WO2012083547A1 - Anti-verticillium wilt gene of gossypium barbadense and its use - Google Patents

Abstract

Provided are an anti- verticillium wilt protein of gossypium barbadense and the encoding gene as well as their use in preventing verticillium wilt of plants. The method of producing the said protein by DNA recombinant technology is also provided.

Description

 Island cotton against Verticillium wilt disease gene and its application

Technical field

 The present invention is in the field of biology, and in particular, the present invention relates to a Verticillium dahliae resistance gene and a protein product expressed therefrom which are isolated from a constructed Sea Island cotton cDNA library by molecular cloning techniques. The invention also relates to the use of such genes and proteins for improving the resistance of plants to Verticillium wilt. Background technique

 Verticillium dahliae (Verticillium dahliae, Latin name Verticillium dahliae L.) is a typical soil-borne pathogen that causes disease in more than 170 crops such as cotton. Although many varieties of cotton resistant to (resistant) yellow wilt have been bred over the years. However, there is no significant difference in lint loss caused by planting disease-resistant or disease-resistant products and planting susceptible varieties in severely infected or severely ill areas of Verticillium wilt. The main reason is that the resistance of resistant varieties mainly comes from the existing resistant varieties of upland cotton, and there is no resistance gene in the existing upland cotton varieties.

 Chinese scientists have successively transformed the genes such as chitinase and peroxidase into cotton susceptible varieties through Agrobacterium-mediated genetic transformation, which partially improved the resistance of cotton to Verticillium wilt, but for Verticillium wilt No significant resistance. So far, the anti-Verticillium gene against Verticillium dahliae has not been reported.

 Therefore, there is an urgent need to develop new resistance to Verticillium wilt genes from plant varieties derived from non-terrestrial cotton. The use of molecular biology to isolate the resistance to Verticillium wilt from the island cotton resistant to Verticillium wilt, and the use of molecular breeding methods for crop resistance to Verticillium wilt can effectively solve the above problems, and is also an important way to carry out breeding against Verticillium wilt. Summary of the invention

 It is an object of the present invention to provide a novel protein against Verticillium wilt and a gene encoding the polypeptide of this protein. Another object of the invention is to use the proteins and genes to increase the resistance of plants to Verticillium wilt.

 After extensive and intensive research, the present inventors firstly isolated a new GbVel gene from a constructed island cotton cDNA library by molecular cloning method, and confirmed by experiments that it has high resistance to Verticillium wilt and is transferred to plants. This can lead to an increase in the resistance of plants to Verticillium wilt. The present invention has been completed on this basis.

 In a first aspect of the invention, there is provided a novel isolated protein, designated GbVel protein, characterized by comprising a polypeptide of the amino acid sequence set forth in SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or Its active derivatives.

 The GbVel protein further comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2 formed by substitution, deletion or addition of one or more (; preferably 1-20) amino acid residues, and having resistance to Verticillium wilt.

 In a second aspect of the invention, a novel resistance to Verticillium wilt disease gene having a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding the above GbVel protein polypeptide; (b) a polynucleotide complementary to the polynucleotide (a).

 Preferably, a resistance to Verticillium wilt gene having a polynucleotide sequence as shown in SEQ ID NO: 1.

 More preferably, an anti-Verticillium wilt gene having a polynucleotide sequence comprising:

 (a) a sequence of positions 202 to 3063 in SEQ ID NO: 1, or

 (b) Sequences from 1 to 1500 in SEQ ID NO: 1.

 According to a third aspect of the present invention, there is provided a vector comprising the above-mentioned resistance to Verticillium wilt, the polynucleotide of the gene comprising the polynucleotide of any one of the above.

 The invention also relates to a genetically engineered host cell comprising the vector. The host cell may be a host cell transformed or transduced by the vector, or may be a host cell directly transformed or transduced by the above-mentioned resistance to Verticillium wilt.

