GhLAC14-3 gene of cotton and application of GhLAC14-3 gene in regulation and control of verticillium wilt resistance of plants

Abstract

The invention discloses a cotton GhLAC14-3 gene and application of the cotton GhLAC14-3 gene in regulation and control of verticillium wilt resistance of plants. According to the invention, the GhLAC14-3 gene is separated from cotton, the nucleotide sequence of the GhLAC14-3 gene is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein of the GhLAC14-3 gene is shown as SEQ ID No.2. The invention further performs preliminary research on the function of the cotton GhLAC14-3 gene in cotton verticillium wilt resistance, and the result shows that after the GhLAC14-3 gene in cotton is silenced, the verticillium wilt resistance of a cotton seedling plant is obviously reduced; after the GhLAC14-3 gene is over-expressed in arabidopsis thaliana, the verticillium wilt resistance of the arabidopsis thaliana is remarkably improved, so that the resistance of a GhLAC14-3 gene positive regulation plant to verticillium dahliae is determined. The invention has application prospects in the aspects of improving the verticillium wilt resistance of plants or cultivating verticillium wilt resistant plant varieties and the like.

Description

Technical Field

[0001] The present invention relates to a disease resistance-related gene separated from cotton and its use, and in particular to a disease resistance-related gene separated from cotton. GhLAC14-3 Gene and its use in regulating plant resistance to Verticillium wilt, cotton GhLAC14-3 Genes and their areas of use. Background Art

[0002] Cotton is an important economic crop and the world's most important natural fiber source, accounting for about 35% of the world's total fiber. Compared with synthetic fibers, cotton is a renewable resource with important environmental and social benefits. Verticillium dahliae ). Verticillium dahliae mutates quickly, has a complex pathogenic mechanism and is widely distributed, so Verticillium wilt is called the "cancer" of cotton and is one of the serious diseases of cotton. The molecular mechanism of Verticillium dahliae pathogenicity is relatively complex, and two hypotheses are generally recognized, namely the "duct blockage hypothesis" and the "toxin hypothesis". In addition, Verticillium dahliae also secretes a large number of cell wall degrading enzymes, including pectinase, cellulase, etc. These pathogenic factors of Verticillium dahliae affect the pathogenicity of the pathogen to a certain extent.

[0003] The process of cotton resistance to Verticillium wilt requires the participation of a series of related genes and proteins. With the completion of cotton genome sequencing, a series of Verticillium wilt resistance genes have been discovered and identified: an ATP binding protein gene was cloned from G. raymondii ABCF5 , and verified through VIGS technology that it is a negative regulatory factor in cotton resistance to Verticillium wilt (Dong Q, Magwanga RO, Cai X, et al. RNA-sequencing, physiological and rnai analyses provide insights into the response mechanism of the abc-mediated resistance toVerticillium dahliae infection in cotton[J]. Genes, 2019, 10(2): 110). GhCyP3 It can inhibit the U-box E3 ubiquitin ligase GhPUB17, causing GhPUB17 to lose its disease resistance function (Qin T, Liu S, Zhang Z, et al. GhCyP3improves the resistance of cotton to Verticilliumdahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17[J]. Plant Molecular Biology, 2019, 99(4-5): 379-393). Plant laccase (LAC) is located in the exoplasm and is involved in the synthesis and polymerization of lignin. It is reported that LAC regulates the lignin synthesis pathway, which is involved in a variety of plant stress resistance processes, including cotton's resistance to Verticillium wilt: RNA-seq sequencing analysis of the expression profile of cotton disease resistance genes inoculated with Verticillium dahliae showed that the expression level of LAC was increased (Xu L, Zhu L, Tu L, et al. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticilliumdahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry[J]. Journal of Experimental Botany, 2011, 62(15): 21-5607). Overexpression of cotton in Arabidopsis GhLAC15 The cotton laccase gene can enhance resistance to Verticillium wilt by increasing the degree of lignification of the cell wall and increasing the lignin composition (Zhang Y, Wu L, Wang X, et al. GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants[J]. Molecular Plant Pathology, 2019,20(3): 309-322). Gene GhLac1 Overexpression of leads to increased lignification and increased resistance to Verticillium dahliae (Hu Q, Min L, Yang X, et al. Laccase GhLac1modulates broad-spectrum biotic stress tolerance via manipulatingphenylpropanoid pathway and jasmonic acidsynthesis[J]. Plant Physiology,2018, 176(2): 1808-1823). Therefore, exploring genes related to cotton resistance to Verticillium wilt has application prospects in improving cotton resistance to Verticillium wilt or cultivating Verticillium wilt-resistant plant varieties. Summary of the invention

[0004] One of the objects of the present invention is to provide a method for separating cotton from GhLAC14-3 Genes and their encoded proteins; The second object of the present invention is to provide a GhLAC14-3 Gene expression cassette or recombinant expression vector; The third object of the present invention is to GhLAC14-3 Gene, GhLAC14-3 Genes encoding proteins containing GhLAC14-3 The gene expression box or expression vector is used to regulate plant resistance to Verticillium wilt or to cultivate Verticillium wilt-resistant plant varieties.

