Cotton GhATG1t gene and its use in regulating plant resistance to Verticillium wilt

Abstract

The invention discloses cotton GhATG1t Gene and its use in regulating plant resistance to Verticillium wilt. The present invention is isolated and obtained from cotton GhATG1t The gene, whose nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence of its encoded protein is shown in SEQ ID No.2. The present invention further utilizes virus-induced gene silencing technology and transgenic overexpression Arabidopsis technology to GhATG1t The preliminary study on the function of genes in cotton resistance to Verticillium wilt found that GhATG1t Gene silencing significantly reduced the resistance of cotton seedlings to Verticillium wilt. GhATG1t Overexpression of the gene in Arabidopsis significantly improved the resistance of Arabidopsis to Verticillium wilt. GhATG1t The gene positively regulates the resistance of plants to Verticillium dahliae; the invention has application prospects in improving the resistance of plants to Verticillium wilt or cultivating Verticillium wilt-resistant plant varieties.

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. GhATG1t Gene and its use in regulating plant resistance to Verticillium wilt, cotton GhATG1t Genes and their areas of use. Background Art

[0002] Upland cotton ( Gossypium hirsutum ) belongs to the Malvaceae family ( Malvaceae ) Cotton Gossypium ) plant, is one of the important economic crops. Verticillium wilt is a soil-borne fungal disease that seriously affects the growth and development of cotton plants. Verticillium dahliae ) is caused by Verticillium wilt. To date, no effective fungicide has been used to prevent infection with Verticillium dahliae, and common upland cotton varieties rarely show resistance to Verticillium dahliae. Nowadays, studies have shown that transgenic methods have successfully provided cotton with resistance to pathogenic fungi. Therefore, discovering more genes related to cotton wilt resistance and exploring the corresponding mechanisms are of great significance for the breeding of new disease-resistant cotton varieties.

[0003] Many genes related to resistance to Verticillium wilt have been identified. These resistance genes can be divided into two categories: one is the resistance gene that directly resists the invasion of pathogens, such as GhCDPK28-A10 (Zhang Man, Zhang Jin, Zhang Xinyu, et al. Upland cotton GhNAC1 Cloning and functional analysis of the gene against Verticillium wilt[J]. China Agricultural Science and Technology Herald, 2023, 25: 35-44.) and GhNPR1 (Zeng Yanming, Hu Guang, Jia Pei, etc. Cotton WRKY7 Gene cloning and analysis of resistance to Verticillium wilt [J]. Acta Agriculturae Boreali-Sinica, 2022, 37: 191-200.), etc. The proteins encoded by these genes can recognize pathogens and trigger a series of defense responses, such as producing antibacterial substances and activating the expression of immune-related genes. Some genes also improve disease resistance by regulating the plant immune system, such as GhNAC1 (Lei Yu, Zhang Zhennan, Hu Guang, et al. Cotton WRKY22 Gene isolation and analysis of resistance to Verticillium wilt[J]. Acta Agriculturae Boreali-Sinica, 2018, 33: 160-168) WRKY7 (Liu ZX, Sun Y, Ye Z, et al. Identification and bioinformatics analysis of VOZ transcription factorgene family members in Mangosteen[J]. Journal of Southern Agriculture, 2021, (7): 1762-1770) and WRKY22 (Kaur K, Megha S, Wang ZP, et al.Identification and expression analysis of C2H2-zinc finger protein genes reveal their role in stress tolerance in Brassica napus [J]. Canadian Science Publishing, 2022, 66(5): 91-107) and other transcription factors can regulate the expression of immune-related genes when cotton is infected with Verticillium dahliae, thereby enhancing cotton's resistance to Verticillium wilt. Therefore, mining cotton Verticillium wilt resistance-related genes has application prospects in improving cotton Verticillium wilt resistance or cultivating Verticillium wilt-resistant plant varieties. Summary of the invention

[0004] One of the purposes of the present invention is to isolate and obtain genes and their encoded proteins related to regulating Verticillium wilt resistance from cotton;

[0005] The second object of the present invention is to provide an expression cassette, an expression vector or a recombinant host bacteria containing the gene isolated from cotton and related to regulating resistance to Verticillium wilt;

[0006] The third purpose of the present invention is to apply the genes isolated from cotton to regulate plant resistance to Verticillium wilt or to cultivate Verticillium wilt-resistant plant varieties.