 According to a fourth aspect of the present invention, there is provided a method for producing a polypeptide having activity against Verticillium wilt, the method comprising: (a) cultivating the transformed or transduced host cell under conditions suitable for expression; (b) from the above A polypeptide having activity against Verticillium wilt is isolated from host cell culture.

 According to a fifth aspect of the present invention, a method for improving resistance of a plant to Verticillium wilt, comprising the steps of:

 (a) preparing an Agrobacterium carrying the vector of claim 6;

 (b) contacting the plant cell or tissue or organ with the Agrobacterium of step (a), thereby transferring the gene of any one of claims 3 to 5 into the plant cell and integrating it into the chromosome of the plant cell;

 (c) selecting plant cells or tissues or organs into which the gene has been transferred;

 (d) regenerating the plant cell or tissue or organ obtained in the step (c).

 In the present invention, the terms "GbVel protein", "GbVel polypeptide" or "anti-Verticillium protein" are used interchangeably and refer to a protein or polypeptide having an anti-Verticillium wilt protein GbVel amino acid sequence (SEQ ID NO: 2). . They include GbVel proteins with or without the initial methionine.

 As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .

 As used herein, "isolated GbVel protein or polypeptide" means that the GbVel polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art can purify using standard protein purification techniques.

GbVel protein.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, either chemically synthesized or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). According to the reorganization of the production plan Mainly, the polypeptide of the invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also or may not include an initial methionine residue.

 The invention also includes fragments, derivatives and analogs of the GbVel protein. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the native GbVel protein of the invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues May or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusing a mature polypeptide with another compound, or (iv A polypeptide formed by the addition of an amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence used to purify the polypeptide). These fragments, derivatives and analogs are within the purview of those skilled in the art, as described herein.

 In the present invention, the term "GbVel polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 2 which is resistant to Verticillium dahliae activity. The term also encompasses variant forms of the sequence of SEQ ID NO. 2 that have the same function as the GBVE1 protein. These variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions And/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, adding one or more amino acids at the end of C and/or N does not usually alter the function of the protein. The term also encompasses active fragments and active derivatives of the GbVel protein.

 Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to GbVel DNA under conditions of high or low stringency, And a polypeptide or protein obtained using an antiserum against the GbVel polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising a GbVel polypeptide or a fragment thereof. In addition to the nearly full length polypeptide, the present invention also encompasses soluble fragments of the GbBVE1 polypeptide. Typically, the fragment has at least about 10 contiguous amino acids of the GbVel polypeptide 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.

 The invention also provides analogs of GbVel proteins or polypeptides. The difference between these analogs and the native GbVel polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques.

In the present invention, "GbVel protein conservative variant polypeptide" means up to 10, preferably up to 8, more preferably up to 5, optimally up to 3 compared to the amino acid sequence of SEQ ID NO: 2. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1. Table 1 Amino Acid Replacement Table

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1, or may be a degenerate variant. As used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence set forth in SEQ ID NO: 1.

 Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and Non-coding sequence.

 The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polypeptide, or may also include a polynucleotide having additional coding and/or non-coding sequences.

 The present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .

The invention also relates to hybridization to the sequences described above and having at least 50%, preferably at least 70% between the two sequences, More preferably, at least 80% identical polynucleotides. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2xSSC, 0.1% SDS, 60 °C; or (2) hybridization Denaturing agents such as 50% (; v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences It is better to have a hybridization when it is more than 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding GbVel proteins.

 The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

 The full-length sequence of the GbVel nucleotide of the present invention or a fragment thereof can be usually obtained by a method of artificial synthesis, PCR amplification, or recombinant method. For example, full sequence synthesis is first performed according to the sequence of SEQ ID NO: 1.

 For the PCR amplification method, the primers can be designed according to the disclosed nucleotide sequences, especially the open reading frame sequences, and amplified by using the artificial full-length GbVel nucleotide sequence or a fragment thereof as a template. About the sequence.

 Once the relevant sequences have been obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

 In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.

