CN119372251A - Cotton GhLAC14-3 gene and its use in regulating plant resistance to Verticillium wilt - Google Patents

Abstract

The present invention discloses a cotton GhLAC14‑3 gene and its use in regulating plant resistance to Verticillium wilt. The present invention isolates and obtains the GhLAC14‑3 gene from cotton, and its nucleotide sequence is shown in SEQ ID No.1, and its amino acid sequence of the encoded protein is shown in SEQ ID No.2. The present invention further conducts a preliminary study on the function of the cotton GhLAC14‑3 gene in cotton resistance to Verticillium wilt, and the results show that silencing the GhLAC14‑3 gene in cotton significantly reduces the ability of cotton seedlings to resist Verticillium wilt; overexpressing the GhLAC14‑3 gene in Arabidopsis significantly improves the ability of Arabidopsis to resist Verticillium wilt, thereby determining that the GhLAC14‑3 gene positively regulates the resistance of plants to Verticillium dahliae. The present invention has application prospects in improving plant resistance to Verticillium wilt or cultivating Verticillium wilt-resistant plant varieties.

Description

Cotton GhLAC14-3 gene and application thereof in regulating and controlling verticillium wilt resistance of plants

Technical Field

The invention relates to a disease-resistant related gene separated from cotton and application thereof, in particular to GhLAC-14-3 gene separated from cotton and application thereof in regulating and controlling verticillium wilt resistance of plants, belonging to the field of cotton GhLAC-3 gene and application thereof.

Background

Cotton is an important commercial crop, the most important natural fiber source in the world, accounting for about 35% of the total fibers in the world. Compared with synthetic fibers, cotton is a renewable resource and has important environmental and social benefits. Cotton verticillium is mainly caused by verticillium dahliae (Verticillium dahliae). Verticillium dahliae has rapid variation, complex pathogenic principle and wide distribution, so verticillium wilt is called as "cancer" of cotton, and is one of serious diseases of cotton. The pathogenic molecular mechanisms of Verticillium dahliae are relatively complex, and it is widely accepted that the two hypotheses are the "catheter blockage hypothesis" and the "toxin hypothesis", respectively. In addition, verticillium dahliae secrete a large amount of cell wall degrading enzymes including pectase, cellulase and the like. The pathogenicity of these species of verticillium dahliae is affected to some extent by the pathogenic factors of the species.

A series of related genes and proteins are needed in the verticillium wilt resistance process of cotton, and along with the completion of cotton genome sequencing, a series of verticillium wilt resistance genes are successively excavated and identified, namely an ATP binding protein gene ABCF5 is cloned from Raymond cotton, and through a VIGS technology, the negative control factor （Dong Q, Magwanga R O, Cai X, et al. RNA-sequencing, physiological and rnai analyses provide insights into theresponse mechanism of the abc-mediated resistance to Verticillium dahliae infection in cotton[J]. Genes, 2019, 10(2): 110）. gene GhCyP which is the verticillium wilt resistance of cotton can inhibit U-box E3 ubiquitin ligase GhPUB17, so that GhPUB loses disease resistance （Qin T, Liu S, Zhang Z, et al.GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17[J]. Plant Molecular Biology, 2019, 99(4-5): 379-393）. plant Laccase (LAC) is positioned in an exosome to participate in synthesis and polymerization of lignin. LAC has been reported to regulate lignin synthesis pathway involved in various plant resistance processes including resistance reaction of cotton to verticillium dahliae by analysis of expression profile of disease resistance gene of cotton inoculated with verticillium dahliae by RNA-seq sequencing, and increased expression level （Xu L,Zhu L, Tu L, et al. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry[J]. Journal of Experimental Botany,2011, 62(15): 21-5607）. over-expression of upland cotton GhLAC15 gene in Arabidopsis can enhance resistance to verticillium dahliae （Zhang Y, Wu L, Wang X, et al. The cotton laccase gene 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 by increasing cell wall lignification degree and increasing lignin composition to cause increased lignification, resulting in increased resistance of plants to verticillium dahliae （Hu Q, Min L, Yang X, et al. Laccase GhLac1 modulates broad-spectrum biotic stress tolerance via manipulating phenylpropanoid pathway and jasmonic acidsynthesis[J]. Plant Physiology, 2018, 176(2): 1808-1823）. and thus, excavation of verticillium dahliae resistance related gene of cotton has application prospect in improving verticillium dahliae resistance of cotton or cultivating verticillium dahliae resistant plant variety and the like.

Disclosure of Invention

It is an object of the present invention to provide GhLAC-14-3 gene isolated from cotton and its encoded protein;

It is a second object of the present invention to provide an expression cassette or recombinant expression vector comprising GhLAC gene;

The third purpose of the invention is to apply GhLAC gene 14-3, coded protein of GhLAC gene 14-3, expression cassette or expression vector containing GhLAC gene 14-3 to regulate and control verticillium wilt resistance of plants or to cultivate verticillium wilt resistant plant varieties.