[0007] To achieve the above object, the technical solution adopted by the present invention includes:

[0008] One aspect of the present invention is to provide a gene related to regulating Verticillium wilt resistance isolated from upland cotton. GhATG1t Gene, the GhATG1t The nucleotide sequence of the gene is selected from any one of (a)-(d):

[0009] (a) a polynucleotide sequence as shown in SEQ ID No.1; (b) a polynucleotide that can hybridize with the complementary sequence of SEQ ID No.1 under stringent hybridization conditions; (c) a polynucleotide that has at least 90% or more homology with the polynucleotide as shown in SEQ ID No.1; (d) a mutant in which one or more bases are deleted, substituted or inserted based on the polynucleotide as shown in SEQ ID No.1, and the mutant still has the function or activity of regulating resistance to Verticillium wilt.

[0010] 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.

[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. GhATG1t The amino acid sequence of the encoded protein of the gene is shown in SEQ ID No.2.

[0013] The present invention also discloses a GhATG1t A recombinant expression vector of a gene; preferably, the recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector; most preferably, it is a recombinant plant expression vector.

[0014] The present invention further discloses a GhATG1t The invention relates to a recombinant host cell or a recombinant bacterium containing a gene; wherein the recombinant bacterium includes but is not limited to recombinant Escherichia coli or a recombinant eukaryotic cell; the recombinant eukaryotic cell includes but is not limited to recombinant fungi or recombinant plant cells.

[0015] Those skilled in the art can construct the gene knockout vector by conventional gene editing technology or gene knockout vector construction method. GhATG1t Gene editing vectors or constructing gene editing vectors according to conventional methods in the art GhATG1t The recombinant plant expression vector of the gene is well known to those skilled in the art; for example, GhATG1t The gene is operably connected to an expression control element to obtain a recombinant plant expression vector that can express the gene in plants; the recombinant plant expression vector comprises a promoter, GhATG1t The CDS sequence and terminator of the gene; the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter, and the terminator sequence can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. The vector may also contain a selective marker gene for selecting transformed cells, which is used to select transformed cells or tissues. The marker gene includes: genes encoding antibiotic resistance and genes conferring herbicide resistance, etc. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein, etc.

[0016] Another aspect of the present invention is to provide GhATG1tThe application of genes in regulating plant resistance to Verticillium wilt, including: GhATG1t Genes are regulated at the transcription and translation levels in plants to regulate plant resistance to Verticillium wilt; accordingly, by changing GhATG1t Genes can be regulated at the transcription and translation levels to achieve resistance to Verticillium wilt or by changing GhATG1t The regulation of Verticillium wilt resistance at the transcription and translation levels of genes in plants should fall within the protection scope of the present invention.

[0017] How to increase or decrease GhATG1t The expression level of the gene in cotton or plants can be achieved by various conventional technical means by those skilled in the art; for example, by constructing GhATG1t Gene overexpression vector, transformed into plants by Agrobacterium-mediated genetic transformation, obtained GhATG1t Gene overexpression strains can improve plant resistance to Verticillium wilt; or CRISPR and VIGS methods can be used to knock out or interfere with the GhATG1t Genes or other plant GhATG1t Homologous genes in cotton GhATG1t Genes or other plants GhATG1t Homologous genes are deleted, mutated or reduced GhATG1t The expression level of the gene reduces the resistance of cotton to Verticillium wilt.

[0018] As a preferred embodiment of the present invention, the present invention provides a method for improving plant resistance to Verticillium wilt, comprising: constructing GhATG1t Gene overexpression vector; GhATG1t The gene is overexpressed in plants, and the resulting transgenic plants have improved resistance to Verticillium wilt.

[0019] As another preferred embodiment of the present invention, the present invention provides a method for cultivating plant varieties resistant to Verticillium wilt, comprising: constructing GhATG1t Gene overexpression vector; GhATG1t The gene is overexpressed in the plant; and a transgenic plant variety with improved resistance to Verticillium wilt is screened from the obtained transgenic positive plants.