 At present, DNA sequences encoding the proteins of the present invention (or fragments and derivatives thereof) have been obtained by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

 The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or GbVel gene coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.

 The polynucleotide sequences of the present invention can be used to express or produce recombinant GBVE11 polypeptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:

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

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

 (3). Separating and purifying the protein from the culture medium or the cells.

In the present invention, the GbVel protein polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" Refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In summary, 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 DNA sequence encoding the GbVel gene and appropriate transcriptional/translational 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 mR A synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

 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; insect cells, and the like.

 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 a gene.

 It will be apparent to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. Transformed plants can also 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 conventional methods to obtain plants resistant to resistance to Verticillium wilt.

 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 extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These parties 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, ultra-treatment, ultra-centrifugation, 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.

 In another aspect, the present invention also encompasses polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, which are specific for the DNA of the GbVel gene or a polypeptide encoded by the fragment thereof. Preferably, those antibodies which bind to a GbVel protein gene product or fragment but which do not recognize and bind to other non-related antigen molecules. The antibodies of the present invention include those capable of binding to and inhibiting the GbVel protein, as well as those which do not affect the function of the GbVel protein. Also included in the invention are antibodies that bind to a modified or unmodified form of the GbVel protein gene product.

 The present invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, or chimeric antibodies.

 Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, purified

The GbVel protein gene product or its antigenic fragment can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing a GbVel protein or an antigenic fragment thereof can be used to immunize an animal to produce an antibody. Such monoclonal antibodies can be prepared using hybridoma technology. The various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the GbVel protein gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. An antibody that binds to an unmodified form of the GbVel protein gene product can be produced by immunizing an animal with a gene product produced in E.C3⁄4/0; an antibody that binds to a post-translationally modified form (eg, glycosylation or phosphorylation) The protein or polypeptide) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell such as yeast or insect cells. An antibody against the GbVe 1 protein can be used to detect the GbVe 1 protein in a sample.

 Production of polyclonal antibodies can be immunized with GbVel proteins or polypeptides, such as rabbits, mice, rats, and the like. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.

 The invention also relates to a test method for quantifying and localizing the level of GbVel protein. These assays are well known in the art and include FISH assays and radioimmunoassays.

 A method for detecting the presence or absence of a GbVel protein in a sample is detected by using a specific antibody of the GbVel protein, which comprises: contacting the sample with a GbVel protein-specific antibody; observing whether an antibody complex is formed, and forming an antibody complex means The GbVel protein is present in the sample.

 A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as "gene chip") for expression analysis of genes. R A-polymerase chain reaction using GbVel protein-specific primers (RT-PCRM extra-amplification also detects transcripts of GbVel protein.

In one embodiment of the invention, an isolated polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is provided. The polynucleotide of the present invention is isolated from the constructed island cotton cDNA library using in situ hybridization. Its sequence is shown in SEQ ID NO: 1, which comprises a polynucleotide sequence of 3106 bases in length, which is The reading frame is located at position 202-3163 and encodes a 953 amino acid full-length GbVel protein (SEQ ID NO: 2). The GbVel protein of the present invention has the following characteristics: A) The invasive function against Verticillium dahliae is unclear, and the resistance is not reported on plants such as cotton; B) The structure is new, and there is no GbVel protein-encoding gene at the nucleic acid level and has been reported. Homology, up to 20% homology to other RA inhibitory protein families at the amino acid level, does not contain existing patent protection sequences and mutation sites.

 GbVel protein provides a new way to improve the resistance of plants to Verticillium wilt, and thus has great application prospects. By introducing the GbVel gene into crop varieties (such as cotton, tomato, and other cotton or solanaceous crops), and changing the resistance of the existing excellent crop varieties to resistance to Verticillium wilt, cotton, tomato, and other cotton genus resistant to Verticillium wilt can be obtained. Solanaceae crop varieties, improve resistance to Verticillium dahliae, especially Vertici Uium dahliae, and solve practical problems in agricultural production.

