1 protein of the present invention may be selected from: (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) an amino group defined in (a) (a) Derived protein in an acid sequence which has been substituted, deleted or added with one or several amino acids and has activity to improve crop seed traits.

The variant forms include (but are not limited to): one or more (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually Within 20, preferably less than 10, more preferably less than 5; amino acids. For example, in the art, when substituted with similar or similar amino acids, the function of the protein is generally not altered. As another example, the addition of one or more amino acids at the C-terminus and/or the N-terminus generally does not alter the function of the protein. For example, the RDL1 protein of the present invention may or may not include an initial methionine residue and is still improved. Activity of crop seed traits.

The random mutagenesis can be carried out by irradiation or exposure to a mutagen, and the protein in (b) above can also be obtained by site-directed mutagenesis or other known molecular biology techniques. The transgenic plant can be constructed using a coding sequence encoding the protein, and whether the seed trait in the transgenic plant is improved to screen and identify the resulting protein (see, for example, the method of the present invention;).

The protein of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. The term also encompasses active fragments and active derivatives of the RDL1 protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, sequences encoded by sequences that hybridize to the RDL1 protein coding sequence under high or low stringency conditions. A protein, or a polypeptide or protein obtained using an antiserum against the RDL1 protein. Other polypeptides, such as fusion proteins comprising the RDL1 protein or a fragment thereof, can also be used in the present invention. In addition to the nearly full length polypeptide, the present invention also encompasses soluble fragments of the RDL1 protein. Typically, the fragment has at least about 10 contiguous amino acids of the RDL1 protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100. A contiguous amino acid. Crop seeds and their traits

As used herein, "crop" refers to a plant of economic value in agriculture, agriculture, such as grain, cotton, oil, etc., whose economic value is mainly embodied in the seed of the plant. Crops include, but are not limited to, dicotyledonous plants or monocotyledons. Preferred monocot plants are grasses, more preferably rice, wheat, barley, corn, sorghum, and the like. Preferred dicot plants include, but are not limited to, Malvaceae, Brassicaceae, and the like, more preferably cotton, oilseed, etc., most preferably cotton.

In the present invention, the properties of the seed include, but are not limited to, seed volume, seed weight, seed fiber length, and/or strength of seed fibers, and the like. The improvement of the seed trait refers to an increase in seed volume, an increase in seed weight, an increase in seed fiber, and/or an increase in seed fiber strength, as compared with the seed produced by the unmodified plant. In the present invention, the terms "seed finger" or "hundred grain weight" are used interchangeably and refer to the weight per 100 seeds, which reflects the seed size and fullness of the seed.

Also provided in the invention is a method of producing a crop seed having improved traits, the method comprising: The expression level of the RDL1 gene (preferably the cotton RDL1 gene;) in the crop is high, that is, the improvement is achieved by increasing the RDL1 gene expression level or the RDL1 protein content in the crop. Those skilled in the art can select an appropriate modification method depending on the intended purpose. For example, a transgenic method can be employed, which generally includes the steps of constructing a vector into which the RDL1 gene is transferred, transferring to a crop, and breeding. Vector, host and transgenic plants

The present invention also relates to a vector comprising the RDL1 gene, and a host cell genetically engineered using the vector, and a transgenic plant which highly expresses RDL1 by transgene.

The coding sequences of the present invention can be used to express or produce recombinant RDL1 proteins by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the RDL1 protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) Separating and purifying proteins from a culture medium or a cell.

In the present invention, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In general, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.

Methods well known to those skilled in the art can be used to construct expression vectors containing the RDL1 coding sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In the present invention, pEGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121 or the like is preferably used.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like. In the present invention, Agrobacterium is preferably used as a host cell.

When a polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene. It will be apparent to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. The terms "transgenic crop", "transformant" or "transformed plant" are used interchangeably herein to refer to a cell, an organ or a plant obtained by a conventional transgenic method and which has been transferred into the RDL1 gene of the present invention and stably expresses the RDL1 protein. .

The transformed plant can be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method. For transformed plant cells, tissues or organs, plants can be regenerated by a conventional method to obtain plants having improved disease resistance. The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction;) and the cells are cultured for a further period of time.