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises:

in one aspect of the present invention, there is provided GhLAC-14-3 gene related to controlling verticillium wilt resistance isolated from upland cotton, the nucleotide sequence of GhLAC-14-3 gene is selected from any one of (a) to (d):

(a) A polynucleotide shown in SEQ ID No. 1;

(b) A nucleotide sequence capable of hybridizing under stringent hybridization conditions to the complement of the polynucleotide sequence of SEQ ID No. 1;

(c) A nucleotide sequence having at least 90% or more homology to the polynucleotide shown in SEQ ID No. 1;

(d) A mutant with deletion, substitution or insertion of one or more bases based on the nucleotide shown in SEQ ID No.1, and the mutant still has the function or activity of regulating and controlling plant verticillium wilt resistance.

The percentage of sequence homology described in the present invention can be obtained by well known bioinformatics algorithms, including Myers and Miller algorithms, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul algorithms, as is well known to those skilled in the art.

In addition, one skilled in the art can optimize the nucleotide sequence shown in SEQ ID No.1 to enhance expression efficiency in plants, e.g., can use preferred codons of the target plant to synthesize a polynucleotide to enhance expression efficiency in the target plant.

The present invention can also be a mutant obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No.1, or by performing missense mutation of one or more base pairs.

The nucleotide sequence of the GhLAC-3 gene can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of GhLAC-14-3 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 a function of regulating verticillium resistance of plants.

In addition, the nucleotide sequence described in the present invention may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

The chimeric genes or expression cassettes obtained by chimeric or connecting the genes shown in SEQ ID No.1 and other genes belong to the protection scope of the invention, and recombinant expression vectors containing the chimeric genes or expression cassettes also belong to the protection scope of the invention.

Another aspect of the invention provides a coding protein of GhLAC-3 gene related to verticillium wilt resistance regulation, which is separated from upland cotton, and the amino acid sequence of the coding protein is shown as SEQ ID No. 2.

Another aspect of the invention is the use of GhLAC gene 14-3, protein encoded by GhLAC gene 14-3, expression cassette containing GhLAC gene 14-3 or recombinant plant expression vector for regulating plant verticillium resistance or for breeding verticillium resistant plant varieties.

In a preferred embodiment of the invention, the modulation of plant verticillium resistance is an increase in plant verticillium resistance.

For reference, the invention provides an embodiment, namely, the expression quantity or activity of the GhLAC14-3 protein related to plant verticillium wilt resistance is improved by over-expressing the GhLAC protein coding gene related to plant verticillium wilt resistance in plants, so that the plant verticillium wilt resistance is improved.

A preferred embodiment of the invention is a method for increasing verticillium wilt resistance or breeding a plant variety resistant to verticillium wilt comprising overexpressing GhLAC gene in a plant, enhancing the expression level of GhLAC14-3 gene or enhancing the function or activity of GhLAC14-3 protein, e.g., by ligating GhLAC14-3 gene derived from upland cotton with an expression regulatory element to obtain a recombinant plant expression vector expressing the gene in a plant, transforming the recombinant plant expression vector into a plant, and overexpressing GhLAC14-3 gene in the plant to obtain a transgenic plant having increased verticillium wilt resistance.

The invention provides a GhLAC-3 gene plant recombinant expression vector, which comprises a GhLAC-3 gene derived from upland cotton, and an expression regulatory element connected to obtain the recombinant plant expression vector, wherein the recombinant plant expression vector can consist of a 5 'end non-coding region, a nucleotide shown as SEQ ID NO.1 and a 3' non-coding region, wherein the 5 'end non-coding region can comprise a promoter sequence, an enhancer sequence or/and a translation enhancing sequence, the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter, and the 3' non-coding region can comprise a terminator sequence, an mRNA cleavage sequence and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells, for selection of transformed cells or tissues. The marker gene includes a gene encoding antibiotic resistance, a gene conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also includes phenotypic markers such as beta-galactosidase and fluorescent protein.

Transformation protocols and protocols for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, high velocity ballistic bombardment, and the like. In certain embodiments, the upland cotton GhLAC-3 gene may be provided to plants using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al PLANT CELL reports 1986.5:81-84).

In a preferred embodiment of the present invention, a method for reducing verticillium wilt resistance in a plant comprises mutating GhLAC' 14-3 gene in the plant to reduce the expression level of GhLAC14-3 gene or to cause a defect in the normal function of GhLAC14-3 protein.

The mutation comprises substitution, deletion and/or addition of one or more nucleotides on the nucleotide sequence of GhLAC gene or its promoter. Preferably, the mutation can be obtained by means of physical mutagenesis, chemical mutagenesis, gene editing, including but not limited to radiation mutagenesis, space breeding, etc., chemical mutagenesis including mutagenesis caused by treatment with a mutagen such as EMS, etc., and gene editing including but not limited to zFN, TALE, and/or CRISPR/Cas, etc.

The GhLAC-3 gene in the plant can be knocked out or mutated by a conventional method such as conventional gene knockout or gene editing technology, for example, a GhLAC-14-3 gene knocked out vector is constructed or a GhLAC-14-3 gene CRISPR/Cas9 gene editing vector is constructed by the gene editing technology, and GhLAC-3 gene in the plant can be knocked out or mutated by the conventional method, and all the methods are well known to the skilled person.