 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 Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Other aspects of the invention will be apparent to those skilled in the art from this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS:

 Figure 1 is a schematic diagram showing the construction of a genetic transformation vector of the disease resistance gene GbVel. In the figure: LB and RB are the left and right border sequences of T-DNA, respectively; CaMV35S is the tobacco mosaic virus 35S promoter;

 Hpt is hygromycin

 Ve represents the disease resistance gene GbVel; NOS is the terminator.

 Figure 2 is a PCR detection of cotton seedlings (TO) leaves transgenic with GbVel gene, in which: M is DL2000 Marker: 2000, 1000, 750, 500, 250 and 100 bp; Ve is a positive control (GbVel plasmid); WT is non-transgenic Cotton seedlings; 1-8 are transformed cotton seedlings.

 Fig. 3 is a Southern hybridization analysis of cotton plants transgenic with GbVe 1 gene, in which: P is a gene encoding plasmid against verticillium wilt; 1 to 6 are transgenic plants, respectively. detailed description

Example 1 Cloning of CDNA fragment against Verticillium wilt

 (1) Construction of cDNA library in the root of island cotton

 The construction method of the library was carried out by the method of Stratagene's cDNA library construction kit, and the specific steps of the method are as follows.

The island cotton variety Pima-90 can be used for cotton extraction after being treated with Verticillium dahliae (V. serrata) for 2 hours. Total RNA of flowers. 0.5 g of seaweed roots of seaweed cotton were weighed, ground to a fine powder with liquid nitrogen, and dispensed into two 1.5 ml eppendorf tubes. Add 1 ml of TRIZOL to each tube and shake vigorously to make the mixture evenly. Leave it at room temperature for 5 min. It was then centrifuged at 12,000 g for 10 min at 4 ° C and the supernatant was aspirated into a clean 1.5 ml eppendorf tube. Add 0.2 ml of chloroform to each tube, shake vigorously for 15 s, and let stand for 2 to 3 min at room temperature. It was then centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was transferred to a clean 1.5 ml eppendorf tube, an equal volume of isopropanol was added, mixed by inversion, and allowed to stand at room temperature for 10 min. Centrifuge at 4 ° C, 12,000 g for 10 min. The supernatant was discarded, washed with 1 ml of 75% ethanol, and centrifuged at 7,500 g for 5 min at 4 °C. Discard the supernatant, dry at room temperature for 15-20 min, then dissolve in an appropriate amount of R A-free water (55~60 ° C water bath for 10 min to fully dissolve RA). The obtained RA was used for the cDNA synthesis reaction.

 Total RNA was purified to mRNA according to the QIAGEN mRNA Purification Kit, and immediately subjected to single-strand cDNA synthesis and double-stranded cDNA synthesis according to the method provided in the ClonTECHTM kit.

BM25.8 and XL1-Blue were separately applied to solid plates of kanamycin (50 mg/L) and tetracycline (50 mg/L) resistant LB, and cultured overnight in a 37 ° C incubator. Single colonies were inoculated onto the corresponding LB+ MgS04 medium and cultured overnight at 37 °C. This plate can be used for future experimental sources. The cDNA was ligated to the vector λΤιίρ1Εχ2 according to the following gradient. l ^Control Insert+1.5^ ddH ₂ 0 was used as a control group to test the ligation efficiency, and the control was linked to other linked reactions in the table at 16 ° C in a water bath overnight.

Each tube-ligated product was subjected to a λ phage packaging reaction to obtain a cDNA library having a number of clones of about 1 to 2 χ 10 ^δ .

 (2) Isolation of the resistance to Verticillium wilt by in situ hybridization

 The obtained λ phage was plated in a number of about 10,000 clones per petri dish (20 cm in diameter), and the total number of plates was 20. A circular nylon membrane having a diameter of 20 cm was used for orientation labeling, and the labeled nylon membrane was used to photocopy the above-mentioned plate, and the phage clone on the plate was photocopied onto the nylon membrane.

