Application of GhTLP19 protein in regulation and control of cotton verticillium wilt-resistant bacteria and method for regulating and controlling cotton verticillium wilt-resistant bacteria
阅读说明：本技术 GhTLP19蛋白在调控棉花抗黄萎病菌中的应用及调控棉花抗黄萎病菌的方法 (Application of GhTLP19 protein in regulation and control of cotton verticillium wilt-resistant bacteria and method for regulating and controlling cotton verticillium wilt-resistant bacteria ) 是由 栗战帅 范术丽 张朝军 王慧颖 马启峰 乔凯凯 于 2021-08-26 设计创作，主要内容包括：本发明提供了一种GhTLP19蛋白在调控棉花抗黄萎病菌中的应用及调控棉花抗黄萎病菌的方法,涉及生物技术领域,本发明通过对棉花内GhTLP19蛋白的研究,首次鉴定了棉花植物GhTLP19蛋白在调控棉花响应黄萎病菌中的应用,为棉花抗病品质的遗传改良提供了有效的途径。通过使棉花包含GhTLP19蛋白的编码基因或含有所述编码基因的生物材料和/或使棉花过表达GhTLP19蛋白,能够显著增强棉花对黄萎病菌的耐受性,为培育抗黄萎病菌的棉花新品种提供了宝贵的基因资源。(The invention provides application of GhTLP19 protein in regulation and control of verticillium wilt bacteria of cotton and a method for regulating and controlling verticillium wilt bacteria of cotton, relates to the technical field of biology, and firstly identifies application of GhTLP19 protein of cotton plants in regulation and control of response of cotton to verticillium wilt bacteria through research on GhTLP19 protein in cotton, and provides an effective way for genetic improvement of disease resistance quality of cotton. The cotton contains the coding gene of the GhTLP19 protein or the biological material containing the coding gene and/or the cotton over-expresses the GhTLP19 protein, so that the tolerance of the cotton to verticillium wilt bacteria can be obviously enhanced, and valuable gene resources are provided for breeding new varieties of verticillium wilt bacteria resistant cotton.)
The application of GhTLP19 protein in regulating and controlling verticillium wilt resistance of cotton;
the amino acid sequence of the GhTLP19 protein is shown in SEQ ID No. 1.
2. The use as claimed in claim 1, wherein the cotton is rendered verticillium wilt resistant by over-expressing GhTLP19 protein in cotton.
The application of the coding gene of the GhTLP19 protein or the biological material containing the coding gene in regulating and controlling verticillium wilt resistance of cotton;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
4. The use as claimed in claim 3, wherein the cotton is rendered verticillium wilt resistant by including in the cotton a gene encoding the GhTLP19 protein or a biological material containing said encoding gene.
5. Use according to claim 3 or 4, wherein the biological material comprises a gene expression cassette, an expression vector or a host cell.
6. A method for regulating and controlling verticillium wilt resistance of cotton is characterized by comprising the following (a) and/or (b):
(a) allowing cotton to comprise a gene encoding GhTLP19 protein or biological material containing the encoding gene;
(b) over-expressing the GhTLP19 protein in cotton;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
7. The method of claim 6, wherein the cotton comprises a gene encoding GhTLP19 protein and/or the cotton overexpresses GhTLP19 protein by transferring the gene encoding GhTLP19 protein into the cotton.
8. The method as claimed in claim 7, wherein the coding gene of GhTLP19 protein is constructed on an expression vector, then agrobacterium is transformed by the obtained recombinant expression vector, the stem segment is divided by the embryogenic organ of recipient cotton infected by the transformed agrobacterium, and after callus is cultured, differentiation culture is continued to obtain the transgenic cotton strain.
9. The method of claim 8, wherein the expression vector comprises pBI 121.
10. The method of claim 8, wherein the embryonic organ of the recipient cotton comprises the hypocotyl.
Technical Field
The invention relates to the technical field of biology, in particular to application of GhTLP19 protein in regulation and control of cotton verticillium wilt resistance and a method for regulating and controlling cotton verticillium wilt resistance.