[0020] In the present invention, any plant transformation method can be used to transform the recombinant plant expression vector constructed by the present invention into the tissues and cells of the target plant to obtain a transformant, and then the transformant is regenerated by a plant tissue culture method to obtain a complete plant and its asexual line or its offspring. The transformation method includes Agrobacterium-mediated genetic transformation, protoplast transformation, plant virus vector, microinjection, electric shock method, etc.

[0021] 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.

[0022] The present invention utilizes virus-induced gene silencing (VIGS) technology and transgenic overexpression Arabidopsis technology to treat cotton GhATG1t The preliminary study on the function of genes in cotton resistance to Verticillium wilt found that GhATG1t After gene silencing, the cotton plant's resistance to Verticillium wilt was significantly reduced; GhATG1t Overexpression of the gene in Arabidopsis thaliana significantly improved the resistance of transgenic Arabidopsis thaliana to Verticillium wilt. GhATG1t The gene positively regulates plant resistance to Verticillium wilt caused by Verticillium dahliae.

[0023] The term definition of the invention

[0024] 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.

[0025] 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.

[0026] 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.

[0027] "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℃. T m 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 mStringent 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0033] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0034] 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

[0035] Figure 1 for GhATG1t Heatmaps for identification of gene expression patterns.

[0036] Figure 2 For target fragment amplification and pTRV2- GhATG1t Results of recombinant plasmid construction and identification; A: amplification of target VIGS fragment, where M: DL8000 DNA marker; lines 1: amplification in 'Xinlu Zao 27' GhATG1t VIGS fragment; B: pTRV2- GhATG1t PCR detection of recombinant plasmid bacterial solution, where M: DL8000 DNA marker; lines 1: PCR of recombinant plasmid bacterial solution.

[0037] Figure 3 for GhATG1t Resistance identification results of gene-silenced cotton plants; A: Silence GhCLA1 The leaves turned white after the gene was added, bar=3 cm; B: Real-time quantitative PCR detection GhATG1t Silencing efficiency; C: pTRV:: after 10 days of V991 infection 00 and pTRV:: GhATG1t Plant phenotypic identification, bar=3 cm; D: 10 days after inoculation, pTRV:: 00 and pTRV:: GhATG1t Statistics of disease index of cotton plants; E: 10 days after inoculation, pTRV:: 00 and pTRV:: GhATG1t Determination of relative fungal biomass of cotton plants; ** indicates significant difference at the P<0.01 level.

[0038] Figure 4 For transfer GhATG1t Phenotypic observation results of Arabidopsis thaliana inoculated with Verticillium dahliae; A: GhATG1t Phenotypic observation of V991 inoculated Arabidopsis thaliana, bar=3 cm; B: RT-PCR detection GhATG1t Expression; C: disease index statistics; D: fungal biomass statistics; ** indicates significant difference at the P<0.01 level.

[0039] Figure 5 The results of toxicity and self-activation verification of the bait protein GhATG1t; A: pGBKT7 and pGBKT7- GhATG1t Yeast growth on SD / -Trp medium; B: pGBKT7-P53 / pGADT7-SV40-T, pGBKT7 / pGADT7, pGBKT7- GhATG1t The growth conditions of yeast after co-transfection with pGADT7 on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade media respectively.

[0040] Figure 6 These are the verification results of the interaction between the bait protein GhATG1t and the interacting protein GhBsr-d1L1; A: Y2H method verified the interaction between GhATG1t and GhBsr-d1L1; B: LCI method verified the interaction between GhATG1t and GhBsr-d1L1; C: BiFC method verified the interaction between GhATG1t and GhBsr-d1L1. DETAILED DESCRIPTION

[0041] 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.

[0042] Experimental materials, vectors and strains

[0043] Upland cotton variety 'Xinluzao 27'; transgenic Arabidopsis recipient genotype is Colombia wild type (Col-0) cotton VIGS vectors pTRV1, pTRV2, pTRV2:: GhCLA1 ; 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 Key Laboratory of Ecological Adaptation and Evolution of Extreme Environment Organisms in Xinjiang, College of Life Sciences, Xinjiang Agricultural University.