 The nylon membrane was acid-denatured (0.25 mol / L HC1 solution for 5 minutes), alkali denaturing (0.4 mol / L NaOH solution for 10 minutes) and neutralization reaction (0.5 mol / L Tris-HCl (pH 7.5), 1.5 mol It can be used for southern hybridization in /L NaCl solution and 10 min in distilled water and 3 times in distilled water.

The amount of 100 ng of probe was added per 200 cm ^{2 of} nylon membrane. The probe was labeled according to the following reaction system. Before the probe was labeled, the probe was bathed in boiling water at 100 ° C for 10 min, and then placed on ice for 10 min to fully denature the probe. The probe labeling system is: fully denatured DNA 5-15 L, dNTPs (dATP, dTTP, dGTP) 6μΙ^, Klenow fragment 2units, labeling buffer 5 L, random primer 4 L, added with sterile water to make up 5 (^L system, Centrifuge quickly and mix. Add 32P isotope in 2 L per tube, and let stand for 5 hr to fully label the DNA. The well-labeled probe is denatured and used.

Southern hybridization was carried out according to the method of Molecular Cloning 2nd Edition. After the end of the hybridization, the hybridization membrane was wrapped with a plastic wrap to measure the radioactivity on the membrane. The sensitized front screen, the hybridization film, the X-ray film and the sensitized rear screen were placed in the dark sputum in turn, and the X-ray film was washed after 7-10 days of self-development in a -70 ° C refrigerator. After the hybridization membrane was taken out, the moisture on the intensifying screen was blotted with absorbent paper and air-dried for 30 min. (3) Sequencing verification of cDNA fragments of resistance to Verticillium wilt gene

 Positive cloned phage corresponding to the hybridization results of the nylon membrane were picked. The insert fragment carrying the positive clone was transfected into E. coli DH5ct according to the method shown in the Stratagene cDNA Library Construction Kit. The strain of Escherichia coli can be sequenced after plasmid extraction. Example 2 Artificial Synthesis of GbVel Protein Gene Against Verticillium Wilt

According to the completed nucleotide sequence containing the 3106 bp coding region, a single-stranded oligonucleotide fragment having a sticky end of about 150-200 bp in length was synthesized from the positive and the sub-chain sequences, respectively, in eight segments. Eight complementary single-stranded oligonucleotide fragments each having a one-to-one correspondence between the positive and the sub-strands were annealed to form eight double-stranded oligonucleotide fragments with sticky ends. The double-stranded oligonucleotide fragment was ligated and assembled into a complete GbVel gene by T ₄ DNA ligase. The synthetic DNA fragment contains the nucleotide sequence of positions 202 to 3063 in SEQ ID: 1 and the Xbal and Sacl sites are contained at both ends of the synthetic gene.

 The artificially synthesized 5' and 3' end cleavage sites were used as Xbal and Sacl site GbVel genes for the construction of the plant expression vector of the GbVel protein gene against yellow wilt. (Fig. 1) Example 3 Construction of plant expression vector of GbVel protein gene resistant to Verticillium wilt

 The specific method for constructing the plant expression vector of the GbVel protein gene against Verticillium wilt is as follows (Fig. 1):

 A. pBI121 and pCAMBIA2301 were digested with Hindlll and EcoRI, and pBI121 with p35S-GUS-Nos-ter fragment was ligated into pCAMBIA230J to form intermediate vector p35S-2301-GUS;

 B. Double-cutting p35S-2301-GUS with Xbal and Sacl and the above-mentioned synthetic GbVel gene, replacing with GbVel

P35S-2301-GUS correspondingly cleaves the GUS to obtain a plant expression vector for the GbVel gene against Verticillium wilt. It is then transferred to the Agrobacterium system for transformation of cotton. Example 4 Construction of Transgenic Cotton Resistant to Verticillium Wilt Disease Gene Using Agrobacterium-mediated Transformation

 The Agrobacterium containing the gene of interest was streaked on a suitable resistant LB medium, cultured at 28 ° C for 2 days, and a single colony was picked and inoculated into 50 ml (concentration of 50 mg/L kanamycin) LB liquid medium. in. The medium was incubated at 200 rpm for 28-48 hours on a shaker at 28 degrees. When the OD600 reached between 0.3 and 0.8, the cells were collected by centrifugation at 3000 g. The collected cells were diluted to between OD600 = 0.2-0.4 using 1/2 MS liquid medium.