It is known to those skilled in the art that the main principle of CRISPR/Cas gene editing systems or gene editing methods is to find the location where gene editing is to be performed, i.e. to target DNA sequences, in the host genome by means of a nucleic acid fragment called guide-RNA (gRNA), and then cleave the DNA by means of Cas proteins. In the present application, the Cas protein includes, but is not limited to, cas9, cas12a, cas12j, cas12e, cas13, and/or Cas14, among others.

The interference GhLAC-3 gene or the normal function of the promoter can be achieved by adopting RNA interference technology (RNAi) to interfere the normal expression of the GhLAC-3 protein coding gene or the promoter thereof or cause the normal function to be defective, and the RNA interference technology is a conventional technology in the field, and the RNA interference technology specifically combines the 21-23bp short-chain double-stranded RNA (siRNA) or long-chain double-stranded RNA (dsRNA; double-STRAND RNA) with the mRNA homologous region expressed by the target gene to degrade the mRNA and achieve the effect of inhibiting the gene expression.

Plants described herein include, but are not limited to, monocotyledonous or dicotyledonous plants. Most preferably, the plant is cotton or Arabidopsis.

A series of differential expression genes (DIFFERENTIALLY EXPRESSED GENES, DEGs) are screened by a bioinformatics method through transcriptome analysis on the arabidopsis infected with the verticillium dahliae. The gene GhLAC-3 with obvious homology with DEG is obtained through NCBI website comparison, and the laccase protein is coded, the induction expression analysis of the gene is carried out on the verticillium dahliae to determine the response of the gene to verticillium dahliae infection, the function of the gene in verticillium resistance of cotton is initially explored by further utilizing subcellular localization, VIGS, arabidopsis genetic transformation and other molecular biological technologies, and the result shows that the gene GhLAC-3 can positively regulate and control the resistance of cotton to verticillium.

Definition of terms in connection with the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the preferred methods, devices, and materials are now described.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoroamidites, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.

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 as used herein also refers to the concept of similar functional properties between different polynucleotide molecules, e.g. promoters with similar functions may have homologous cis-elements. Polynucleotide molecules are homologous when they hybridize specifically under specific conditions to form duplex molecules. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule may be used as a probe or primer to identify another polynucleotide molecule that shares homology.

The term "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. In general, a probe will hybridize to its target sequence to a greater degree of detectability under stringent conditions than to other sequences (e.g., at least 2-fold over background. Stringent hybridization conditions will be sequence-dependent, and longer sequences will be different under different environmental conditions.) by controlling the stringency or wash conditions of hybridization, target sequences 100% complementary to the probe can be identified, detailed guidance for nucleic acid hybridization can be found in reference （Tijssen, Techniques in biochemistry and molecular biology hybridization with nucleic probes, "Overview ofprinciples of hybridization and the strategy of nucleic acid assays. 1993)., which is typically selected to be about 5-10℃ C.T _m below the thermal melting point (T _m) of the specific sequence at a defined ionic strength pH (at a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence (at a defined ionic strength, pH and nucleic acid concentration) is present in excess of the target sequence. 50% of the probe is occupied at a defined ionic strength, conditions can be those wherein salt concentration is below about 1. M sodium ion concentration at a pH of 7.0 to 8.3, typically about 0.35℃sodium ion concentration, and hybridization can be about 50℃or more than 30% of the other nucleic acid hybridization (at a defined ionic strength, such as 5-10℃or 50℃or more than 30% of the nucleic acid) is included in the condition of hybridization, and hybridization can be accomplished by addition of a stable hybridization agent such as a nucleotide (at least 50℃or at least 50% of a defined ionic strength) at a defined ionic strength) or at a nucleic acid concentration of the nucleic acid concentration is not present in excess of the equilibrium condition, 5 XSSC and 1% SDS at 42℃or 5 XSSC, 1% SDS at 65℃washed in 0.2 XSSC and 0.1% SDS at 65 ℃. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.

"Plurality" as used herein generally means 2 to 8, preferably 2 to 4, substitutions means substitution of one or more amino acid residues with different amino acid residues, deletions means reduction of the number of amino acid residues, i.e., lack of one or more amino acid residues, respectively, therein, insertions means changes in the amino acid residue sequence which result in addition of one or more amino acid residues relative to the native molecule.

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

The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest), 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 contiguous polynucleotide molecule, and more preferably are contiguous. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

The term "recombinant plant expression vector" means one or more DNA vectors used to effect transformation of plants, and these vectors are often referred to in the art as binary vectors. 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, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "transformation" refers to a process of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" means the transcription and/or translation of an endogenous gene or transgene in a plant cell.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, e.g., direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate 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.