[0044] 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 (h). 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). The growth conditions were indoor cultivation at 28°C, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0045] Test reagents

[0046] The total RNA extraction kit for polysaccharide and polyphenol plants was purchased from Hangzhou Bioray Technology Co., Ltd.; the plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the DNA Marker and reverse transcription kit MonScriptTMRTIII all-in-oneMix (with dsDNase) were purchased from Monad Biotechnology Co., Ltd.; the seamless cloning kit pEASY®-UniSeamless Cloning and Assembly Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the 2×Taq PCR Mix was purchased from Beijing Aidelai Biotechnology Co., Ltd.; the restriction endonuclease Eco R. Bam HI was purchased from Thermo Fisher Scientific; the fluorescence quantitative kit 2x ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; the synthesis of primers used in PCR and DNA sequencing were completed by Xinjiang Youkang Biotechnology Co., Ltd.

[0047] Primer sequences

[0048] The primer sequences used in the following test examples of the present invention are shown in Table 1.

[0049] Table 1 Primers used in this experiment

[0050]

[0051] Note: Lowercase letters represent homology arms

[0052] 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. GhATG1t , which encodes a serine / threonine protein kinase associated with autophagy; GhATG1t This gene is a new gene whose function has not been reported in cotton. GhATG1t The nucleotide sequence of the gene is shown in SEQ ID No.1:

[0053]

[0054] Should GhATG1t The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2:

[0055] MEPQAMAAMPYLLEPFTIGDYLLKSKLRESSVSIVWKAETKSSGEEVAVKQVFLSKLNKHLANCLDCELNFLSSVNHPNIIRLLQVLQSESCLFLVLEFCAGGNLTSYIRRYGRVQEQLARRFMQQLGAGLEVLQSHHIIHRDLKP SEQ IDNo.2).

[0056] Serine / threonine protein kinases (STPKs) are an important class of enzymes that regulate a variety of biological processes in cells by catalyzing the phosphorylation of serine or threonine residues on proteins. These processes include but are not limited to metabolism, transcription, cell division, cell motility, programmed cell death, immune response, and nervous system function. Studies have shown that ATG1t is involved in the regulation of autophagy in a variety of plants, but there is insufficient evidence for its direct involvement in biotic stress responses.

[0057] Test Example 1 GhATG1t Gene expression pattern identification assay

[0058] 1 Test methods

[0059] For identification GhATG1t The expression pattern of the gene during the pathogen infection of cotton was detected by real-time fluorescence quantitative qRT-PCR in the roots, stems and leaves of cotton at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 hpi (Hours post infection). The specific experimental method is as follows:

[0060] The 'Xinluzao 27' cotton was cultured in the soil of the growth chamber for 15 days, with water treatment as the control group. The spore concentration reached 10 7 cfu / mL of V991 bacterial solution was used as the experimental group GhATG1tqRT-PCR relative expression analysis of genes. After the roots were immersed in water or V991 spore suspension at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 hpi, the root, stem and leaf tissues were collected, and RNA was extracted using the polysaccharide and polyphenol plant total RNA extraction kit, and cDNA was prepared using the reverse transcription kit MonScriptTMRTIII all-in-oneMix. NCBI was used to design qRT-PCR primers. GhATG1t -qRT-F / R, with GhUBQ7 As an internal reference, the primers were GhUBQ7 -qRT-F / R (Table 1), three technical replicates were performed for each sample. -ΔΔCt Methods The gene expression level was calculated, where the Ct value was the cycle threshold value.

[0061] 2 Test results

[0062] GhATG1t The results of gene expression pattern identification showed that GhATG1t The gene expression levels in different parts showed significant changes. The root genes showed an overall downward trend, with the largest changes at 2 hpi and 48 hpi, decreasing to 0.11 times and 0.01 times that of the control group; the stem genes showed an overall upward trend, with the largest changes at 0.5 hpi and 12 hpi, increasing to 21 times and 15 times the original; the gene expression level in the leaves increased to 15 times that of the control group after about 2 hpi, and decreased to 0.01 times that of the control group at 8 hpi ( Figure 1 ). According to the results GhATG1t The expression of the gene was induced by infection with Verticillium dahliae, and the gene expression level changed significantly in the early stage of infection.