(1) Co-cultivation of explants and Agrobacterium: Take the hypocotyls of the above 5-6 days old seedlings, cut into small pieces of about 0.5-0.8 cm long, and agrobacterium liquid diluted in 1/2 MS liquid medium. Soak for 15 minutes, take out, blot the bacterial liquid on the surface of hypocotyls with filter paper, transfer to solid CB2.1 medium without any antibiotics, 30-40 hypocotyls per dish, at 25 °C Co-culture for 50-60 hours. (2) Induction of callus: The co-cultured hypocotyls were washed 3-4 times with sterile water containing cef 250 mg/l, and the hypocotyls were blotted with sterile filter paper to dry the water on the hypocotyl surface. The hypocotyls were transferred to CB3.1 medium for 3-4 weeks at 28 ° C - 30 ° C (16 h light / 8 h dark cycle light culture). Small callus can be seen at approximately 3 weeks and then subcultured every other month until the callus reaches the appropriate amount.

 (3) Induction of embryogenic callus and somatic embryos: Transfer a certain amount of callus into CB4 medium,

Incubate at 28 °C - 30 °C (16h light / 8h dark cycle light culture), subculture every other month until somatic embryos appear.

 (4) Germination of somatic embryos: Transfer somatic embryos to CB5 medium, let them germinate and grow into seedlings.

 (5) Obtainment of transgenic plants: Transfer of seedlings (2-4 cm long, shoots with leaves) Transfer to CB6 medium (requires larger container), 28 ° C - 30 ° C (16 h light / 8 h dark cycle) Light culture) grow for 4 months. The transgenic plants with better roots were directly transferred to small pots containing wet soil, and cultured in a culture chamber at 28 ° C to 30 ° C (16 h light / 8 h dark, circulating light culture) for 2 weeks, and then transferred. Small pots into the greenhouse. The transgenic plants are watered daily, fertilized, etc. until the cotton bolls mature. Then, the seeds were collected and the seeds were stored at 4 °C.

 (6) PCR identification of transgenics:

 The total DNA of the plant was obtained by extracting the total DNA of the plant. The total DNA of 1.5μ1 was used as a template to carry out PCR amplification with primers (2400-2420bp, 3000-3025bp). A total of 126 sterile cotton seedlings were detected, among which Twelve positive plants (T0) with specific bands were detected, and the electrophoresis results of PCR products of some plants are shown in Fig. 2.

 A total of 40 transgenic cotton plants with resistance to Verticillium wilt were obtained. Example 5 Identification of Transgenic Plants with Resistance to Verticillium Wilt in Cotton Using Southern Hybridization Method

 Southern blot analysis of the integration of target genes in the island cotton genome, as follows:

 (1) Digestion and electrophoresis of control and transgenic plant DNA

 After digestion at 37 °C for 24 to 48 h, 5 products were electrophoresed, and under UV light, the enzyme was completely cut, and the enzyme was completely digested in the lane, and then the enzyme was cleaved in a freeze-dried centrifuge. Concentrate to 50 on the machine, add 6 Loading buffer, and electrophoresis on 1% agarose gel. Electrophoresis was first performed at 120 V for 20 min. After all the DNA ran out of the wells, the voltage was adjusted to 2 V/cm and electrophoresed for 5 h.

(2) Transfer film After the electrophoresis is finished, the gel is removed, the excess rubber block is cut off, the length and width are measured, the upper right corner is cut off as a mark, and then the de-salting solution is placed for 10-15 min for depurination treatment, at which time the color of bromophenol blue turns yellow. .