The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Advantages of the invention

The advantages of the invention are:

(1) A new use of the RDL1 gene and protein, which can be used to effectively improve the quality of crop seeds; (2) A transgenic plant having improved traits with improved seed size, weight, fiber length, fiber strength, etc. , providing excellent raw materials for the production and processing of grain, cotton, oil, etc.;

(3) It provides a new way to improve the seed traits of plants, and thus has great application prospects. Example

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1. Isolation of RDL1 cDNA

Extraction of cotton RNA by cold phenol method: 2 g of material (fibers on the surface of the ovule 9 days after flowering of the upland cotton), ground into powder in liquid nitrogen, transferred to a 50 ml centrifuge tube, and added to 8 1111 extraction buffer (1 13⁄45'11 ( 1, 50 mM) EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24: 1), shake and mix, place on ice for 1 h, mix once every 10 minutes. Centrifuge at 13000 g for 20 minutes at °C. Repeat phenol: chloroform: isoamyl alcohol extraction 2~4 times, and finally extract once with chloroform: isoamyl alcohol (24: 1). Take the supernatant and add 1/2 volume. High-salt solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol, mix, place at -70 °C for 1 h. 4, C, 13000 g Centrifuge for 20 minutes, remove the supernatant, precipitate The water was treated with 1 ml of DEPC, centrifuged at 13,000 g for 10 minutes at 4 ° C. The supernatant was transferred to a 1.5 ml Enppendorf tube, and 1/3 volume of 8 M LiCl and volume of NaAC was added and allowed to stand overnight at -20 °C. Centrifuge at 13000 g for 20 minutes at ° C. The supernatant was removed, and the precipitate was washed twice with 1 ml of 70% ethanol, and the resulting precipitate was dried at room temperature and dissolved in 100 to 200 L of DEPC-treated water.

According to the sequence of AY641990, a pair of specific primers (containing an enzyme cleavage site and a protective base;) were synthesized, and the primer was used to carry out a PCR reaction using RDL1 cDNA as a template:

RDLl-S-BamHI:

5'-CGGGATCCATGAAGGTTCTCTCCCCAATTCTTGCT-3' (SEQ ID NO: 7); RDLl-A-SacI:

5'-CCGGAGCTCTTACTTAGGGACCCAAACAATGTG-3' (SEQ ID NO: 8).

PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes; then denaturation at 94 °C for 30 seconds, renaturation at 56 °C for 30 seconds, extension at 72 °C for 1 minute for 35 cycles; and finally extension at 72 °C for 10 minutes. After subcloning, the sequence was confirmed by sequencing. The results showed that the resulting sequence was identical to the AY641990 sequence. Example 2. Construction of RDL1 and GFP fusion vector and transformation of Agrobacterium

The GFP gene used was derived from pEGFP-1 (Clontech, Palo Alto, USA). A suitable restriction site is introduced by PCR. The stop codon for GFP was removed and a non-folding sequence of 6 amino acids (GPGGGG) was added.

GS 1: 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO: 9)

TCGTCCATGCC-3' (SEQ ID NO: 10) was used as a primer, pEGFP-1 vector was used as a template, and the GFP coding region was amplified with Pyrobest DNA polymerase (purchased from TAKARA). After the gel was recovered, it was digested with Smal and Ncol. Between the EcoRV and the Ncol site of pET32c, a GFP-32c intermediate vector was constructed.

The target fragment was amplified with Pyrobest DNA polymerase, and the corresponding cleavage site BamHI and

Sacl (see Example 1), after reading the reading frame, double-digesting into the corresponding sites of the GFP-32C intermediate vector, constructing a GFP-target intermediate vector, and then amplifying the fusion fragment with Pyrobest DNA polymerase. At the same time, the Xbal and Kpnl restriction sites were introduced, and the 35S:GFP-RDL 1 transgene vector (referred to as RG, the vector diagram is shown in Figure 1) was generated by double digestion into the 35S promoter of the modified pCAMBIA2301, and verified by sequencing.

Agrobacterium tumefaciens was transformed by freeze-thaw method. Take a single colony LBA4404 or GV3101 (Invitrogen) in 3 ml LB medium (25 g/ml rifamycin Rif and 50 μ _δ /ηι1 kanamycin Kan or gentamicin Gen), 28 °C, Incubate overnight at 220 rpm. 21111 broth was added to 5011111^ medium (25 ₈ /11111^ and 5 (^ ₈ /11110611), 28 ° C, 220 rpm, and cultured to OD _{6 (K)} = 0.5 (about 6 hours). After 30 minutes, centrifuge at 5000 g for 5 minutes at 4 ° C. Resuspend the pellet in 10 ml of 0.15 M NaCl, centrifuge at 5000 g for 5 minutes at 4 ° C. Resuspend the pellet in 1 ml of ₂₀ mM CaCl ₂ and dispense For 50 μΐ/tube, frozen in liquid nitrogen, preserve competent cells at -70 ° C. Mix the target gene binary vector and 50 μΐ/tube competent cells, place on ice for 30 minutes, and freeze for 1 minute in liquid nitrogen. The bacterial solution was thawed in a °C water bath for 5 minutes, and 1 ml of LB medium was added, and cultured at 28 ° C, 220 rpm for 2 to 4 hours. 50 to 100 μl of LB medium plate (25 g/ml Rif, 50 g/ Mg Gen and 50 g/ml kanamycin Kan or hygromycin Hyg), 2 days later, single colonies were identified for PCR. Example 3. Plant transformation and screening of transgenic progeny

