Application of GbTCP20 gene in improving cotton resistance to verticillium wilt

Abstract

The application discloses application of a GbTCP20 gene in improving cotton Verticillium wilt resistance and belongs to the technical field of biotechnology.The GbTCP20 gene disclosed by the application encodes a TCP transcription factor.The application provides a full-length ORF nucleotide sequence and an amino acid sequence of the GbTCP20 in a Gossypium hirsutum var. hirsutum L. variety Hai7124.The expression of the gene is significantly up-regulated by a Verticillium albo-atrum Eulge et Hunter induction.Silencing of the GbTCP20 expression in cotton weakens the Verticillium wilt resistance.Overexpression of the cotton GbTCP20 in Arabidopsis thaliana enhances the Verticillium wilt resistance.The cotton GbTCP20 positively regulates lignin biosynthesis, and the combination of molecular biology technology and plant physiology experiments reveals that the GbTCP20 positively regulates lignin biosynthesis to endow the cotton with Verticillium wilt resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology and relates to GbTCP20 gene and application of the gene in improving cotton Verticillium wilt resistance. The full-length ORF of the gene is 885 bp, which is a TCP transcription factor encoding 294 amino acids. RT-qPCR analysis shows that the gene is significantly up-regulated after cotton root tissue is induced by Verticillium dahliae. Plant TCP protein is a typical transcription factor, which is widely involved in regulating plant growth and development, stress response and other life processes. The application discloses a new regulation mechanism of cotton GbTCP20, inhibits the expression of GbTCP20 gene encoding cotton TCP20 protein, inhibits the expression of GbCOMT gene, inhibits the lignin synthesis pathway, thereby inhibits the deposition of lignin, greatly weakens the physical defense of secondary cell wall, and reduces the resistance of cotton to Verticillium wilt. Overexpression of GbTCP20 gene in Arabidopsis significantly improves the disease resistance of plants. The gene is used in biotechnology to determine that the gene has a positive regulation on lignin synthesis in the process of plant Verticillium wilt resistance, expands the understanding of the role of TCP protein in plant Verticillium wilt resistance, and has significant breeding value. BACKGROUND

[0002] The TCP family is a plant-specific transcription factor, whose name is derived from four genes: TEOSINTE BRANCHED 1 (TB1) in maize, CYCLOIDEA (CYC) in snapdragon, and PROLIFERATING CELL FACTORS 1 and 2 (PCF1 and PCF2) in rice. TCP proteins are characterized by a conserved non-canonical domain near the N-terminus, consisting of about 60 amino acid residues with a helix-loop-helix structure, similar to eukaryotic bHLH transcription factors. In Arabidopsis, the TCP family is divided into class I TCP and class II TCP. Both classes can target GC-rich sequences in promoters, with class I targeting GGNCCCAC elements and class II targeting GTGGNCCC elements. TCPs are known to regulate plant biotic stress responses. Class I TCP subfamily members, including TCP8, TCP9, TCP14, TCP15, TCP19, TCP20, and TCP21, and class II TCP subfamily members, including TCP10, TCP13, and TCP17, positively regulate plant resistance by directly targeting bacterial or fungal effectors. Meanwhile, TCP8, TCP9, TCP20, and TCP21 proteins increase salicylic acid (SA) content to respond to pathogen invasion by regulating the expression of AtlCS1. In addition, TCP9 and TCP20 inhibit JA synthesis by negatively regulating the expression of the jasmonic acid (JA) biosynthesis gene LOX2. However, class IICINCINNATA (CIN) / TCP members positively regulate JA synthesis by directly binding to the promoter of LOX2. TCP14 also triggers Arabidopsis systemic acquired resistance (SAR) by interacting with NPR1. TCP transcription factors TCP8, TCP14, and TCP15 interact with the Arabidopsis immune adaptor factor SRFR1. Therefore, the functional role of TCPs in plant immunity is increasingly attracting attention.

[0003] Lignin, as an important secondary metabolite in the process of plant growth and development, plays an important role in the process of plant growth and immune defense. On the one hand, during the process of lignification of plant cell wall, lignin as a structural material fills in the cell wall components, enhancing the mechanical support and compression resistance of the cell. At the same time, lignin is an important component of the formation of the vascular tissue conduit, participating in the transportation of water and inorganic salt in the plant. On the other hand, lignin, as an insoluble complex phenolic polymer, its hydrophobic structure as a physical barrier to resist the invasion of external pathogens and pests. It has been reported that there are differences in vascular structure between different disease-resistant cotton varieties, and the rate and total amount of lignin and lignin-like polymer synthesis and deposition are significantly different between resistant and susceptible varieties. For example, the expression of genes related to lignin synthesis in Gossypium barbadense is up-regulated after Verticillium dahliae induction, and the total amount of lignin in vascular tissue is significantly accumulated. Silencing of GhCOMT gene in Gossypium hirsutum inhibits the lignin synthesis pathway activated by Verticillium dahliae invasion, resulting in Gossypium hirsutum more susceptible to Verticillium dahliae. At present, transcription factors related to lignin synthesis in model plant Arabidopsis have been identified and reported, and NAC and MYB are two major transcription factor families that regulate lignin synthesis. Among them, NAC transcription factors are divided into VND, NST, SND and WND four categories, which are located in the upstream of the lignin synthesis regulation network. TCP family has rarely been reported to regulate lignin synthesis, and only one report shows that Arabidopsis TCP4 indirectly regulates lignin synthesis by targeting the promoter of VND7. In recent years, some stress-induced transcription factors related to lignin synthesis in cotton have been reported. Gossypium hirsutum transcription factor GhMYB4 inhibits lignin synthesis by negatively regulating the expression of GhC4H-1, GhC4H-2, Gh4CL-4 and GhCAD-3 genes, enhancing the resistance of cotton to Verticillium dahliae. Gossypium hirsutum transcription factor GhWRKY1-like enhances the resistance of cotton to Verticillium dahliae by positively regulating the lignification process and S-type lignin synthesis. The cotton TCP family members revealed in this study have a direct positive regulation of lignin synthesis, which broadens the previous understanding.