Background
Cotton is mainly planted in warmer areas and is the main economic crop in more than 30 countries such as china, usa and the like. Verticillium wilt, a gram of cotton, is a disease in which Verticillium dahliae (Verticillium dahliae) infests the root system of cotton through soil and causes vascular bundle blockage. When meeting a proper environment in soil, the micro sclerotium of the verticillium dahliae can rapidly germinate and grow a large amount of hypha, the hypha can be wound on the root of cotton, invade through a root cap or an injured root, and then propagate in gaps among epidermal cells through longitudinal growth. After the successful propagation, the horizontal growth is continued, a large number of spores generated by the propagation can be expanded to each tissue of the host, so that the microtubule tissue is browned, the catheter is blocked and secretes toxin, the leaves of the host are yellowed, wilted and shed, and finally the host dies. When cotton is attacked by verticillium wilt, the completion of the cotton growing period and the quality of fiber are affected. Therefore, it is necessary to breed a new variety of cotton having resistance to verticillium wilt bacteria.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide application of GhTLP19 protein in regulation and control of verticillium wilt resistance of cotton so as to at least alleviate one of technical problems in the prior art.
The invention also aims to provide the application of the coding gene of the GhTLP19 protein or the biological material containing the coding gene in regulating and controlling verticillium wilt resistance of cotton.
The invention also aims to provide a method for regulating and controlling verticillium wilt-resistant bacteria of cotton.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides application of GhTLP19 protein in regulation and control of verticillium wilt-resistant bacteria of cotton;
the amino acid sequence of the GhTLP19 protein is shown in SEQ ID No. 1.
Further, the verticillium wilt resistance of cotton is realized by enabling the cotton to over-express GhTLP19 protein.
The invention also provides the application of the coding gene of the GhTLP19 protein or the biological material containing the coding gene in regulating and controlling verticillium wilt resistance of cotton;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
Further, the verticillium wilt resistance of cotton is realized by enabling the cotton to contain a coding gene of GhTLP19 protein or biological materials containing the coding gene.
Further, the biological material includes a gene expression cassette, an expression vector or a host cell.
In addition, the invention also provides a method for regulating and controlling verticillium wilt resistance of cotton, which comprises the following steps (a) and/or (b):
(a) allowing cotton to comprise a gene encoding GhTLP19 protein or biological material containing the encoding gene;
(b) over-expressing the GhTLP19 protein in cotton;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
Further, the coding gene of the GhTLP19 protein is transferred into cotton so that the cotton contains the coding gene of the GhTLP19 protein and/or the cotton overexpresses the GhTLP19 protein.
Further, the coding gene of the GhTLP19 protein is constructed on an expression vector, then the obtained recombinant expression vector is used for transforming agrobacterium, the transformed agrobacterium is used for infecting embryonic organs of receptor cotton to divide stem segments, and after callus is cultured, differentiation culture is continued to obtain the transgenic cotton plant.
Further, the expression vector comprises pBI 121.
Further, the embryonic organs of the recipient cotton include the hypocotyl.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, through research on GhTLP19 protein in cotton, the application of GhTLP19 protein in cotton plant in regulation and control of response of cotton to verticillium wilt is identified for the first time, and an effective way is provided for genetic improvement of cotton disease resistance quality. The cotton contains the coding gene of the GhTLP19 protein or the biological material containing the coding gene and/or the cotton over-expresses the GhTLP19 protein, so that the tolerance of the cotton to verticillium wilt bacteria can be obviously enhanced, and valuable gene resources are provided for breeding new varieties of verticillium wilt bacteria resistant cotton.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the expression pattern analysis of GhTLP19 provided by the present invention. (A) Expression level of GhTLP19 after CCRI36 was infected with Verticillium dahliae V991 (FPKM). (B) qRT-PCR measures the expression level of GhTLP19 after CCRI36 is treated with verticillium dahliae V991. The standard error is calculated for 3 biological replicates. UBQ7 was used as an internal reference gene.
FIG. 2 is a graph showing that inhibition of expression of GhTLP19 provided by an embodiment of the present invention makes cotton more susceptible to verticillium wilt. (A) phenotype of VIGS (CLCrV: GhTLP19) and control (CLCrV:00) lines after Verticillium dahliae treatment. (B) Expression levels of GhTLP19 in plants CLCrV:00 and CLCrV: GhTLP19 treated with Verticillium dahliae. (C) After V991 inoculation, the accumulation of verticillium wilt bacteria in plants of CLCrV:00 and CLCrV: GhTLP 19. (D) And (3) detecting the fungal biomass in plants CLCrV:00 and CLCrV: GhTLP19 after verticillium wilt bacterium inoculation. (E) Disease indices of CLCrV:00 and CLCrV: GhTLP19 plants after inoculation with Verticillium dahliae. L11, L12 and L13 represent CLCrV, GhTLP19-11, -12 and-13 respectively. Error bars represent the mean ± s.d. of three independent experiments. Statistical significance at the 0.01 and 0.05 probability levels, respectively.