Drawings

FIG. 1 is a graph of the fluorescent quantitative expression analysis of GhLAC-3 genes, wherein P <0.01 level difference is very significant, and P <0.05 level difference is significant;

FIG. 2 is a map of subcellular localization of GhLAC-3 protein in tobacco leaves;

FIG. 3 is a diagram showing the construction of an electrophoresis verification of GhLAC-14-3 gene pTRV2 vector, wherein FIG. 3-A is a diagram showing the amplification of GhLAC-14-3 PCR silencing fragment, 1 is a diagram showing the amplification of GhLAC-3 VIGS fragment, M is 2000 DNA Marker, FIG. 3-B is a diagram showing the detection and electrophoresis verification of colony PCR, 1-3 is a diagram showing the identification of recombinant plasmid bacterial liquid, M is 2000 DNA Marker;

FIG. 4 is a graph of resistance to verticillium wilt of GhLAC14-3 gene-silenced cotton plants, wherein FIG. 4-A is a graph of leaf phenotype identification after silencing GhCLA1 gene, bar=2 cm, FIG. 4-B is a graph of qRT-PCR detection GhLAC-3 silencing efficiency, FIG. 4-C is a graph of plant phenotype identification after 20D infection with Verticillium dahliae V991, bar=0.2 cm, FIG. 4-D is a graph of statistical indices of conditions of pTRV2:00 and pTRV2: ghLAC:14-3 cotton plants after 20D inoculation, and FIG. 4-E is a graph of pTRV2:00 and pTRV2: ghLAC:14-3 cotton plants relative to fungal biomass determination, wherein P <0.01 level difference is significant, and P <0.001 level difference is significant;

FIG. 5 is a graph for identifying the resistance of the over-expressed GhLAC-3 Arabidopsis thaliana inoculated with Verticillium dahliae, wherein FIG. 5-A is a model graph of the Pcambai 1304 vector, FIG. 5-B is a graph for RT-PCR detection GhLAC-3, FIG. 5-C is a graph for observing the phenotype of the over-expressed GhLAC-14-3 Arabidopsis thaliana inoculated with Verticillium dahliae V991, bar=2 cm, FIG. 5-D is a graph for statistics of disease index, and FIG. 5-E is a graph for statistics of fungal biomass, which shows that the difference in P <0.01 level is significant;

FIG. 6 is a graph of toxicity and self-activation activity of bait protein GhLAC-3, wherein FIG. 6-A is a graph of pGBKT7-lam+pGADT7-T at SD/-Trp/-Leu plate growth, FIG. 6-B is a graph of pGBKT7-53+pGADT7-T at SD/-Trp/-Leu plate growth, FIG. 6-C is a graph of pGBKT 7-GhLAC-3+pGADT 7 at SD/-Trp/-Leu plate growth, FIG. 6-D is a graph of pGBKT7-lam+pGADT7-T at SD/-Trp/-His/-Ade plate growth, FIG. 6-E is a graph of pGBKT7-53+pGADT7-T at SD/-Trp/-His/-Lee plate growth, and FIG. 6-F is a graph of pGBKT 7-GhLAC-3+pGADT at SD/-Trp/-His/-Lee plate growth;

FIG. 7 is a graph of results of interaction verification of GhLAC-3 with candidate protein GhMAPKKK2, wherein FIG. 7-A is a graph of yeast two-hybrid verification of GhLAC-3 and GhMAPKKK2, FIG. 7-B is a graph of BiFC verification of GhLAC-14-3 and GhMAPKKK2, and FIG. 7-C is a graph of LCI verification of GhLAC-14-3 and GhMAPKKK 2.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.

Test materials, vectors and strains

The upland cotton variety ' XLZ ', the transgenic Arabidopsis acceptor genotype is Columbia wild type (Colombia wild type, col-0), cotton VIGS vectors pTRV1, pTRV2 and pTRV2 are CLA1, verticillium dahliae strong pathogenic bacteria V991, escherichia coli DH5 alpha strain and Agrobacterium GV3101 competent cells, and the test materials, vectors and strains are all stored in a crop functional genomics and molecular improvement laboratory of Xinjiang agricultural university student's life sciences.

Plant growth conditions

Arabidopsis thaliana was grown under conditions of 24℃light for 16 hours (h) and 21℃darkness of 8 h on Murashige-Skoog (MS) agar medium. After 7 days (d), seedlings were transferred to sterilized turfy soil. The upland cotton growing condition is that seeds with full seeds are selected, washed by sterile water and soaked in a greenhouse at 37 ℃ for 24-36 h, the white seeds are planted in flowerpots containing a mixture of vermiculite and black soil (1:2) after autoclaving, three cotton plants are planted in each flowerpots, the growing condition is 28 ℃ indoor culture, and the illumination period is 16: 16 h under illumination and 8: 8 h under darkness.

The growth condition of the Nicotiana benthamiana is that the Nicotiana benthamiana is cultivated in a room at 25 ℃, the illumination period is 16 h of illumination and 8 of darkness h.

Test reagent

Kanamycin, gentamicin, MES, acetosyringone, mgCl ₂ and a culture medium are all domestic analytical reagents, a polysaccharide polyphenol plant total RNA extraction kit is purchased from Hangzhou Bori technology company, ecoR I, bamH I, ncoI, speI and other restriction endonucleases are purchased from Simer femto (Thermo) company, taqDNA polymerase, T4 DNALIGASE, IN-Fusion ligase, RNaseA, high-fidelity polymerase TRANSSTAR KD Plus, a reverse transcription kit, a fluorescence quantification kit, an agarose gel recovery kit, a DNA molecular weight Marker and the like are purchased from Beijing full-scale gold biotechnology Co. The synthesis of primers used in PCR and the sequencing of DNA were all performed by Shanghai Biotechnology Co.