 The gel was transferred to a new tray, rinsed twice with deionized water, and then transferred to denaturant for at least 50 min for DNA denaturation.

 After the denaturation was completed, the gel was transferred to another new tray, rinsed twice with deionized water, and then transferred to the neutralizing solution for 30 min.

 The glass plate was placed on a ceramic square plate and two Whatman 3 MM filter papers were placed as a siphon bridge. A sufficient amount of transfer buffer lOx SSC was added to the plate to drive out the bubbles in the siphon bridge and the glass plate. A nitrocellulose filter of the same length and width as the gel size was cut off and immersed in a lOx SSC solution for 3-5 min.

 Place the gel face down, place it in the center of the siphon bridge, and use a glass rod to drive out the air bubbles between the filter paper and the block. A nitrocellulose membrane is placed over the gel and the chamfers are aligned. Seal the four sides of the gel with Parafilm to prevent short circuits. Place three layers of 3 MM filter paper on the membrane and use a glass rod to drive out the bubbles. Cover the filter paper with a multi-layer absorbent paper, place a glass plate of the appropriate size on the top, and place a 500 g weight on top of the glass plate. Transfer the film for 3-5 days, and replace the absorbent paper continuously. After the transfer was completed, the colloid was removed, stained with EB for 10 min, and UV-detected to check the transfer effect. The nylon membrane was clamped with 3 MM filter paper, allowed to stand at room temperature for 30 min, and then fixed at 80 ° C for 2-3 h. The nylon membrane was wrapped in tinplate and stored at 4 ° C for later use.

 (3) Probe preparation

 A specific primer was designed based on the 3'-end relative non-conserved region of the disease resistance gene, and a 400 bp fragment (2800-3200) was amplified as a probe for Southern blot detection.

 The probe labeling procedure is as follows: Take 20 Cross-linker and dilute to the working concentration with 80 water (provided in the kit). The working fluid can be stored for one week at 2-8 °C. The DNA used for labeling was diluted to 10 ng^L with water supplied in the kit. The salt concentration in the nucleic acid must be as low as possible, not exceeding 50 mmol/L. Take 10 diluted DNA samples in a trace of Eppendorf^l^ tube and denature in a boiling water bath for 5 min. Immediately place the sample on ice for 5 min, gently centrifuge on a microcentrifuge, and collect the mixture to the bottom of the tube. Add 10 Reaction buffer to the cooled DNA sample, mix gently and place on ice.

 Add 2 Labeling reagent and mix gently. Add 10 Cross-linker working solution, mix thoroughly, gently centrifuge on a microcentrifuge, and collect the mixture to the bottom of the tube. The reaction mixture was incubated at 37 ° C for 30 min. The marked probe can be used immediately or placed on ice for 2 h. Long-term storage requires the addition of 50% glycerol (v/v).

 (4) Crossing:

Take the required volume of hybridization buffer and preheat to 55 °C. The amount of Buffer is generally 0.25 mL/cm2 hybridization membrane. For large hybrid membranes, the amount of Buffer can be reduced to 0.125 mL/cm2; the membrane is placed in the Hybridization buffer and pre-hybridized in the hybridization oven for at least 15 min; the labeled probe is added to the pre-hybrid Buffer, typically per ml of Buffer. Add 5-10 Ng probe; hybridized overnight in a 55 °C hybridization oven. The stringency can be adjusted by changing the hybridization temperature (50-75 °C).

 (5) Wash film:

Preheat the Primary wash buffer to 55 °C in an amount of 2-5 mL/cm ² ; Carefully transfer the fiber membrane to the Primary wash buffer, rinse at 55 °C for 10 min; rinse with a Primary wash buffer at 55 °C. 10 min; Transfer the fiber membrane to a clean container, add excess Secondary wash buffer, rinse at room temperature for 5 min; rinse again with the Secondary wash buffer for 5 min.