[0005] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention is to provide a gene related to regulating Verticillium wilt resistance isolated from upland cotton. GhLAC14-3 Gene, the GhLAC14-3 The nucleotide sequence of the gene is selected from any one of (a)-(d): (a) the polynucleotide shown in SEQ ID No. 1; (b) a nucleotide sequence that can hybridize with the complementary sequence of the polynucleotide sequence of SEQ ID No. 1 under stringent hybridization conditions; (c) a nucleotide sequence having at least 90% homology with the polynucleotide represented by SEQ ID No. 1; (d) A mutant in which one or more bases are deleted, substituted or inserted based on the nucleotide sequence shown in SEQ ID No. 1, and the mutant still has the function or activity of regulating plant resistance to Verticillium wilt.

[0006] The percentage of sequence homology described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0007] In addition, those skilled in the art can optimize the nucleotide sequence shown in SEQ ID No. 1 to enhance the expression efficiency in plants; for example, the preferred codons of the target plant can be used for optimization to synthesize the polynucleotide to enhance the expression efficiency in the target plant.

[0008] The present invention can also be used to obtain mutants by deleting one or several amino acid residue codons in the DNA sequence shown in SEQ ID No. 1, or performing missense mutation of one or several base pairs.

[0009] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to GhLAC14-3 The nucleotide sequence of the gene is mutated. GhLAC14-3 Nucleotides with 75% or higher identity to the nucleotide sequence of the gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating plant resistance to Verticillium wilt.

[0010] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0011] The chimeric gene or expression cassette obtained by chimerizing or connecting the gene shown in SEQ ID No. 1 of the present invention with other genes belongs to the protection scope of the present invention; the recombinant expression vector containing the chimeric gene or expression cassette also belongs to the protection scope of the present invention.

[0012] Another aspect of the present invention is to provide a gene related to regulating Verticillium wilt resistance isolated from upland cotton. GhLAC14-3 The amino acid sequence of the encoded protein of the gene is shown in SEQ ID No.2.

[0013] Another aspect of the present invention is to GhLAC14-3 Gene, GhLAC14-3 Genes encoding proteins containing GhLAC14-3 The gene expression box or recombinant plant expression vector is used to regulate plant resistance to Verticillium wilt or to cultivate Verticillium wilt-resistant plant varieties.

[0014] In a preferred embodiment of the present invention, the regulation of plant resistance to Verticillium wilt is to improve plant resistance to Verticillium wilt.

[0015] For reference, the present invention provides an embodiment, namely, by overexpressing a gene encoding a GhLAC14-3 protein associated with plant resistance to Verticillium wilt in a plant, thereby increasing the expression level or activity of the GhLAC14-3 protein associated with plant resistance to Verticillium wilt, thereby improving plant resistance to Verticillium wilt.

[0016] A preferred embodiment of the present invention is a method for improving plant resistance to Verticillium wilt or cultivating Verticillium wilt-resistant plant varieties, comprising: overexpressing in a plant GhLAC14-3 Gene, GhLAC14-3 The expression of the gene is increased or the function or activity of the GhLAC14-3 protein is enhanced; for example, GhLAC14-3 The gene is connected to the expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into plants, so that GhLAC14-3 The gene is overexpressed in plants, and the resulting transgenic plants have improved resistance to Verticillium wilt.

[0017] For reference, the present invention provides a GhLAC14-3 Gene plant recombinant expression vector, comprising: GhLAC14-3 The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.1 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0018] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0019] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell being transformed. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment. In certain embodiments, various transient transformation methods may be used to transform upland cotton. GhLAC14-3 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0020] A preferred embodiment of the present invention is a method for reducing plant resistance to Verticillium wilt, comprising: GhLAC14-3 Gene mutation GhLAC14-3 Reduced gene expression may cause defects in the normal function of the GhLAC14-3 protein.