FIG. 3 shows that overexpression of GhTLP19 improves the resistance of cotton to verticillium dahliae. (A) Phenotype of WT, OE-GhTLP19 and Anti-GhTLP19 plants under Verticillium dahliae treatment. (B) After verticillium wilt is inoculated, hypha are accumulated in the stems of WT, OE-GhTLP19 and Anti-GhTLP19 plants. (C) After inoculation of verticillium dahliae. Disease indices of WT, OE-GhTLP19 and Anti-GhTLP19 plants. Error bars represent the mean ± s.d. of three independent experiments. Statistical significance at the 0.05 probability level, respectively.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to the first aspect of the invention, the application of GhTLP19 protein in regulating and controlling verticillium wilt resistance of cotton is provided;
the amino acid sequence of the GhTLP19 protein is shown in SEQ ID No. 1.
According to the invention, through research on GhTLP19 protein in cotton, the application of GhTLP19 protein in cotton plant in regulation and control of response of cotton to verticillium wilt is identified for the first time, and an effective way is provided for genetic improvement of cotton disease resistance quality.
In some preferred embodiments, the invention also finds that the tolerance of cotton to verticillium wilt bacteria can be remarkably enhanced by enabling cotton to overexpress GhTLP19 protein, and provides valuable gene resources for breeding new varieties of cotton resistant to verticillium wilt bacteria.
According to the second aspect of the invention, the application of the coding gene of the GhTLP19 protein or the biological material containing the coding gene in regulating and controlling verticillium wilt resistance of cotton is provided;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
Wherein, the expression "having" means that the nucleotide sequence of the gene encoding the GhTLP19 protein can be the nucleotide sequence shown in SEQ ID NO.2 only, or can be composed of the nucleotide sequence shown in SEQ ID NO.2 and other nucleotide sequences, such as the nucleotide sequence encoding functional units for protein purification, fluorescent protein markers, DNA binding sites and the like, or encoding elements having a regulating effect on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers or transcription factor binding sites and the like; the "having" may also mean that the nucleotide sequence shown as SEQ ID NO.2 is discontinuous in the gene encoding the GhTLP19 protein, but cDNA of the nucleotide sequence shown as SEQ ID NO.2 can be produced.
In some preferred embodiments, the invention also finds that the tolerance of cotton to verticillium wilt bacteria can be remarkably enhanced by enabling the cotton to contain the coding gene of the GhTLP19 protein or the biological material containing the coding gene, and valuable gene resources are provided for breeding new varieties of cotton resistant to verticillium wilt bacteria.
Preferably, the biological material comprises a gene expression cassette, an expression vector or a host cell.
According to a third aspect of the present invention, the present invention also provides a method for regulating and controlling verticillium wilt resistance of cotton, which is characterized by comprising the following (a) and/or (b):
(a) allowing cotton to comprise a gene encoding GhTLP19 protein or biological material containing the encoding gene;
(b) over-expressing the GhTLP19 protein in cotton;
the amino acid sequence of the GhTLP19 protein is shown as SEQ ID No. 1;
the coding gene of the GhTLP19 protein has a nucleotide sequence shown as SEQ ID No. 2.
Because the coding gene of the GhTLP19 protein is hardly expressed when the cotton is not infected by verticillium wilt, the normal cotton contains the coding gene of the GhTLP19 protein, which can play a corresponding role in preventing and improving the tolerance of the cotton to the verticillium wilt.
Meanwhile, the GhTLP19 protein is found to improve the tolerance capability of cotton to verticillium wilt through research, so that the over-expression of the GhTLP19 protein in cotton can also play a role in regulating and controlling verticillium wilt resistance of cotton.
In the present invention, "and/or" means that cotton can be made to contain a gene encoding GhTLP19 protein or a biological material containing the gene alone, or cotton can be made to overexpress GhTLP19 protein on the basis of making cotton contain a gene encoding GhTLP19 protein or a biological material containing the gene.
In some preferred embodiments, the cotton comprises a gene encoding GhTLP19 protein and/or the cotton overexpresses GhTLP19 protein by transferring the gene encoding GhTLP19 protein into cotton.
The cotton contains the coding gene of the GhTLP19 protein and/or the cotton over-expresses the GhTLP19 protein in a transgenic mode, and the method is simple and easy in process, short in period and high in regulation efficiency.
Alternatively, the expression vector carrying the gene encoding GhTLP19 protein may be transferred into cotton by conventional biotechnological methods such as Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation, but the invention is not limited thereto.
In some preferred embodiments, the gene encoding the GhTLP19 protein is constructed into an expression vector, then agrobacterium is transformed with the obtained recombinant expression vector, the stem segments of the embryonic organs of recipient cotton are infected with the transformed agrobacterium, and after callus is cultured, differentiation culture is continued to obtain transgenic cotton plants.