Primer sequences

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

TABLE 1 primers used in this study

Note that lowercase letters are homology arms.

In the early stage of the test, a large number of differentially expressed genes were found by transcriptomic analysis of Arabidopsis thaliana inoculated with Verticillium dahliae (DIFFERENTIALLY EXPRESSED GENES). The highly homologous gene GhLAC-3 in cotton genome, which codes for a plant laccase involved in lignin synthesis and polymerization, is obtained by bioinformatic analysis and from NCBI website, ghLAC14-3 gene is a new gene which has not been reported to function in cotton.

The nucleotide sequence of GhLAC14-3 gene is shown in SEQ ID No.1 ：ATGGATAAAAACCAAAAACATTACAAGGAAAGAGAGGGGAGACCCGAAGAACACAGATAAACATAGAACCAAAAACAAGAAAAACATGGGTTCTGAAAAGCAAGGGTTTATATGGTTATCAGGGCTTTTGTTTCTGAATATCCTTGTGTTATCCACAGCTGATGTCCATTATTACGAGTTTTTTTTGCAAGAATCCCAGTTCACTAAGCTGTGTAGCACGAAGAGCATCTTGACCGTCAATGGCAGCTTTCCAGGGCCTGAGATTCGGGTTCGCAGAGGGGACACAGTTTTTGTCAACGTCCACAATCAAGGAAACCATGCTGTATCCCTCAAGTGGGAGGGCGTTAAGGGTTCAATTGATGGTTCGAATGAGTTGATTCAGCCAGGGAGAAACTTCACTTACAAGATAGAGTTAAAGGGTGAAATAGGAACTCTATGGTGGCACGCCACCAGTGCTTGGGCTGCGGCAACCGTCCACGGTGCCTTTGTCATTTCGCCGGCAGCGAATGAAGACTATCCTTTTCCCGCACCTACTTCTGACCAAACAATTATACTTGGGCAATGGTTCAAGCAAGAGTTAACAGAAGGTGATAAAACCATAGCTCCTGGCCGAGCAGATGCTTACACTATCAATGGCCATCCCGGAGAAACTTATGGATGCAGCAACGATACAACTTTTGAGATGCAAGTAGATTACGAGGGCCTTTACCTTGTTCGCGTAATAAATGCCGTTGTCAATGAAACAATGGTGTTTGGCGTAGCATCCCACAGCTTCACCATTGTCGGACAAAATGGGGCTTACACCAAACGTTCCTTTACAAATTCTCTAACCCTAGCACCCGCCCAAGTTGTTGACGTTCTGTTGTGTGCAAACGTAAACGTTGGCCATTATTACATCACTGCTCGACCTTCTTCTGGCACATATATTACCAACGGAATTCTACGATATCTCACCACTAGTTCTTAATTTAGTCAAACAGTTTTTCATTGCTAAAGATGAATAATGTTGAAGTTCAAATAAGAGCTCAATTAAAATATAGGTGTGAAAATTGTTGTATTATTTAAATATGTATAATATATTTTAAAATAAAATACATATTACTTATTAATGTTTTTGAGTTTTGAGGAATGGTCATTTTCCGTTTTAATGCATAAAA（SEQ ID No.1）.

The amino acid sequence of the protein coded by GhLAC gene is shown as SEQ ID No.2 ：MGSEKQGFIWLSGLLFLNILVLSTADVHYYEFFLQESQFTKLCSTKSILTVNGSFPGPEIRVRRGDTVFVNVHNQGNHAVSLKWEGVKGSIDGSNELIQPGRNFTYKIELKGEIGTLWWHATSAWAAATVHGAFVISPAANEDYPFPAPTSDQTIILGQWFKQELTEGDKTIAPGRADAYTINGHPGETYGCSNDTTFEMQVDYEGLYLVRVINAVVNETMVFGVASHSFTIVGQNGAYTKRSFTNSLTLAPAQVVDVLLCANVNVGHYYITARPSSGTYITNGILRYLTTSS（SEQ ID No.2）.

Test example 1 GhLAC14-3 Gene expression Pattern identification test

1. Test method

Verticillium dahliae V991 strain solution 1mL was added to CM medium (6 g/L yeast extract, 6 g/L acid hydrolyzed casein and 10 g/L sucrose), cultured at 25℃with 200rpm, cultured at 4-5 d and counted for spore concentration using a hemocytometer. When the spore concentration reached 2×10 ⁷ cfu/mL, mycelium was filtered off with 4 layers of gauze, spore liquid was collected, cotton roots were soaked in V991 spore liquid, RNA of root tissues was extracted and reverse transcribed into cDNA after soaking to 0 hours post-incubation (hpi), 0.5 hpi, 1 hpi, 2 hpi, 4 hpi and 8 hpi, respectively. qRT-PCR was performed using cotton housekeeping gene GhUBQ as an internal reference gene, with 3 replicates using the primer sequences shown in Table 1. According to Ct values of the target gene and the reference gene, the expression quantity of the target gene is calculated by using a 2 ^-ΔΔCt method, wherein the Ct value is a cycle threshold value.