[0021] The mutations include GhLAC14-3 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing; physical mutagenesis includes but is not limited to radiation mutagenesis, space breeding, etc.; chemical mutagenesis includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes but is not limited to ZFN, TALE and / or CRISPR / Cas, etc.

[0022] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. GhLAC14-3 Gene knockout mutation, such as constructing GhLAC14-3 Gene knockout vectors or constructed using gene editing technology GhLAC14-3 CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants GhLAC14-3 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0023] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the targeted DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0024] The interference GhLAC14-3 The normal expression or normal function of the gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the GhLAC14-3 protein or its promoter or to cause defects in its normal function. RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short-chain double-stranded RNA (siRNA) or a long-chain double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0025] The plants described in the present invention include but are not limited to: monocotyledonous plants or dicotyledonous plants. Most preferably, the plant is cotton or Arabidopsis thaliana.

[0026] The present invention screened a series of differentially expressed genes (DEGs) by bioinformatics methods by performing transcriptome analysis on Arabidopsis thaliana infected with Verticillium dahliae. A gene with significant homology to DEG was obtained by comparing it with the NCBI website. GhLAC14-3 , and found that it encodes laccase protein; the present invention performs dahliae Verticillium induced expression analysis on the gene to determine its response to dahliae Verticillium infection; the present invention further uses subcellular localization, VIGS, Arabidopsis genetic transformation and other molecular biological techniques to preliminarily explore the function of the gene in cotton resistance to Verticillium wilt. The results show that the gene GhLAC14-3 It can positively regulate cotton's resistance to Verticillium wilt.

[0027] Definitions of terms used in this invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

[0028] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-strand or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences and explicitly specified sequences of conservative modifications thereof. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0029] The term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide position identity (i.e., sequence similarity or identity). The term homology used herein also refers to the concept of similar functional properties between different polynucleotide molecules, such as promoters with similar functions may have homologous cis elements. When polynucleotide molecules specifically hybridize to form duplex molecules under specific conditions, they are homologous. Under these conditions (referred to as stringent hybridization conditions), a polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule with a common homology.

[0030] "Stringent hybridization conditions" as used herein refer to conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times greater than the background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or washing conditions. For detailed guidance on nucleic acid hybridization, reference may be made to the literature (Tijssen, Techniques in biochemistry and molecular biology hybridization with nucleic probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are typically selected to be lower than the thermal melting point (T ) of a specific sequence at a specified ionic strength pH. m ) about 5-10℃. Tm The temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T m Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60° C. for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42° C.; or 5×SSC, 1% SDS, incubated at 65° C., washed in 0.2×SSC and washed in 0.1% SDS at 65° C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer.

[0031] The "multiple" mentioned in the present invention generally means 2-8, preferably 2-4; the "replacement" mentioned refers to replacing one or more amino acid residues with different amino acid residues respectively; the "deletion" mentioned refers to a reduction in the number of amino acid residues, that is, the lack of one or more amino acid residues therein respectively; the "insertion" mentioned refers to a change in the amino acid residue sequence, and the change results in the addition of one or more amino acid residues relative to the natural molecule.

[0032] The term "promoter" refers to a polynucleotide molecule which in its natural state is located upstream or 5' of the translation start codon of an open reading frame (or protein coding region) and is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0033] The term "operably linked" refers to a first polynucleotide molecule (e.g., a promoter) being linked to a second transcribable polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single continuous polynucleotide molecule, and more preferably are adjacent. For example, if a promoter regulates or mediates transcription of a target gene in a cell, then the promoter is operably linked to the target gene.