Wherein, expression vector refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host.
pBI121 is preferably used as the expression vector of the present invention.
Preferably, the embryonic organ of the recipient cotton comprises the hypocotyl.
The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.
Experimental materials:
the cotton materials selected in the experiment are the middle cotton institute 36 and the middle cotton institute 24, the cotton materials are planted in the key laboratory test field (Anyang white wall) of the cotton biology institute of the Chinese academy of agricultural sciences, and the management measure is normal field management.
Experimental reagents and consumables:
enzyme and kit:GXL DNApolymerase high-fidelity enzyme, a fluorescence quantification kit, an RNA reverse transcription kit, a gel recovery kit and a PCR product purification kit are purchased from Takara bioengineering, Dalian, Co., Ltd;the Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small quantity extraction kit is purchased from magenta company; restriction enzymes were purchased from NEB; DNAmarker, plant Total RNA extraction kit purchased from TIANGEN corporation.
Other drugs: agarose is a Spanish original product, peptone, yeast extract, chloroform, isoamylol, ethanol, isopropanol, sodium chloride and the like are domestic analytical purities, ampicillin and the like are purchased from Bao bioengineering Dalian Co., Ltd, and Escherichia coli competent cells are purchased from Beijing Tiangen Biochemical technology company.
Culture medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), and 10g/L sodium chloride (NaCl); LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of agar powder, and the volume is fixed to 1L;
LB selective medium: before LB plate, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 deg.C, shaking up and plating.
The main apparatus is as follows: PCR amplification apparatus (BIO-RAD), high speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR apparatus (ABI7500), electric heating constant temperature incubator (Shanghai Sensin), constant temperature culture oscillator (Shanghai Zhicheng), and artificial climate chamber.
Example 1: expression pattern analysis of cotton gene GhTLP19
Analysis of expression profiles of cotton CCRI36 infected by verticillium wilt pathogens in the early stage of the laboratory shows that GH _ A05G1964(GhTLP19) is hardly expressed when the cotton is not infected by the verticillium wilt pathogens, but the expression level in roots is obviously up-regulated after the cotton is infected by the verticillium wilt pathogens, the expression trends of homologous genes GH _ D05G2004 on a Dt subgroup are similar, but the trend of the homologous genes GH _ D19 is not obvious, and the expression trends of other 4 genes are not changed (A in figure 1).
Taking spore liquid of verticillium dahliae V991 out of a refrigerator at-80 ℃, then uniformly coating on a PDA (days post antistesis) culture medium, placing in the dark for 6 days at 25 ℃, picking up normal and good hyphae, transferring to Czapek's liquid culture, and culturing for about 5 days at 25 ℃ by a shaking table at 150 rpm. The culture was filtered through 8 layers of gauze, and then the spore concentration was counted on a hemocytometer and adjusted to 2X 10 with distilled water7one/mL. 10mL of the prepared verticillium dahliae spore solution is absorbed by the root of each seedling. Then placing in a nutrition pot in a greenhouse at 25 ℃ with the illumination period of 16h illumination/8 h darkness. Control was treated with clear water. Sampling roots of a treatment group and a control group at 0h, 6h, 12h, 24h and 48h after infection respectively, extracting RNA and carrying out reverse transcription of cDNA, obtaining a CDS sequence of GhTLP19 from CottonFGD, designing a primer, and carrying out fluorescence quantitative PCR. B in FIG. 1 is a graph showing the expression pattern of GhTLP 19.
Cloning process: taking roots of the materials of the treatment group and the control group in different treatment periods, quickly freezing the roots in liquid nitrogen, grinding the roots in the liquid nitrogen, and storing the roots in a refrigerator at the temperature of-80 ℃ for later use; extracting total RNA of plants: RNA extraction is carried out by adopting an RNA extraction kit of TIANGEN company; synthesizing cDNA according to instructions of a reverse transcription kit FSQ-201 of Toyobo; and diluting the reverse transcription product cDNA solution by 6 times to be used as a PCR reaction template and carrying out fluorescence quantification. The fluorescent quantitative primers are as follows:
qrtGhTLP19-F：GCAGTCAAGGCAGTTGGTGGTA(SEQ ID NO.3)；
qrtGhTLP19-R：ATATTCCGGCGTGTTGAAGGCA(SEQ ID NO.4)。
and (3) PCR reaction system:
PCR reaction procedure:
cloning of cotton GhTLP19 Gene: the gene sequence of GhTLP19 is obtained from CottonFGD, a primer is designed, and the CDS sequence of GhTLP19 is amplified from root cDNA of cotton plant 36 in upland cotton obtained in the above step, the open reading frame is 735bp, 244 amino acids are coded, the relative molecular weight of the protein is 25.48kDa, and the isoelectric point is 4.601.