2. Test results

The change in expression of GhLAC-3 gene after induction of Verticillium dahliae V991 was analyzed by fluorescent quantitative PCR, and the qRT-PCR results are shown in FIG. 1. The GhLAC14-3 genes have extremely obvious difference in gene expression quantity when the verticillium bacteria infects 0.5 hpi, 4 hpi and 8 hpi compared with the control group at the same time, the expression quantity is improved by 3-5 times compared with the control group, the GhLAC14-3 genes can respond to V991 to induce expression, the initial infection period is obviously improved, and the GhLAC14-3 genes are presumed to play a role in the pathogen invasion process.

Test example 2 GhLAC14-3 subcellular localization test

1. Test method

CDS sequence primers of GhLAC14-3 genes were designed and inserted into the pCAMBIA1304-GFP vector (NcoI and SpeI cleavage linearized), the correctly sequenced plasmids were transformed into GV3101 Agrobacterium competent cells, and fluorescence was observed under confocal microscopy after injection of tobacco epidermal cells 48-72 h, with pH2B-mCherry and PIP2A-mCherry as Marker controls.

2. Test results

Tobacco leaves were transformed with Agrobacterium to transiently express GhLAC-3-GFP and GFP in tobacco cells, while Marker targeting proteins were used to determine the cellular localization of GhLAC-14-3, the results are shown in FIG. 2. The fluorescence of empty GFP is distributed in different organelles of the whole cell, green fluorescence is visible at the cell membrane of the tobacco leaf epidermal cell expressing GhLAC-14-3-GFP fusion protein, and the fluorescence is overlapped with the red fluorescence of a cell membrane positioning Marker to be yellow fluorescence, which proves that GhLAC-14-3 protein is positioned at the cell membrane.

Test example 3 GhLAC14-3 Gene cotton silencing plant verticillium wilt resistance identification test

1. Test method

The sequence of the silencing fragment of GhLAC-3 gene was designed using SGN-VIGS website (https:// VIGS. Solgenemics. Net /), and the upstream and downstream primers of the sequence were designed, and the specific primer sequences are shown in Table 1. The cDNA of cotton leaf is used as template, the PCR technology is used to amplify the silent fragment sequence, the In-Fusion technology is used to insert corresponding fragment In the corresponding position of TRV2 vector (EcoRI/BamHI digestion linearization), and the TRV2:: ghLAC-3 plasmid is used to transform GV3101 Agrobacterium competent cell to infect cotton. When the leaves of positive control plant pTRV2: ghLAC14-3 show obvious albino phenotype, qRT-PCR was performed to detect the gene silencing efficiency.

And (3) inoculating the silencing plant with verticillium dahliae, observing the phenotype difference between the silencing plant and a control plant after 20 d, and carrying out stem cutting detection on the silencing plant and the control plant by adopting a leaf grading method to count the disease index. And detecting the biomass of the verticillium dahliae in the plant body through the ITS/VE1 primer.

The disease classification and disease index were calculated as follows:

The disease classification comprises 5 grades, wherein 0 grade is healthy plants, 1 grade is yellowing occurs around 0% -25% of leaf veins, 2 grade is that 25% -50% of leaves are chlorosis or wilting, 3 grade is that 50% -75% of leaves are chlorosis or wilting, and 75% -100% of leaves are chlorosis or wilting.

Disease index = Σ (disease grade x disease grade number)/(study number x 4) ×100%.

2. Test results

2.1 Construction of a recombinant vector of VIGS

The cotton leaf cDNA is used as template to amplify the silencing target sequence of GhLAC-3 gene, the electrophoresis result is shown in figure 3-A, the detected product length is 378bp, the method is in line with the expectation, the clone sequencing is carried out on the product, and the result is consistent with the target sequence. The PCR detection result of the recombinant vector is shown in the figure 3-B, the electrophoresis result shows that the fragment size is about 378bp, and the sequencing comparison analysis is carried out to determine that pTRV2: ghLAC14-3 vector construction is successful.

2.2 Verification of Gene function results by VIGS silencing GhLAC-3 Gene

PTRV2, pTRV2: ghCLA1 and pTRV2: ghLAC14-3 were mixed with pTRV1 Agrobacterium in equal proportions, respectively, and the cotton cotyledons were infected. The phenotypic observation results are shown in FIG. 4-A, and the albinism phenomenon appears in positive control plants of the infected pTRV2: ghCLA1 after 20 d, which indicates that the VIGS vector can work normally in the plant body. The expression level of the target gene GhLAC-3 of the positive control plant detected by the qRT-PCR technology is shown as a figure 4-B, and compared with the pTRV 2:00 of the control group, the expression level of GhLAC-3 of the silent plant is obviously lower than that of the control group, which indicates that the GhLAC14-3 gene silent plant is successfully obtained. The analysis result of phenotype of 20 d of the silencing plant after being inoculated with verticillium dahliae is shown in fig. 4-C, compared with that of a control plant pTRV 2:00, the yellowing degree of leaves of the silencing plant pTRV2: ghLAC14-3 is serious to be withered, and the browning degree of vascular bundles of the silencing plant is shown to be deeper than that of the control plant pTRV 2:00 by observing the pTRV2: ghLAC14-3 through longitudinal cutting of stems. The plant disease index and the plant in-vivo fungal biomass statistical result are shown in the figure 4-D and the figure 4-E, and the pTRV2: ghLAC14-3 gene silencing plant disease index and the plant in-vivo fungal biomass are obviously higher than those of a control plant pTRV 2:00. Taken together, the GhLAC-14-3 gene plays an important role in the process of resisting the infection of the verticillium dahliae by cotton.