[0034] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0035] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0036] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0037] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 for GhLAC14-3 Gene fluorescence quantitative expression analysis diagram; ** indicates extremely significant difference at P<0.01 level, and * indicates significant difference at P<0.05 level; Figure 2 This is the subcellular localization map of GhLAC14-3 protein in tobacco leaves; Figure 3 for GhLAC14-3 Electrophoresis verification diagram of gene pTRV2 vector construction; among them, Figure 3 -A is GhLAC14-3 PCR silent fragment amplification diagram, 1: amplification GhLAC14-3 VIGS fragment, M: 2000 DNA Marker; Figure 3 -B is the electrophoresis verification diagram of colony PCR detection, 1-3: PCR identification of recombinant plasmid bacterial solution, M: 2000 DNA Marker; Figure 4 for GhLAC14-3 Identification of resistance to Verticillium wilt in gene-silenced cotton plants; Figure 4 -A for silence GhCLA1 Leaf phenotype identification diagram after gene, bar = 2 cm; Figure 4 -B is a graph showing the silencing efficiency of GhLAC14-3 detected by qRT-PCR; Figure 4-C is the phenotypic identification of plants 20 days after infection with Verticillium dahliae V991, bar = 0.2 cm; Figure 4 -D is 20 days after inoculation, pTRV2: 00 and pTRV2:: GhLAC14-3 Statistical chart of cotton plant disease index; Figure 4 -E: 20 days after inoculation, pTRV2: 00 and pTRV2:: GhLAC14-3 Relative fungal biomass determination of cotton plants; ** indicates significant difference at the P<0.01 level, *** indicates significant difference at the P<0.001 level; Figure 5 For overexpression GhLAC14-3 Arabidopsis thaliana inoculated with Verticillium dahliae resistance identification diagram; Figure 5 -A is the Pcambia1304 vector model diagram; Figure 5 -B is RT-PCR detection GhLAC14-3 Expression diagram; Figure 5 -C for overexpression GhLAC14-3 Phenotypic observation of Arabidopsis thaliana inoculated with Verticillium dahliae V991, bar = 2 cm; Figure 5 -D is the disease index statistical chart; Figure 5 -E is the statistics of fungal biomass; ** indicates significant difference at the P<0.01 level; Figure 6 Figure 2 is a graph showing the toxicity and self-activation activity of the bait protein GhLAC14-3; Figure 6 -A is the growth state diagram of pGBKT7-Lam+pGADT7-T on SD / -Trp / -Leu plate; Figure 6 -B is a diagram showing the growth status of pGBKT7-53+pGADT7-T on SD / -Trp / -Leu plate; Figure 6 -C is a diagram showing the growth status of pGBKT7-GhLAC14-3+pGADT7 on SD / -Trp / -Leu plate; Figure 6 -D is the growth status diagram of pGBKT7-Lam+pGADT7-T on SD / -Trp / -Leu / -His / -Ade plate; Figure 6 -E is a diagram showing the growth status of pGBKT7-53+pGADT7-T on SD / -Trp / -Leu / -His / -Ade plates; Figure 6 -F is a graph showing the growth status of pGBKT7-GhLAC14-3+pGADT7 on SD / -Trp / -Leu / -His / -Ade plates; Figure 7 This is a diagram showing the interaction verification results between GhLAC14-3 and the candidate protein GhMAPKKK2; Figure 7-A is a yeast two-hybrid validation diagram of GhLAC14-3 and GhMAPKKK2; Figure 7 -B is the BiFC validation diagram of GhLAC14-3 and GhMAPKKK2; Figure 7 -C is the LCI verification diagram of GhLAC14-3 and GhMAPKKK2. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0040] Experimental materials, vectors and strains Upland cotton variety 'XLZ35'; transgenic Arabidopsis recipient genotype is Colombia wild type (Col-0); cotton VIGS vectors pTRV1, pTRV2, pTRV2:: CLA1 ; The highly pathogenic strain of Verticillium dahliae V991; Escherichia coli DH5α strain; Agrobacterium tumefaciens GV3101 competent cells; The above experimental materials, vectors and strains are all stored in the Crop Functional Genomics and Molecular Improvement Laboratory, College of Life Sciences, Xinjiang Agricultural University.

[0041] Plant growth conditions The growth conditions for Arabidopsis thaliana were: on Murashige-Skoog (MS) agar medium, 24°C light for 16 hours (h), 21°C dark for 8 hours. After 7 days (d), the seedlings were transferred to sterilized peat soil. The growth conditions for upland cotton were: seeds with full grains were selected, washed with sterile water and soaked in a 37°C greenhouse for 24-36 hours, and white seeds were planted in pots containing a mixture of autoclaved vermiculite and black soil (1:2). Three cotton plants were planted in each pot. The growth conditions were 28°C indoor culture, and the light cycle was 16 hours of light and 8 hours of darkness.

[0042] The growth conditions of Nicotiana benthamiana were: 25°C indoor cultivation with a photoperiod of 16 h of light and 8 h of darkness.

[0043] Test reagents Kanamycin, gentamicin, MES, acetosyringone, MgCl2 and culture medium were all domestic analytical grade reagents; the polysaccharide and polyphenol plant total RNA extraction kit was purchased from Hangzhou Biori Technology Co., Ltd.; Eco RI, Bam H Ⅰ, Nco Ⅰ and Spe Restriction endonucleases such as Ⅰ were purchased from Thermo Fisher Scientific, and TaqDNA polymerase, T4 DNA Ligase, In-Fusion ligase, RNaseA, high-fidelity polymerase TransStar KD Plus, reverse transcription kit, fluorescence quantitative kit, agarose gel recovery kit, and DNA molecular weight marker were all purchased from Beijing Quanshijin Biotechnology Co., Ltd. The synthesis of primers used in PCR and DNA sequencing were completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0044] Primer sequences The primer sequences used in the following test examples of the present invention are shown in Table 1.