According to TaKaRaGXL DNApolymerase Hi-Fi enzyme Specification, PCR reaction system as follows:
the PCR amplification procedure was: 3min at 98 ℃; 10s at 98 ℃; 15s at 56 ℃; 1min at 68 ℃ for 35 cycles; 10min at 68 ℃. The primer sequences are as follows:
GhTLP19-F：ATGGCGATTTCCTTTGGG(SEQ ID NO.5)；
GhTLP19-R：TTAGCTGGTTGGACAAAATGTG(SEQ ID NO.6)。
then, performing gel cutting recovery on the target fragment by using a gel recovery kit; recovering the gum from the productConnecting a T vector to construct and transform escherichia coli by using an Ultra One Step Cloning Kit; overnight culture at 37 ℃ and then shake culture at 37 ℃ after picking the monoclonal from an ampicillin resistant LB culture medium; and (3) carrying out PCR verification on the bacterial liquid, selecting a positive clone sample, sending the sample to sequencing of biological and biological technologies, and adding 60% of glycerol into the bacterial liquid with the correct sequencing for preservation at-70 ℃.
Example 2: VIGS silencing GhTLP19 reducing cotton tolerance to verticillium dahliae
The full medium cotton plant 36 seeds are selected and planted in a phytotron, and the photoperiod and the temperature conditions are as follows: irradiating for 16h at 28 ℃; dark 8h, 22 ℃. After the cotyledon of the seedling is flattened and the first true leaf is exposed (about 10 days), the VIGS bacterial liquid injection test is carried out.
Construction of VIGS silencing vector: the silencing vector pCLCrVA is subjected to double enzyme digestion by SpeI and AscI, and the digestion product is subjected to gel recovery. The silencing fragment of GhTLP19 was amplified from 36 cDNAs from Mitsuwonus gossypii by PCR using the following primers:
GhTLP19clcrv-F:CAAAATGGCATGCCTGCAGACTAGTTTGACTGCT ACGGCCATGTT(SEQ ID NO.7)；
GhTLP19clcrv-R:GAATTCACTAGACCTAGGGGCGCGCCCAGTATGG CCTGGTTCCCTG(SEQ ID NO.8)。
constructing the cloned fragment into an enzyme-digested pCLCrVA vector by using an infusion method, transferring a plasmid with correct sequencing into agrobacterium, performing colony PCR, and preserving bacterial liquid glycerol with a correct strip to 80 ℃.
The vectors CLCrV: GhTLP19, empty vectors CLCrV:00, CLCrVB were transformed into Agrobacterium LBA4404 competence. And (4) using LB culture medium containing corresponding resistance for expansion and shaking, and centrifuging and discarding the supernatant after the bacterial liquid is shaken to be orange. With resuspension (10mM MgCl)210mM MES, 200mM acetosyringone) of activated Agrobacterium OD600Adjusted to 1.5. Mixing CLCrV: GhTLP19 with CLCrVB, CLCrV:00 with CLCrVB, and CLCrV: GhPDS with CLCrVB at a ratio of 1:1, standing at room temperature for 2 hours, and then penetrating into cotton cotyledons fully expanded by CCRI36 by using a 1mL syringe. After overnight dark treatment, the cells were transferred to a greenhouse at 25 ℃ for 16h light/8 h dark cycle. When CLCrV GhPLDS plants show phenotype, sampling identifies the silencing efficiency of the target gene. Followed by verticillium wilt treatment as described in example 1. Control was treated with clear water. At least 30 plants were investigated for statistical disease index per treatment. The main stems were cut open 20 days after inoculation for observation of the hypha content. Simultaneously taking the treated and the control leaves to extract and reverse transcribe the total RNA, and utilizing a qRT-PCR methodThe biomass of verticillium dahliae in leaves was determined using the following primers:
V-qPCR-F：AACAACAGTCCGATGGATAATTC(SEQ ID NO.9)；
V-qPCR-R：GTACCGGGCTCGAGATCG(SEQ ID NO.10)。
after infection, the CLCrV: GhTLP19 plants showed marked necrosis, yellowing and detached leaves, whereas the control plants showed less pronounced phenomena (FIG. 2). In addition, the accumulation of hyphae in the stems of VIGS plants was significantly higher than that of the control group (fig. 2). The Disease Index (DI) of CLCrV: GhTLP19 plants was significantly higher than that of the control group (FIG. 2). Thus, silencing of GhTLP19 has been shown to reduce cotton tolerance to verticillium dahliae.