[0063] Test Example 2 GhATG1t Test on the resistance identification of silent cotton plants to Verticillium wilt

[0064] 1 Test methods

[0065] Design specific primers by Primer5 GhATG1t -VIGS-F / R (Table 1), amplified using KOD DNA polymerase (Toyobo, Shanghai, China) GhATG1t coding region, and then using Eco RI and Bam HI enzyme was connected to the pTRV2 vector to obtain pTRV2:: GhATG1t Vector, pTRV2:: GhATG1t The plasmid was transformed into Agrobacterium GV3101, and the gene was silenced by Agrobacterium infection. GhATG1tAgrobacterium was used to infect 7-day-old cotyledons of 'Xinluzao 27' with pTRV2:: 00 As a negative control, pTRV2:: GhCLA1 As a positive control. GhATG1t -qRT-F / R primer pair was used to extract leaf samples for qPCR detection of silencing effect (Table 1).

[0066] Using the cotton inoculation method of Experimental Example 1, the root tissues of the experimental and control cottons were immersed in the spore solution for 5 minutes (min) and then replanted into the nutrient soil. The cotton phenotype was observed after 10 days and the disease index was calculated using the leaf grading method. Cotton genomic DNA was extracted, and the fungal biomass was detected in the ribosomal RNA gene ITS1 and ITS2 regions (Z29511) in the genomic DNA of Verticillium dahliae. The primers used were Vd-ITS-F / R, and the internal reference gene was GhUBQ7 (Table 1). qRT-PCR reactions were performed on an ABI7500 Fast instrument and data were analyzed by 2 -ΔΔCt Method analysis (Su X, Wu S, Liu L, et al. Potential Antagonistic Bacteria against Verticillium dahliae Isolated from Artificially Infested Nursery[J]. Cells, 2021,10(12): 3588).

[0067] 2 Test results

[0068] 2.1 Construction of VIGS recombinant vector

[0069] The cDNA of cotton variety 'Xinluzao 27' was amplified with Eco RI and Bam HI restriction site GhATG1t ( Figure 2 A). The amplified target fragment (300 bp) was sequenced and compared with the target gene CDS library. The gene sequence was consistent with the target sequence and was used for the construction of VIGS vector. Eco RI and Bam The VIGS vector pTRV2 digested with HI was connected and transformed into E. coli, and then identified by bacterial liquid PCR, which was consistent with the expected gene fragment size ( Figure 2 B), indicating that the VIGS recombinant plasmid was successfully constructed. After further transformation of Agrobacterium competent GV3101, cotton VIGS gene silencing experiment was carried out.

[0070] 2.2 VIGS Silencing GhATG1t Verification of gene function results

[0071] One week after the injection of the bacterial solution, GhCLA1 The true leaves of the control plants showed bleaching ( Figure 3 A) Five silenced and five control plants were selected, and leaf RNA was extracted for Real-time quantitative PCR detection. GhATG1t Gene silencing efficiency ( Figure 3 B), the results show GhATG1t The gene is silenced with a silencing efficiency of 70%. GhATG1t The seedling function of the gene was studied. Ten days after the control and silenced plants were inoculated with V991, the control group showed yellowing of individual leaves with the leaf margins curling downwards. GhATG1t The silent group showed more severe symptoms of Verticillium wilt, such as plant wilting and leaf shedding. GhATG1t The cotton plants in the silenced group and the control group were cut open at the cotyledon junction for observation. Under a stereo microscope, it was observed that the vascular bundles of the cotton plants in the silenced group turned brown, with more obvious brown stripes ( Figure 3 C). At the same time, GhATG1t The disease index and relative fungal biomass of the silenced plants were significantly higher than those of the control group ( Figure 3 D-3E). Therefore, GhATG1t The gene plays a positive regulatory role in cotton's resistance to Verticillium wilt.