[0045] Table 1 Primers used in this study

[0046] Note: Lowercase letters represent homology arms.

[0047] In the early stage of this experiment, a large number of differentially expressed genes were found through transcriptomic analysis of Arabidopsis inoculated with Verticillium dahliae. The highly homologous genes in the cotton genome were obtained from the NCBI website through bioinformatics analysis. GhLAC14-3 , which encodes a plant laccase involved in the synthesis and polymerization of lignin, GhLAC14-3 The gene is a new one and its function has not been reported in cotton.

[0048] Should GhLAC14-3

[0049] Should GhLAC14-3 The amino acid sequence of the protein encoded by the gene is SEQ ID Shown in No.2: MGSEKQGFIWLSGLLFLNILVLSTADVHYYEFFLQESQFTKLCSTKSILTVNGSFPGPEIRVRRGDTVFVNVHNQGNHAVSLKWEGVKGSIDGSNELIQPGRNFTYKIELKGEIGTLWWHATSAWAAATVHGAFVISPAANEDYP FPAPTSDQTIILGQWFKQELTEGDKTIAPGRADAYTINGHPGETYGCSNDTTFEMQVDYEGLYLVRVINAVVNETMVFGVASHSFTIVGQNGAYTKRSFTNSLTLAPAQVVDVLLCANVNVGHYYITARPSSGTYITNGILRYLTTSS (SEQ ID No.2).

[0050] Test Example 1 GhLAC14-3 Gene expression pattern identification assay 1 Test methods Take 1 mL of Verticillium dahliae V991 bacterial suspension and add it to CM medium (6 g / L yeast extract, 6 g / L acid hydrolyzed casein and 10 g / L sucrose), culture at 25°C, 200 rpm, and culture for 4-5 days. Count the spore concentration using a hemocytometer. When the spore concentration reaches 2×10 7 cfu / mL, filter out the mycelium with 4 layers of gauze, collect the spore solution, and soak the cotton roots in the V991 spore solution. After soaking for 0 hours post-incubation (hpi), 0.5 hpi, 1 hpi, 2 hpi, 4 hpi, and 8 hpi, extract the RNA from the root tissue and synthesize cDNA by reverse transcription. QUR qRT-PCR was performed for the internal reference gene. The primer sequences were shown in Table 1. Three replicates were set. According to the Ct values ​​of the target gene and the internal reference gene, 2 -ΔΔCt Methods The expression level of the target gene was calculated, where the Ct value was the cycle threshold value.

[0051] 2 Test results Quantitative PCR analysis GhLAC14-3 The expression changes of genes after induction by Verticillium dahliae V991, qRT-PCR results are shown in Figure 1 shown. GhLAC14-3The gene expression levels at 0.5 hpi, 4 hpi and 8 hpi of Verticillium dahliae infection were significantly different from those in the control group at the same time points, and the expression levels were 3-5 times higher than those in the control group, indicating that GhLAC14-3 The gene can respond to V991-induced expression transiently and increase significantly at the early stage of infection. GhLAC14-3 Genes play a role in the infection process of pathogens.

[0052] Experimental Example 2 GhLAC14-3 subcellular localization assay 1 Test methods design GhLAC14-3 CDS sequence primers of the gene and in pCAMBIA1304- GFP Vector ( Nco Ⅰ and Spe The target fragment was inserted into the target fragment after restriction endonuclease I digestion and linearization. The correctly sequenced plasmid was transformed into GV3101 Agrobacterium competent cells. Fluorescence was observed under a confocal microscope 48-72 h after injection into tobacco epidermal cells, with pH2B-mCherry and PIP2A-mCherry as marker controls.

[0053] 2 Test results Agrobacterium was used to transform tobacco leaves. GhLAC14-3-GFP The cellular localization of GhLAC14-3 was determined by transiently expressing GFP in tobacco cells and using marker localization proteins. Figure 2 The fluorescence of the empty GFP is distributed in different organelles throughout the cell. Green fluorescence can be seen at the cell membrane of the epidermal cells of tobacco leaves expressing the GhLAC14-3-GFP fusion protein, and it overlaps with the red fluorescence of the cell membrane localization marker to form yellow fluorescence, proving that the GhLAC14-3 protein is localized at the cell membrane.