Example 3: GhTLP19 transgenic cotton responding to verticillium wilt infection
The CDS of GhTLP19 cloned in example 1 was subjected to overexpression and construction of an interference vector.
The Plasmid extraction procedure was slightly modified with reference to the EasyPure @ Plasmid MiniPrep Kit of all-type gold.
1) 5mL of E.coli which had been amplified was centrifuged at 10000 Xg for 1min, and the supernatant was decanted.
2) Adding 250 μ L of RB solution (RNaseA added into RB before use, preserving at 2-8 deg.C), suspending, shaking and mixing.
3) Add 250. mu.L of LB solution and gently invert 5 times to completely lyse the cells (within 5 minutes).
4) Add 350. mu.L of NB and gently invert 5 times upside down, after complete formation of a precipitate, let stand at room temperature for 2 min.
5) Centrifugation at 12000 Xg for 5min, and aspiration of only the supernatant to the spin column. Fast separation at 12000 Xg for 1min, and waste liquid is discarded.
6) 650. mu.L of WB solution (80 ml of absolute alcohol was added and mixed), was quickly detached at 12000 Xg for 1 minute, and the waste liquid was discarded.
7) Repeat step 6).
8) Centrifuged at 12000 Xg for 2min, and then allowed to stand at room temperature for 30 min.
9) Place the column in a clean EP tube and drip preheated ddH into the center of the column2O(pH>7.0)30μL，65℃Standing at room temperature for 15 min.
10) Centrifuging at 10000 Xg for 2min, eluting plasmid, and storing in a refrigerator at-20 deg.C.
Construction of overexpression and interference vectors:
construction of overexpression vectors: the extracted pBI121 binary vector is subjected to double digestion by BamHI and SacI, and is cloned from a T vector with correct sequencing by using a primer with a joint of GhTLP19 gene by overexpression and interference (the interference vector is full-length interference, namely, a CDS sequence of a target gene is reversely inserted into the overexpression vector). The product of enzyme cutting and gene cloning is recovered by cutting Gel by using an easy pure @ Quick Gel Extraction Kit, and the specific steps are as follows:
1) the DNA of interest is excised from the agarose gel, transferred to a centrifuge tube and weighed, calculated as 100mg of gel equals 100. mu.L.
2) GSB (3 times the gel volume) was added to an EP tube and mixed in a 55 ℃ water bath with inversion every 2 minutes.
3) Transferring to centrifugal column, standing at room temperature for 1min, centrifuging at 10000 Xg for 1min, and removing waste liquid.
4) Adding 650 μ L WB solution into the centrifugal column, centrifuging at 10000 Xg for 1min, and pouring off the waste liquid.
5) The centrifuge is 10000 Xg for fast separation for 2 min.
6) Transferring the centrifugal column into another centrifugal tube, opening the centrifugal column cover, standing for 15min, and adding preheated ddH dropwise into the center of the centrifugal column2O(pH>7.0) 30. mu.L, and standing at room temperature for 15 min.
7) Centrifuging at 10000 Xg for 2min, and storing the recovered product in a refrigerator at-20 deg.C.
Use ofII One Step Cloning Kit the recovered restriction enzyme and PCR products were subjected to seamless Cloning ligation as follows:
after gently mixing by using a pipette tip, the mixture was reacted for 30min at 37 ℃ in a PCR instrument, and then the temperature was reduced to 4 ℃ and the mixture was transferred to ice.
Then the recombinant product is transformed into escherichia coli, and the specific steps are as follows:
1) competent cells of DH5 alpha clones were thawed on ice.
2) All the recombinant products were added to the competent cells in a semi-thawed state taken out of the refrigerator, and the centrifuge tube wall was flicked gently with a finger and placed on ice for 30 min.
3) The mixture was allowed to stand in a 42 ℃ water bath for 45sec and then placed on ice for 2 min.
4) Add 500. mu.L of empty LB liquid medium (1g peptone, 0.5g yeast powder and 1g NaCl to 100mL ddH2O), shaking the bacteria by a shaker at 200rpm and 37 ℃ for 45 min.
5) All the bacterial solutions were gently spread with sterilized pipette tips on solid LB (liquid LB plus 15g/L Agar) plates containing kanamycin resistance which had been preheated to 37 ℃ and air dried.
6) The cells were cultured overnight in an incubator at 37 ℃ while being inverted.