a. Cotton transgenic

The Agrobacterium containing the vector plasmid was cultured on YEB bacterial medium supplemented with kanamycin 50 mg/L, rifampicin 100 mg/L, streptomycin 300 mg/L for 2 to 3 days, and then single colonies were inoculated. The YEB liquid medium containing the same antibiotic was cultured in suspension at 28 ° C, 200 rpm / min on a shaker overnight. The bacterial solution was centrifuged at 4000 rpm/min for 10 minutes, and the pellet was resuspended in 1/2 MS liquid medium containing 30 g/L of glucose and 100 μηιοΙ/L of acetosyringone, and the OD _{6 (K)} value was adjusted to about 0.4 to 0.6. Infected solution is reserved.

Cotton R15 (wild-type upland cotton of tetraploid, as the parent of transgenic) was routinely sterilized and placed in 1/2MS0 medium [1/2MS salt (purchased from DUCHEFA M0221) + 5g/L glucose + 7g/L agar powder, pH6.0], germinated in the dark, after 5~7 days, the hypocotyls of the sterile seedlings were cut into segments of about 1.0 cm as the transformed explants.

The explants are immersed in Agrobacterium liquid for 15 to 20 minutes and transferred to co-culture medium.

MSB1 (MS salt ten B5 organic (an organic mixture containing inositol, nicotinic acid, V _B ^n V _B6 ) ten 30 g / L glucose ten O.lmg / LKT (cytokinin) ten 0.1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 12.2 g/L GeMte (pH curing agent), pH 6.0), after 2 days of dark culture at 22 ° C, the explants were transferred to the medium. Callus induction was performed on MSB2 (MSB1 + 500 mg/L cephalosporin deca 80 mg/L kanamycin;). Explants were induced by resistant callus, callus proliferation and embryogenic callus induction (medium MSB3: MS salt ten B5 organic ten 30 ₈ glucose ten 2.5 ₈ 0611^6, pH 6.0), body Cell Embryogenesis (medium MSB4: MS salt ten B5 organic ten 30 g/L glucose ten 1.0 g/L asparagine ten 2.0 g/L glutamine ten 3.0 g/L Gelrite, pH 6.0; MS salt KNO _{3 is} doubled, NH ₄ NO ₃ ) is removed, and resistant test tube seedlings are regenerated. When the test tube seedlings grow to 3-4 true leaves, they are transplanted into pots and placed in an artificial climate chamber for growth.

b. Transgenic Arabidopsis

Transformation of Arabidopsis plants was carried out using floral dip (Clough and Bent, 1998, Plant J. 16, 735-743). The Agrobacterium culture method is the same as above, and the bacterial cells are centrifuged at 4000 rpm/min for 10 minutes. Resuspend in 500 1111 containing 0.02% 81^61 1^77 in 5% sucrose solution. Soak the aerial part of the plant in the bacterial solution for 5 seconds, place it in a plastic basin, moisturize and protect from light for 16 to 24 hours. T _Q seeds were vernalized at 4 ° C for 2 to 4 days, treated with 20% bleaching water for 15 minutes, and washed with sterile water for 3 to 4 times. Hang on 0.5% agarose (55 °C), spread on 0.6% agar in LB medium (50 g/ml Kan or Hyg), at 22 ° C, continuous light, about one week later, transplant green resistant seedlings Grow in nutrient soil (peat: vermiculite: perlite = 1: 1: 1). Example 4. Molecular biological identification of transgenic plants

a. PCR

DNA extraction was performed using a cold phenol method. Take 2 g of cotton young leaves (transgenic cotton with R15 as a receptor), grind it into powder in liquid nitrogen, transfer to a 50 ml centrifuge tube, and add 8 1111 extraction buffer (1

50 mM EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24: 1), shake and mix, place on ice for 1 h, mix once every 10 minutes. Centrifuge at 13000 g for 20 minutes at 4 °C. Repeat Phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extract once with chloroform: isoamyl alcohol (24: 1). Take the night, add 1/2 volume of high salt solution (0.8 M sodium citrate, 1.2 M NaCl) and 1/2 volume of isopropanol, mix, and let stand at -70 °C for 1 h. After centrifugation at 13,000 g for 20 minutes at 4 ° C, the supernatant was removed, and the pellet was washed twice with 1 ml of 70% ethanol. The precipitate was dried at room temperature and dissolved in 1 ml of sterile water. Centrifuge at 13000 g for 10 minutes at 4 °C. The supernatant was taken and 5 to 10 μM RNase (10 mg/ml) was added at 37 ° C for 30 minutes.