[0054] Test Example 3 GhLAC14-3 Test on the resistance of gene-silenced cotton plants to Verticillium wilt 1 Test methods The SGN-VIGS website (https: / / vigs.solgenomics.net / ) was used to design GhLAC14-3 The silencing fragment sequence of the gene was designed, and the upstream and downstream primers of the sequence were designed. The specific primer sequences are shown in Table 1. The silencing fragment sequence was amplified by PCR technology using cotton leaf cDNA as a template, and the In-Fusion technology was used to amplify the silencing fragment sequence in the TRV2 vector ( Eco RⅠ / Bam Insert the corresponding fragment into the position corresponding to the HⅠ restriction enzyme linearization, and convert TRV2:: GhLAC14-3 The plasmid was transformed into GV3101 Agrobacterium competent cells and infected with cotton. GhLAC14-3 When the leaves showed obvious albino phenotype, qRT-PCR was performed to detect the gene silencing efficiency.

[0055] The silent plants were inoculated with Verticillium dahliae, and the phenotypic differences between the silent plants and the control plants were observed 20 days later. The disease index was calculated using the leaf grading method, and the silent plants and the control plants were tested by stem splitting. The biomass of Verticillium dahliae in the plants was detected using the ITS / VE1 primer.

[0056] The disease classification and disease index calculation are as follows: The disease is graded into 5 levels, where Level 0: healthy plants; Level 1: 0%~25% of leaves show yellowing around veins; Level 2: 25%~50% of leaves are chlorotic or wilted; Level 3: 50%~75% of leaves are chlorotic or wilted; 75%~100% of leaves are chlorotic or wilted.

[0057] Disease index = ∑ (disease level × number of diseased plants) / (number of surveyed plants × 4) × 100%.

[0058] 2 Test results 2.1 Construction of VIGS recombinant vector Using cotton leaf cDNA as template, amplification GhLAC14-3 The target sequence of gene silencing, the electrophoresis results are as follows Figure 3 -A shows that the length of the detected product is 378 bp, which is in line with expectations; the product was cloned and sequenced, and the results obtained were consistent with the target sequence. Figure 3 -B, the electrophoresis results showed that the fragment size was about 378 bp and sequencing comparison analysis confirmed that pTRV2:: GhLAC14-3 The vector was constructed successfully.

[0059] 2.2 VIGS Silencing GhLAC14-3 Gene validation gene function results pTRV2, pTRV2:: GhCLA1 、pTRV2:: GhLAC14-3 Agrobacterium and pTRV1 Agrobacterium were mixed in equal proportions and infected cotton cotyledons respectively. Figure 4 -A, 20 days later, the cells were infected with pTRV2:: GhCLA1 The positive control plants showed albinism, indicating that the VIGS vector can work normally in the plant. The target gene of the positive control plants was detected by qRT-PCR technology. GhLAC14-3 The expression level of Figure 4 -B shows that compared with the control group pTRV2::00, the silenced plants GhLAC14-3 The expression level of GhLAC14-3Gene silenced plants. The phenotypic analysis results of the silenced plants after inoculation with Verticillium dahliae for about 20 days are as follows Figure 4 -C, compared with the control plant pTRV2::00, pTRV2:: GhLAC14-3 The leaves of the gene-silenced plants turned yellow and wilted and fell off severely. The stems were cut longitudinally to observe the pTRV2:: GhLAC14-3 The degree of browning of the vascular bundles of the gene-silenced plants showed that the degree of browning of the vascular bundles of the silenced plants was deeper than that of the control group pTRV2::00. The statistical results of the disease index of the plants and the fungal biomass in the plants are shown in Figure 4 -D with Figure 4 -E, pTRV2:: GhLAC14-3 The disease index and fungal biomass of the gene-silenced plants were significantly higher than those of the control plants pTRV2::00. GhLAC14-3 Genes play an important role in cotton's resistance to infection by Verticillium dahliae.

[0060] Test Example 4 GhLAC14-3 Functional validation test of genes in Arabidopsis 1 Test methods To verify GhLAC14-3 Gene function, will contain pCAMBIA1304- GhLAC14-3 - GFP Agrobacterium carrying the plasmid was used to infect Arabidopsis thaliana, and after obtaining homozygous transgenic Arabidopsis thaliana, the experiment of inoculating Verticillium dahliae was carried out. Wild-type Arabidopsis thaliana and T3 transgenic Arabidopsis thaliana OE-2 and OE-4 were inoculated with Verticillium dahliae V991, and phenotypic observation, disease index and fungal biomass were measured after 14 days.