7) The following day, well-developed single spots were picked with a white pipette tip into 300. mu.L of LB broth with the corresponding resistance and shaken in a shaker at 37 ℃ for 5 h.
8) The positive monoclone is identified by PCR with enzyme of Kangji corporation, 35S universal primer is used in the upstream, target gene downstream primer is used in the downstream, and the specific PCR program is referred to the instruction.
9) Gel electrophoresis results the single clones with bands were sent to Biotechnology Limited for sequencing. The OE-GhTLP19 and Anti-GhTLP19 bacterial liquid was stored in glycerol at-80 ℃ in a refrigerator.
Transforming agrobacterium: transforming the extracted OE-GhTLP19 and Anti-GhTLP19 vector plasmids into agrobacterium tumefaciens LBA4404 competent cells by a freeze-thaw method, wherein the specific transformation process is as follows:
1) adding 500ng plasmid into GV3101 competent cells, flicking the wall of EP tube with finger, and respectively soaking in ice, liquid nitrogen, 37 deg.C water bath and ice for 5 min;
2) adding 700 μ L of empty LB culture solution, shaking at 28 deg.C for 2.5 h;
3) uniformly coating 100 mu L of bacterial liquid on an LB plate which simultaneously contains kanamycin (50mg/L) and rifampicin (50mg/L) by using a yellow gun head, and inverting the bacterial liquid for 2 days at the temperature of 28 ℃;
4) positive clones obtained by colony PCR were transferred to 50mL LB medium containing the corresponding resistance and shaken at 28 ℃.
The activated agrobacterium is referred to Zhang dynasty military Bombycis paper for genetic transformation of cotton, and the specific steps are as follows:
1) transforming the positive clone into an agrobacterium-sensitive strain LBA4404, carrying out amplification culture on agrobacterium, centrifuging, discarding supernatant, adding invasive stain solution (MGL and AS), vibrating to suspend the bacterial solution, shaking at 28 ℃, and activating at least 30min at 200 rpm/min;
2) sterilizing acceptor cotton (CCRI24) seeds with mercuric chloride, cleaning with sterile water, placing in sterile seedling culture medium, and culturing at 30 deg.C for 6 d;
3) cutting the hypocotyl of the acceptor seedling into small stem sections, infecting the small stem sections with activated agrobacterium and drying the small stem sections;
4) flatly laying the hypocotyl in a co-culture medium containing filter paper, and performing dark culture at 20 ℃ for 1-2 d;
5) transferring the hypocotyl into a 2,4-D culture medium, placing the hypocotyl into a light culture chamber, and carrying out subculture for about 20-30 days;
6) growing the callus into rice-grain-shaped particles, transferring the rice-grain-shaped particles into a differentiation culture medium, and further differentiating into embryoids;
7) subculturing the differentiated plantlets into a rooting culture medium until the plantlets grow into plantlets with good and healthy roots;
8) transferring the seedlings into water, hardening the seedlings, and planting the seedlings in a greenhouse after about one week.
The obtained transgenic positive strain is detected, and the primers are as follows:
35S：GACGCACAATCCCACTATCC(SEQ ID NO.11)；
jGhTLP19oe-R：AAAAGCGACCAGACCAACCA(SEQ ID NO.12)；
jGhTLP19anti-R：GGAATATTGCTGCACTGGCG(SEQ ID NO.13)。
transgenic plant detection reaction system
PCR reaction procedure:
the expression level of GhTLP19 in the obtained transgenic positive plant is subjected to qRT-PCR detection, and the fluorescent quantitative process refers to example 1.