The PCR was identified using a GFP-specific primer (same as in Example 2) and the template was a pEGFP-Ι plasmid. The PCR reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; then denaturation at 94 ° C for 30 seconds, renaturation at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute for 35 cycles; last 72 ° C extension for 10 minutes .

b. GUS staining analysis

GUS staining solution (100 mM pH 7.0 phosphate buffer, 50 mM K ₃ [Fe(CN) ₆ ], 50 mM K ₄ [Fe(CN) ₆ ], 10 mM EDTA, 1 mM X-gluc, 0. 1% Triton X-100) soaked in plant material, 37 V, 12 to 24 hours. The 70% ethanol was decolorized and the sample was stored in 70% ethanol. Example 5. Character analysis of transgenic plants

a. Analysis of traits of transgenic cotton

RDL1 transgenic cotton and transgenic female R15 were planted in two places: plant No. 105 and plant No. 117 were planted in Shanghai farm, and plant No. 115 and plant No. 119 were planted in Hainan farm. The T ₂ plants of all transgenic lines were individually harvested from mature cotton, and the consistency of the collected parts was kept as much as possible. 100 seeds were randomly taken from the mature cotton collected from each plant, and 10 or more seeds were used to comb the fibers. The fiber length was measured and the 100 seeds (with or without fiber;) were weighed. The above data is statistically plotted. The data of the No. 115 plant and the No. 119 plant planted on the Hainan farm were 5 statistical results. As shown, the No. 1 and No. 1,054 plants 17 ₂ plants Τ generation seed kernel weight (with fiber) was 19g, R15 is 16g, both having a highly significant difference (p <0.01); As shown in Table 1 , Plant No. 115 and Plant No. 119 T ₂ and T ₃ The fiber length of the generation and the seed finger also showed at least a statistically significant difference with respect to R15. Table 1. Analysis of GFP-GhRDL1 transgenic cotton fiber length and seed index

b. Character analysis of transgenic Arabidopsis thaliana

The simultaneously constructed GhRDL1 transgenic vector (without the GUS gene;) was also transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation, and homozygous transgenic positive plants were obtained by resistance screening and RT-PCR. The next generation of seeds and the simultaneously collected WT seeds were measured for length and width (; n > 50) under a 20-fold magnification dissection microscope and statistical analysis was performed. As shown in Figure 5, the length and width of GhRDL1 transgenic Arabidopsis seeds were statistically significantly different from the length and width of WT seeds, respectively (p < 0.01). All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

1. Use of the plant RDL1 gene or its encoded RDL1 protein for improving crop seed traits.

2. The use according to claim 1, wherein the sequence of the plant RDL1 gene is selected from the group consisting of:

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

(b) a molecule that hybridizes under stringent conditions to (a) a defined sequence and has activity to improve crop seed traits.

3. The use according to claim 1, wherein the sequence of the RDL1 protein is selected from the group consisting of:

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

4. Use according to claim 1 wherein the improvement in crop seed traits comprises: increased seed volume, increased seed weight, seed fiber growth, and/or increased seed fiber strength.

5. Use according to claim 1 wherein the crop is a dicot or a monocot.

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

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

8. A method of preparing a transgenic crop, the method comprising:

(1) providing a vector containing the RDL1 gene;

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

(5) regenerating the plant cells, tissues or organs in step (4),

The seed of the transgenic crop has an improved trait.

9. Use of a transgenic crop prepared by the method of claim 8, wherein the transgenic crop is used to produce a crop seed having an improved trait.

10. A method of producing a crop seed having improved traits, the method comprising: increasing the expression level of an RDL1 gene in the crop.

11. The method of claim 10, comprising preparing a transgenic crop with the method of claim 8 and obtaining a seed thereof.

12. The method according to claim 10, comprising preparing a transgenic crop by the method of claim 8, and crossing the transgenic crop with a non-transgenic crop or other transgenic crop to produce a hybrid progeny, the selected seed having improved Hybrid progeny of traits and obtain their seeds.

13. A transgenic plant, characterized in that the transferred gene comprises a plant RDL1 gene.

The transgenic plant according to claim 13, wherein the sequence of the RDL1 gene is selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5;

(b) a molecule which hybridizes under stringent conditions to (a) a defined sequence and which has activity to improve crop seed traits.

The transgenic plant according to claim 13, wherein the plant is a gramineous plant, a marmoset plant, or a cruciferous plant.

16. The plant according to claim 13, wherein the plant is a Malvaceae cotton plant or a Brassica Brassica crop.

17. The plant of claim 13, wherein the plant is selected from the group consisting of: cotton, canola, rice, wheat, barley, corn, sorghum or Arabidopsis.

18. A method of producing a plant, the method comprising: crossing the transgenic plant of claim 13 with a non-transgenic plant or other transgenic plant to obtain a hybrid progeny comprising the plant RDL1 gene.