Test example 4 functional verification test of transgenic GhLAC14-3 Gene Arabidopsis thaliana

1. Test method

In order to verify the function of GhLAC gene 14-3, agrobacterium containing pCAMBIA1304-GhLAC14-3-GFP plasmid was used to infect Arabidopsis thaliana, and after homozygous transgenic Arabidopsis thaliana was obtained, a Verticillium dahliae inoculation experiment was performed. Phenotype observations, disease indices and determination of fungal biomass were performed after inoculation of wild type Arabidopsis thaliana and T ₃ generation transgenic Arabidopsis thaliana OE-2 and OE-4,14 d with Verticillium dahliae V991.

2. Test results

The agrobacteria containing pCAMBIA1304-GhLAC14-3 shown in FIG. 5-A are used for infecting Arabidopsis thaliana, T ₀ generation plants are subjected to RT-PCR, and the result of the RT-PCR is shown in FIG. 5-B, so that transgenic Arabidopsis thaliana containing GhLAC14-3 genes is obtained. After screening for T ₃ generation homozygous Arabidopsis thaliana, the same was inoculated with Verticillium dahliae V991,14, d, and the phenotypic change was observed, and the results are shown in FIG. 5-C. 14 After d, the leaves of the wild arabidopsis thaliana gradually turn yellow until the whole plant is completely dead, and the leaves of the part of arabidopsis thaliana which is over-expressed with GhLAC gene show typical symptoms of verticillium wilt, but the leaves still remain green. The plant disease index and the plant in-vivo fungal biomass statistical result are shown in the figures 5-D and 5-E, and the GhLAC14-3 gene over-expression plant disease index and the plant in-vivo fungal biomass are obviously lower than those of a control plant. From the above results, ghLAC gene 14-3 plays a positive regulatory role in verticillium wilt resistance of plants.

Test example 5 GhLAC14-3 screening interaction protein test

1. Test method

1.1 GhLAC14 screening for 14-3 interacting proteins

Constructing pGBKT7-GhLAC14-3 plasmid by utilizing an In-Fusion technology, co-transforming the plasmid and pGADT7 plasmid into yeast AH109 competent cells, and sequentially coating the competent cells on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade solid medium plates, wherein pGBKT7-53+pGADT7-T is used as a positive control, and pGBKT7-lam+pGADT7-T is used as a negative control to observe the growth state of the cells. 2 d, and observing the growth state of the yeast in the culture medium, so as to judge the toxicity and the self-activation activity of the pGBKT7-GhLAC14-3 plasmid. After completion, pGBKT 7-GhLAC-3 plasmid and yeast library plasmid were co-transformed into AH109 yeast competent cells, SD/-Trp/-Leu solid medium plates were coated, after cloning was performed at about 2 d, monoclonal spots were grown to SD/-Trp/-Leu/-His/-Ade solid medium after colony had been performed (about 4-5 d), monoclonal were picked, PCR amplified and sequenced, and BlastP alignment was performed on the results in NCBI website to obtain candidate proteins likely to interact with GhLAC14-3 protein.

1.2 Verification of protein interactions by two-hybrid assay (Y2H), in vivo two-molecule complementation assay (BiFC) and in vivo luciferase assay (LCI)

Constructing pGADT7-GhMAPKKK2 vector by utilizing In-Fusion technology, transforming AH109 yeast competent cells with pGBKT7-GhLAC14-3 plasmid for yeast two-hybrid verification, respectively coating on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade+X-alpha-Gal solid culture medium plates, and observing the growth state of the cells. To confirm that candidate protein GhMAPKKK interacted with GhLAC14-3 protein in plants, pYFPNE-GhLAC14-3 and pYFPCE-GhMAPKKK2 vectors were constructed and GV3101 agro-rod competent cells were transformed. And the tobacco cells were mixed and injected in equal proportions with pYFPNE and pYFPCE for a bimolecular fluorescence complementation experiment (Bimolecular Fluorescence Complementation, biFC), and fluorescence reaction was observed under a confocal microscope. Then pLUCN-GhLAC14-3 and pLUCC-GhMAPKKK2 vectors were constructed again and transformed into GV3101 agro-rod competent cells, and luciferase interaction experiments (Luciferase Complementation Assay, LCI) were performed after tobacco injection and fluorescence response was observed with a plant in vivo imager.