[0072] Test Example 3 GhATG1t Functional validation test of genes in Arabidopsis

[0073] 1 Test methods

[0074] To verify GhATG1t The cDNA of cotton 'Xinluzao 27' was used as a template to design specific primers to GhATG1t The gene was cloned and constructed in the pCAMBIA1132 vector. After the vector was sequenced correctly, it was transformed into Agrobacterium and infected with Arabidopsis. After homozygous transgenic Arabidopsis was obtained, the Verticillium dahliae experiment was carried out. V991 was inoculated into non-bolting wild-type Arabidopsis and T3 transgenic Arabidopsis OE1 and OE2, and phenotype observation, disease index and fungal biomass were measured after 11 days.

[0075] 2 Test results

[0076] One week after inoculation, the leaves of wild-type Arabidopsis gradually turned yellow until the whole plant died. GhATG1t Some leaves of the genetically modified Arabidopsis also showed typical symptoms of Verticillium wilt, but some leaves remained normal ( Figure 4 A); Figure 4 B is RT-PCR detection GhATG1t Expression result; transfer GhATG1t The disease index and fungal biomass of the genetically modified Arabidopsis were 0.5 and 0.6 times that of the control group, respectively ( Figure 4 C-4D), prove GhATG1t Overexpression of the gene in Arabidopsis thaliana increased resistance to Verticillium dahliae, demonstrating GhATG1t The gene is a positive regulatory gene that regulates plant resistance to Verticillium wilt.

[0077] Experimental Example 4 GhATG1t screening of interacting proteins

[0078] 1 Test methods

[0079] 1.1 Screening of interacting proteins of GhATG1t

[0080] GhATG1t Encodes a class of serine / threonine protein kinases. The yeast two-hybrid screening library was performed using the mating method to screen GhATG1t interacting proteins in cotton, thereby studying GhATG1t The regulatory mechanism of disease resistance. GhATG1t Transformed into Y2HGold yeast strain, pGBKT7 was used as control to detect yeast toxicity and self-activation. After completion, GhATG1t was screened for interacting proteins in cotton. First, the constructed pGBKT7- GhATG1t The vector and yeast library plasmid were co-transformed into Y2HGold competent cells, inoculated in SD / -Trp / -Leu, and after a white moist single clone grew out, the single clone was streaked to SD / -Trp / -Leu / -His / -Ade, and PCR amplification and sequencing were performed after a single clone grew out.

[0081] 1.2 Verification of protein interactions by two-hybrid assay (Y2H), in vivo luciferase assay (LCI), and in vivo bimolecular complementation assay (BiFC)

[0082] Two-hybrid assay by PCR amplification GhBsr-d1L1 The ORF was recovered and inserted into the pGADT7 yeast expression vector to construct pGADT7- GhBsr-d1L1 The preparation, co-transformation and culture of Y2HGold yeast competent cells were carried out according to the instructions of yeast two-hybrid. GhATG1t and GhBsr-d1L1 The n-LCI and c-LCI vectors were constructed respectively for transformation of Agrobacterium GV1301, co-injection of tobacco leaves and fluorescence detection. In the in vivo bimolecular complementation experiment, GhATG1t was fused to the C-terminus of the YFP protein, and the GhBsr-d1L1 protein was fused to the N-terminus of the YFP protein. The above methods were used for transformation of Agrobacterium GV1301, co-injection of tobacco leaves and fluorescence detection.

[0083] 2 Test results

[0084] 2.1 pGBKT7- GhATG1t Toxicity and self-activation assay results

[0085] The yeast two-hybrid screening library was carried out using the Mating method to screen the GhATG1t interacting proteins in cotton, thereby studying the regulatory disease resistance mechanism of GhATG1t. GhATG1t The recombinant plasmid pGBKT7- GhATG1t The clones could grow on yeast defective medium SD / -Trp plates and had no significant difference in size and quantity from yeast clones containing pGBKT7, indicating that GhATG1t Can be successfully transformed into yeast cells without causing cytotoxicity ( Figure 5 A). pGBKT7-P53 / pGADT7-SV40-T (positive control), pGBKT7 / pGADT7 (negative control), pGBKT7- GhATG1t / pGADT7 (self-activation test group) grew white moist single colonies on SD / -Trp / -Leu nutrient-deficient medium. On SD / -Trp / -Leu / -His / -Ade nutrient-deficient medium, no colonies were produced in the negative control and self-activation test group, and the positive control grew normally, indicating that GhATG1t Failed to activate HIS3 and ADE2 reporter gene expression and had no self-activation effect ( Figure 5 B).