[0061] 2 Test results will contain Figure 5 -pCAMBIA1304 shown in A- GhLAC14-3 The Arabidopsis thaliana was infected with Agrobacterium, and the T0 generation plants were subjected to RT-PCR. The RT-PCR results were as follows: Figure 5 -B, we get GhLAC14-3 After the T3 generation homozygous Arabidopsis was screened, it was inoculated with Verticillium dahliae V991 and the phenotypic changes were observed after 14 days. Figure 5 -C. After 14 days, the leaves of wild-type Arabidopsis gradually turned yellow until the whole plant died. GhLAC14-3 Some leaves of Arabidopsis thaliana with the gene showed typical symptoms of Verticillium wilt, but the leaves remained green. The disease index of the plants and the statistical results of the fungal biomass in the plants are shown in Figure 5 -D with Figure 5 -E, GhLAC14-3The disease index and fungal biomass of the gene overexpression plants were significantly lower than those of the control plants. GhLAC14-3 The gene plays a positive regulatory role in the plant's resistance to Verticillium wilt.

[0062] Experimental Example 5 GhLAC14-3 screening of interacting proteins 1 Test methods 1.1 Screening of GhLAC14-3 interacting proteins Using In-Fusion technology to construct pGBKT7- GhLAC14-3 The plasmid was co-transformed with the pGADT7 plasmid into yeast AH109 competent cells, and then spread on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade solid culture medium plates, with pGBKT7-53+pGADT7-T as a positive control and pGBKT7-Lam+pGADT7-T as a negative control to observe the cell growth status. After 2 days, the growth status of yeast in the culture medium was observed to determine whether pGBKT7- GhLAC14-3 Toxicity and self-activation activity of plasmids. GhLAC14-3 The plasmid was co-transformed with the yeast library plasmid into AH109 yeast competent cells and coated on SD / -Trp / -Leu solid culture medium plates. After about 2 days of growth of clones, single clones were spotted onto SD / -Trp / -Leu / -His / -Ade solid culture medium. After the colonies grew (about 4-5 days), single clones were picked, PCR amplified and sequenced, and the results were compared by BlastP on the NCBI website to obtain candidate proteins that may interact with the GhLAC14-3 protein.

[0063] 1.2 Verification of protein interaction by two-hybrid assay (Y2H), in vivo bimolecular complementation assay (BiFC) and in vivo luciferase assay (LCI) Using In-Fusion technology to construct pGADT7- GhMAPKKK2 vector, and with pGBKT7- GhLAC14-3 The plasmid was transformed into AH109 yeast competent cells for yeast two-hybrid verification, and the cells were spread on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade+X-α-Gal solid culture medium plates, and the cell growth status was observed. In order to determine whether the candidate protein GhMAPKKK2 interacts with the GhLAC14-3 protein in plants, pYFPNE- GhLAC14-3 and pYFPCE- GhMAPKKK2The vector was constructed and transformed into GV3101 Agrobacterium competent cells. Agrobacterium containing pYFPNE and pYFPCE were mixed in equal proportions and injected into tobacco cells for bimolecular fluorescence complementation (BiFC) experiments. The fluorescence reaction was observed under a confocal microscope. GhLAC14-3 With pLUCC- GhMAPKKK2 The vector was used to transform GV3101 Agrocanthus competent cells, and luciferase interaction assay (LCI) was performed after injection into tobacco, and the fluorescence reaction was observed using a plant living imager.

[0064] 2 Test results 2.1 pGBKT7- GhLAC14-3 Toxicity and self-activation assay results The constructed pGBKT7- GhLAC14-3 The plasmid and the pGADT7 empty vector were transformed into yeast AH109 competent cells, which were first spread on SD / -Leu / -Trp solid culture medium plates, with pGBKT7-53+pGADT7-T as the positive control and pGBKT7-Lam+pGADT7-T as the negative control. The results are as follows Figure 6 -A. Figure 6 -B. Figure 6 -C, positive control pGBKT7-53+pGADT7-T, negative control pGBKT7-Lam+pGADT7-T and pGBKT7- GhLAC14-3 +pGADT7 can grow on SD / -Leu / -Trp solid medium plates. GhLAC14-3 The plasmid had no obvious toxicity to yeast. The three groups of bacterial suspension were then spread on SD / -Trp / -Leu / -His / -Ade solid culture medium plates. The results were as follows Figure 6 -D. Figure 6 -E, Figure 6 -F, it was found that only the positive control pGBKT7-53+pGADT7-T could grow on the SD / -Trp / -Leu / -His / -Ade solid medium plate, while the negative controls pGBKT7-Lam+pGADT7-T and pGBKT7- GhLAC14-3 +pGADT7 could not grow on SD / -Trp / -Leu / -His / -Ade solid medium plates, indicating that pGBKT7- GhLAC14-3 The plasmid showed no obvious self-activation in the yeast two-hybrid system. GhLAC14-3 The plasmid can be used for subsequent yeast library screening of interacting proteins.