 (6) Detection:

 Remove the excess Secondary wash buffer from the membrane and place the membrane on a flat SanranWrap with the sample facing up. Place the test reagent on the membrane (30 - 40 L/cm2) and place it for 2-4 min to remove Excess test reagent; lay a layer of clean filter paper in the X-ray clip, put the intensifying screen front screen under the filter paper; wrap the film with plastic wrap, place it on the filter paper and attach it with tape; take a piece in the dark room X-ray films were placed on the hybridization membrane. Then put the screen of the intensifying screen, close the light clip, seal the tape, expose for about 2 h; pour the developer into the square in the dark room, take out the X-ray film, set the developer, and gently shake the liquid. After developing for 3-5 minutes until the black exposure strip appears, immediately transfer the X-ray film to the fixing solution, fix it for about 20 minutes, and take out the X-ray film and rinse it in running water overnight.

 Hybridization experiments have shown that the resistance to Verticillium wilt has been transferred to cotton (Fig. 3). Example 6 Identification of Resistance Levels of Cotton Transgenic Plants Resistant to Verticillium Wilt Disease Using Seedling Inoculation Identification

 In the cotton seedling stage (the number of true leaves is 3-5 leaves), Verticillium dahliae was inoculated, and the resistance identification was carried out by inoculation with alfalfa root.

 The transplanted cotton seedlings were removed from the soil and placed in 100 ml of medium Cl/2MS + 106/ml spore) for 10 minutes, and then the plants were placed in the original culture mash (30 cm in diameter, 30 cm in height) and covered with soil. .

 The above transgenic plants and controls were cultured in a growth incubator having a humidity of 60%, a temperature of 32 ° C, and a light intensity of lOOOOLex.

 After 15 days of seedling growth, the disease resistance classification was analyzed.

 The standards used are shown in Table 3.

 Table 3 Classification criteria for resistance identification of cotton seedlings to Verticillium wilt.

After statistical analysis, it was found that the resistance level of all transgenic plants reached Grade 2 or higher (see Table 4), which was a high resistance level, which also indicated that the transferred GbVel gene was a Verticillium wilt resistance gene.

 Table 4 Analysis of disease resistance of transgenic plants

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

A protein having resistance to Verticillium wilt, which is characterized in that the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or the sequence of SEQ ID NO: 2 has verticillium wilt Resistant polypeptide fragments.

2. A protein according to claim 1 comprising a polypeptide having resistance to Verticillium wilt formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 2 via one or more amino acid residues.

3. A gene resistant to Verticillium wilt, the polynucleotide sequence of which comprises:

 (a) a polynucleotide encoding the polypeptide of claim 1 or 2; or

 (b) a polynucleotide complementary to the polynucleotide (a).

4. A gene resistant to Verticillium wilt, the polynucleotide sequence of which is shown in SEQ ID NO: 1.

5. A gene resistant to Verticillium wilt, the polynucleotide sequence comprising:

 (a) a sequence of positions 202 to 3063 in SEQ ID NO: 1, or

 (b) Sequences from 1 to 1500 in SEQ ID NO: 1.

A vector comprising the polynucleotide of any one of claims 3 to 5.

7. A genetically engineered host cell comprising the vector of claim 6.

8. A method for preparing a polypeptide having resistance to Verticillium wilt, comprising:

 (a) cultivating the host cell of claim 7 under conditions suitable for expression;

 (b) A polypeptide having activity against Verticillium dahliae protein is isolated from the above host cell culture.

9. A method of enhancing resistance to plant verticillium wilt, comprising the steps of:

 (a) preparing an Agrobacterium carrying the vector of claim 6;

 (b) contacting the plant cell or tissue or organ with the Agrobacterium of step (a), thereby transferring the gene of any one of claims 3 to the plant cell and integrating it into the chromosome of the plant cell;

 (c) selecting plant cells or tissues or organs into which the gene has been transferred;

 (d) regenerating the plant cell or tissue or organ obtained in step (c).

10. The method of claim 9 wherein the plant is cotton.