The positive plants obtained were subsequently infected with verticillium wilt bacteria with empty vector control plants, the specific procedure being as in example 1. The rod-cutting experiment and the disease index investigation were then carried out with reference to example 2. The results showed that the over-expressed plants showed less necrosis, yellowing and detached leaves than the control, whereas the interfering plants showed more necrosis, yellowing and detached leaves than the control (FIG. 3). The stem is split from the middle to observe the content of the hypha of the fusarium oxysporum in the stem, and the result shows that the content of the hypha in the stem of the interference plant is the highest and the browning is the most serious; the over-expressed plant has the lowest hypha content and the lowest browning degree in the stalk (figure 3). The disease index statistical result shows that the incidence of disease of the over-expression plants is lower than that of the control group, and the incidence of disease of the interference plants is more significant than that of the control group.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Cotton research institute of Chinese academy of agricultural sciences
Application of <120> GhTLP19 protein in regulation and control of cotton verticillium wilt-resistant bacteria and method for regulating and controlling cotton verticillium wilt-resistant bacteria
<160> 13
<170> PatentIn version 3.5
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<211> 244
<212> PRT
<213> Cotton
<400> 1
Met Ala Ile Ser Phe Gly Ile Tyr Leu Leu Phe Leu Leu Asn Phe Phe
1 5 10 15
Ser Phe Gly Ala Ile Phe Ser Ser Ala Thr Ser Phe Thr Leu Glu Asn
20 25 30
Arg Cys Ser Phe Thr Val Trp Pro Gly Ser Leu Thr Ala Asn Gly Pro
35 40 45
Pro Leu Gly Asp Gly Gly Phe Ala Leu Ala Pro Gly Ser Ser Ser Arg
50 55 60
Leu His Pro Pro Pro Gly Trp Ser Gly Arg Phe Trp Gly Arg Thr Gly
65 70 75 80
Cys Asn Phe Asp Asn Ser Gly Ser Gly Lys Cys Val Thr Gly Asp Cys
85 90 95
Gly Gly Ala Leu Lys Cys Asn Gly Gly Gly Ile Pro Pro Val Ser Leu
100 105 110
Ile Glu Phe Thr Leu Asn Gly His Asp Asn Lys Asp Phe Tyr Asp Ile
115 120 125
Ser Leu Val Asp Gly Tyr Asn Met Ala Val Ala Val Lys Ala Val Gly
130 135 140
Gly Thr Gly Thr Cys Gln Tyr Ala Gly Cys Val Asn Asp Leu Asn Thr
145 150 155 160
Asn Cys Pro Ala Glu Leu Gln Met Met Asp Ser Gly Ser Val Val Ala
165 170 175
Cys Lys Ser Ala Cys Ala Ala Phe Asn Thr Pro Glu Tyr Cys Cys Thr
180 185 190
Gly Ala His Gly Thr Pro Gln Thr Cys Ser Pro Thr Met Tyr Ser Gln
195 200 205
Leu Phe Lys Asn Ala Cys Pro Thr Ala Tyr Ser Tyr Ala Tyr Asp Asp
210 215 220
Ala Thr Ser Thr Met Thr Cys Thr Gly Ala Asp Tyr Leu Ile Thr Phe
225 230 235 240
Cys Pro Thr Ser
<210> 2
<211> 735
<212> DNA
<213> Cotton
<400> 2
atggcgattt cctttgggat ttatcttctt tttctactga atttcttctc atttggggcg 60
atattttctt cagcaacaag ctttacactc gaaaatcgtt gtagtttcac agtatggcct 120
ggttccctga ccgcaaatgg ccctcccctt ggtgatggtg gttttgcatt ggctcctggt 180
tcatcatccc ggctccaccc tccacctggt tggtctggtc gcttttgggg tcgaactggc 240
tgcaatttcg acaactctgg ctcaggaaaa tgtgtaaccg gtgactgtgg tggcgcgtta 300
aagtgcaacg gcggtggcat cccacccgtt tccctgatcg agttcaccct taatggacat 360
gacaataagg acttttatga cattagtctc gtagatggtt acaacatggc cgtagcagtc 420
aaggcagttg gtggtacagg gacttgccaa tatgcaggtt gcgtcaatga cctcaacaca 480
aattgccccg ccgagttaca gatgatggac tcaggttctg tcgtcgcttg taaaagcgcc 540
tgtgctgcct tcaacacgcc ggaatattgc tgcactggcg cacatggtac gcctcagact 600
tgctcaccga caatgtactc acaattgttc aaaaatgcat gccctacggc ttacagttat 660
gcttacgatg acgccactag taccatgact tgcaccgggg cggattatct gatcacattt 720
tgtccaacca gctga 735
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
gcagtcaagg cagttggtgg ta 22
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<400> 4
atattccggc gtgttgaagg ca 22
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence
<400> 5
atggcgattt cctttggg 18
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence
<400> 6
ttagctggtt ggacaaaatg tg 22
<210> 7
<211> 45
<212> DNA
<213> Artificial sequence
<400> 7
caaaatggca tgcctgcaga ctagtttgac tgctacggcc atgtt 45
<210> 8
<211> 46
<212> DNA
<213> Artificial sequence
<400> 8
gaattcacta gacctagggg cgcgcccagt atggcctggt tccctg 46
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
aacaacagtc cgatggataa ttc 23
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence
<400> 10
gtaccgggct cgagatcg 18
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
gacgcacaat cccactatcc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
aaaagcgacc agaccaacca 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence
<400> 13
ggaatattgc tgcactggcg 20