[0065] 2.2 Verification results of interacting proteins pGBKT7- GhLAC14-3 The plasmid was co-transformed with the cotton yeast library plasmid into AH109 yeast competent cells, and then the colonies were grown on a solid medium containing SD / -Trp / -Leu. A single colony was picked and grown on a solid medium containing SD / -Trp / -Leu / -His / -Ade. After a single clone was grown, PCR amplification and sequencing were performed. The sequencing results were aligned by BlastP in NCBI, and the candidate protein GhMAPKKK2 (XM_016855464.2) for the interaction of GhLAC14-3 was obtained.

[0066] The pGADT7 recombinant vector of the candidate protein gene was obtained by In-Fusion technology and then combined with pGBKT7- GhLAC14-3 The plasmid was co-transformed into AH109 competent cells, and finally its growth status was confirmed to be the same as that of the positive control, and the yeast cells turned blue in the medium containing X-α-Gal ( Figure 7 -A).

[0067] Bimolecular fluorescence complementation (BiFC) assay verifies protein interaction results Figure 7 As shown in Figure 5-B, when GhLAC14-3-nYFP and GhMAPKKK2-cYFP were co-expressed in tobacco cells, yellow fluorescence was detected on the cell membrane, whereas no fluorescence was detected when GhLAC14-3-nYFP and cYFP or nYFP and GhMAPKKK2-cYFP or cYFP and nYFP were co-expressed.

[0068] The results of luciferase complementation assay (LCI) to verify protein interaction are as follows Figure 7 As shown in Figure 5-C, fluorescence was detected on the leaf surface when GhLAC14-3-nLCI and GhMAPKKK2-cLCI were co-expressed in tobacco leaves, while no fluorescence was detected when GhLAC14-3-nLCI and cLCI or nLCI and GhMAPKKK2-cLCI were co-expressed, indicating that GhMAPKKK2 interacts with GhLAC14-3.

Claims

1. GhLAC14-3 Gene, GhLAC14-3 Genes encoding proteins or containing GhLAC14-3 Use of a gene expression cassette or a recombinant plant expression vector in regulating plant resistance to Verticillium wilt; wherein, Said GhLAC14-3 The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No. 1; Said GhLAC14-3 The protein encoded by the gene is the amino acid sequence shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that The regulation of plant resistance to Verticillium wilt is to improve the resistance of plants to Verticillium wilt, including: GhLAC14-3 Genes are overexpressed in plants.

3. The use according to claim 1, characterized in that The regulation of plant resistance to Verticillium wilt is to reduce the resistance of plants to Verticillium wilt, including: inhibiting GhLAC14-3 Gene expression or GhLAC14-3 The expression level of the gene in the plant is reduced.

4. The use according to claim 1, characterized in that include: Through regulation GhLAC14-3 Genes in plants regulate plant resistance to Verticillium wilt at the transcription and translation levels.

5. The use according to claim 1, characterized in that: The plant is cotton or Arabidopsis thaliana.

6. A method for improving plant resistance to Verticillium wilt, characterized in that: include: Build contains GhLAC14-3 Gene overexpression recombinant plant expression vector; GhLAC14-3 Gene overexpression recombinant plant expression vector transformed plants, so GhLAC14-3 The gene is overexpressed in plants, and the resulting transgenic plants have increased resistance to Verticillium wilt; wherein the GhLAC14-3 The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.

1.

7. A method for breeding plant varieties resistant to Verticillium wilt, characterized in that: include: Build contains GhLAC14-3 Gene overexpression recombinant plant expression vector; GhLAC14-3 Gene overexpression recombinant plant expression vector transformed plants, so GhLAC14-3 The gene is overexpressed in plants to obtain transgenic positive plants and screen plant varieties with improved resistance to Verticillium wilt; wherein the GhLAC14-3 The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1.

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