[0086] 2.2 Verification results of interacting proteins

[0087] To verify the results of protein interaction, pGADT7- GhBsr-d1L1 Plasmid and pGBKT7- GhATG1t Plasmid co-transformed yeast can grow on SD / -Trp / -Leu / -His / -Ade plates and can decompose X-α-Gal to turn the colonies blue, indicating that this protein interacts with GhATG1t ( Figure 6 A).

[0088] To further verify the results of protein interaction, GhATG1t and GhBsr-d1L1 They were constructed on n-LCI and c-LCI vectors respectively, and fluorescence was observed after injection into tobacco. The appearance of fluorescence indicates that there is an interaction between the two proteins. Experiments have shown that GhATG1t interacts with GhBsr-d1L1 ( Figure 6 B).

[0089] In order to verify the interaction position of GhATG1t and GhBsr-d1L1 in plants, GhATG1t was fused to the C-terminus of YFP protein, and GhBsr-d1L1 protein was fused to the N-terminus of YFP protein; the experimental results showed that GhBsr-d1L1 -YFPN and GhATG1t -YFPC co-transfection can observe yellow fluorescence, and the location of fluorescence is mainly concentrated in the cell nucleus ( Figure 6 C).

Claims

1. GhATG1t The application of genes in regulating plant resistance to Verticillium wilt includes: Through regulation GhATG1t The transcription and translation levels of genes in plants regulate the resistance of plants to Verticillium wilt; wherein the GhATG1t The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1; the plant is cotton or Arabidopsis thaliana; the Verticillium wilt is caused by Verticillium dahliae ( Verticillium dahliae ) caused by plant diseases.

2. Contains GhATG1t The application of a chimeric gene or expression cassette of a gene in regulating plant resistance to Verticillium wilt; wherein, The regulation of plant resistance to Verticillium wilt is to improve the plant resistance to Verticillium wilt, including: GhATG1t A gene is overexpressed in a plant; GhATG1t The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1; the plant is cotton or Arabidopsis thaliana; the Verticillium wilt is caused by Verticillium dahliae ( Verticillium dahliae ) caused by plant diseases.

3. Contains GhATG1t Application of a recombinant expression vector of a gene in regulating plant resistance to Verticillium wilt; wherein, The regulation of plant resistance to Verticillium wilt is to improve the plant resistance to Verticillium wilt, including: GhATG1t A gene is overexpressed in a plant; GhATG1t The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1; the plant is cotton or Arabidopsis thaliana; the Verticillium wilt is caused by Verticillium dahliae ( Verticillium dahliae ) caused by plant diseases.

4. A method for improving plant resistance to Verticillium wilt, characterized in that: including: Build GhATG1t Gene overexpression recombinant plant expression vector; GhATG1t Gene overexpression recombinant plant expression vector transforms plants, GhATG1t The gene is overexpressed in plants, and the resulting transgenic plants have increased resistance to Verticillium wilt; wherein the GhATG1t The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1; the plant is cotton or Arabidopsis thaliana; the Verticillium wilt is caused by Verticillium dahliae ( Verticillium dahliae ) caused by plant diseases.

5. A method for cultivating plant varieties resistant to Verticillium wilt, characterized in that: including: Build GhATG1t Gene overexpression recombinant plant expression vector; GhATG1t Gene overexpression plant expression vector transforms plants, GhATG1t The gene is overexpressed in plants to obtain transgenic positive plants and screen plant varieties with improved resistance to Verticillium wilt; wherein the GhATG1t The nucleotide sequence of the gene is the polynucleotide shown in SEQ ID No.1; the plant is cotton or Arabidopsis thaliana; the Verticillium wilt is caused by Verticillium dahliae ( Verticillium dahliae ) caused by plant diseases.

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