2. Test results

2.1 PGBKT7-GhLAC14-3 toxicity and self-activation detection results

The constructed pGBKT 7-GhLAC-3 plasmid and pGADT7 empty vector are transformed into yeast AH109 competent cells, and the cells are coated on SD/-Leu/-Trp solid medium plates, wherein pGBKT7-53+pGADT7-T is used as a positive control, pGBKT7-lam+pGADT7-T is used as a negative control, and the positive control pGBKT7-53+pGADT7-T, the negative control pGBKT7-lam+pGADT7-T and pGBKT 7-GhLAC-3+pGADT 7 can grow on SD/-Leu/-Trp solid medium plates as shown in the figure 6-A, the figure 6-B and the figure 6-C. The pGBKT7-GhLAC14-3 plasmid was not significantly toxic to yeasts. Three groups of bacterial liquids were then spread on SD/-Trp/-Leu/-His/-Ade solid medium plates, and as a result, as shown in FIG. 6-D, FIG. 6-E and FIG. 6-F, it was found that only the positive control pGBKT7-53+pGADT7-T could grow on SD/-Trp/-Leu/-His/-Ade solid medium plates, while the negative control 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 plasmid had no apparent self-activation in the yeast two-hybrid system. In summary, pGBKT7-GhLAC14-3 plasmid can be used in subsequent yeast library screening interaction protein experiments.

2.2 Verification results of interacting proteins

Co-transforming pGBKT7-GhLAC14-3 plasmid and cotton yeast library plasmid into AH109 yeast competent cells, coating on a plate containing SD/-Trp/-Leu solid culture medium to grow colonies, picking single colonies to grow on the SD/-Trp/-Leu/-His/-Ade solid culture medium, growing monoclonal, and carrying out PCR amplification and sequencing. The sequencing results were BlastP aligned in NCBI to yield GhLAC-3 interaction candidate protein GhMAPKKK2 (xm_ 016855464.2).

PGADT7 recombinant vector of the interacting candidate protein gene was obtained by In-Fusion technique and AH109 competent cells were co-transformed with pGBKT7-GhLAC14-3 plasmid, which finally confirmed that the growth state was the same as positive control and that the yeast cells turned blue In the X- α -Gal-containing medium (FIG. 7-A).

The results of the bimolecular fluorescence complementation assay (BiFC) to verify protein interaction are shown in FIG. 7-B, where yellow fluorescence is detected on the cell membrane when GhLAC-3-nYFP and GhMAPKKK2-cYFP are co-expressed in tobacco cells, and no fluorescence is detected when GhLAC-3-nYFP and cYFP or nYFP and GhMAPKKK2-cYFP or cYFP and nYFP are co-expressed.

The results of the luciferase complementation assay (LCI) to verify protein interaction are shown in FIG. 7-C, fluorescence was detected on the leaf surface when GhLAC-3-nLCI and GhMAPKKK-cLCI were co-expressed in tobacco leaves, whereas no fluorescence was detected when GhLAC-3-nLCI and cLCI or nLCI and GhMAPKKK-cLCI were co-expressed, indicating interaction of GhMAPKKK2 with GhLAC 14-3.

Claims (7)

1. The application of GhLAC14-3 gene, ghLAC gene coding protein, or GhLAC14-3 gene containing expression cassette or recombinant plant expression vector in regulating and controlling plant verticillium resistance, wherein the nucleotide sequence of GhLAC14-3 gene is polynucleotide shown as SEQ ID No. 1;

   the coding protein of GhLAC gene is the amino acid sequence shown as SEQ ID No. 2.
2. 2. The method according to claim 1, wherein the controlling of verticillium wilt resistance in plants is increasing resistance to verticillium wilt in plants, comprising over-expressing GhLAC of the gene 14-3 in plants.
3. 3. The use according to claim 1, wherein the modulation of verticillium resistance in plants is a reduction of verticillium resistance in plants comprising inhibition of GhLAC gene expression in plants or a reduction of GhLAC14-3 gene expression in plants.
4. 4. The method according to claim 1, wherein the plant resistance to verticillium is regulated by regulating the level of transcription and translation of GhLAC gene in the plant.
5. 5. The use according to claim 1, wherein the plant is cotton or arabidopsis.
6. 6. A method for improving the resistance of a plant to verticillium wilt is characterized by comprising the steps of constructing an over-expression recombinant plant expression vector containing GhLAC gene 14-3, transforming the plant with the over-expression recombinant plant expression vector containing GhLAC gene 14-3 to enable the GhLAC gene 3 to be over-expressed in the plant, and improving the resistance of the obtained transgenic plant to verticillium wilt, wherein the nucleotide sequence of the GhLAC gene 14-3 is a polynucleotide shown as SEQ ID No. 1.
7. 7. A method for cultivating a verticillium wilt-resistant plant variety is characterized by comprising the steps of constructing an over-expression recombinant plant expression vector containing GhLAC gene, transforming a plant with the over-expression recombinant plant expression vector containing GhLAC gene 14-3, enabling GhLAC gene 14-3 to be over-expressed in the plant, obtaining a transgenic positive plant, and screening to obtain the plant variety with improved verticillium wilt resistance, wherein the nucleotide sequence of GhLAC gene 14-3 is a polynucleotide shown as SEQ ID No. 1.