[0004] Cotton is one of the important economic crops and the source of textile fibers and seed oil. TCP proteins have been frequently reported to play a role in the transcriptional regulation during cotton development. GhTCP14a and GhTCP22 are key genes that regulate cotton fiber development. Overexpression of GhTCP4 and GhTCP5 in Arabidopsis increases root hair length, root hair and trichome density, and lignin content. GhTCP4 inhibits fiber cell elongation while promoting cell wall thickening. However, the role of TCP proteins in Verticillium dahliae resistance in cotton is not clear. In the present study, we report that a cotton GbTCP20 positively regulates lignin synthesis to confer Verticillium dahliae resistance in cotton, which is different from the reported regulatory mechanism of TCP family in biotic stress.

[0005] Verticillium dahliae Kleb (V. dahliae) is a soil-borne fungal disease that poses a serious threat to the growth of more than 400 plant species, including important economic crops such as rapeseed, tomato, and cotton. In this study, by analyzing the root transcriptome of island cotton (Hai7124) induced by Verticillium dahliae, a TCP transcription factor, GbTCP20 was identified. Compared with control plants, the GbTCP20 silencing group weakened cotton's resistance to Verticillium dahliae. Transcriptome analysis showed that inhibiting GbTCP20 expression significantly suppressed the phenylpropane metabolic pathway, with a significant downregulation of lignin synthesis-related genes. Molecular biology techniques demonstrated that GbTCP20 positively regulates GbCOMT expression, which encodes caffeic acid-O-methyltransferase. Lignin histochemical staining and content analysis showed that inhibiting GbTCP20 expression reduced lignin accumulation. Furthermore, overexpression of GbTCP20 in Arabidopsis thaliana enhanced resistance to Verticillium dahliae. These findings highlight the crucial role of GbTCP20 in enhancing plant resistance to Verticillium wilt and broaden our understanding of the role TCPs play in cotton resistance to Verticillium wilt. Summary of the Invention

[0006] The purpose of this invention is to provide a cotton TCP transcription factor gene (GbTCP20) and its application in improving cotton Verticillium wilt resistance and cultivating new germplasm with improved Verticillium wilt resistance. The full-length cDNA ORF nucleotide sequence and the amino acid sequence of the encoded protein of this gene in the sea island cotton Hai7124 are provided. Using this gene as a target gene, GbTCP20 expression was inhibited by virus-induced gene silencing (VIGS). The role of GbTCP20 in cotton Verticillium wilt resistance was clarified through heterologous overexpression in Arabidopsis thaliana, and it was used to cultivate new germplasm for production application. Furthermore, combining molecular biology techniques with plant physiological experiments revealed that GbTCP20 positively regulates lignin biosynthesis, conferring cotton Verticillium wilt resistance.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention seeks protection for the use of the GbTCP20 gene as shown in SEQ ID NO.1 or biological materials related to the GbTCP20 gene in the following (a1) or (a2) or (a3):

[0009] (a1) Regulates plant disease resistance;

[0010] (a2) Cultivating new plant germplasm with enhanced disease resistance;

[0011] (a3) Cultivate new plant germplasm with reduced disease resistance.

[0012] Further, the biological material related to the GbTCP20 gene is a biological material described in (b1) or (b2) or (b3) below:

[0013] (b1) the protein GbTCP20 encoded by the GbTCP20 gene;

[0014] (b2) a biological material containing the GbTCP20 gene;

[0015] (b3) a biological material for silencing, interfering with, or inhibiting the GbTCP20 gene.

[0016] Further, the protein GbTCP20 encoded by the GbTCP20 gene in (b1) has an amino acid sequence as shown in SEQ ID NO. 2.

[0017] Further, the biological material containing the GbTCP20 gene in (b2) is at least one of (c1) to (c6) below:

[0018] (c1) an expression cassette containing the GbTCP20 gene;

[0019] (c2) a recombinant vector containing the GbTCP20 gene, or a recombinant vector containing the expression cassette of (c1);

[0020] (c3) a recombinant microorganism containing the GbTCP20 gene, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2);

[0021] (c4) a transgenic plant cell line containing the GbTCP20 gene, or a transgenic plant cell line containing the expression cassette of (c1), or a transgenic plant cell line containing the recombinant vector of (c2);

[0022] (c5) a transgenic plant tissue containing the GbTCP20 gene, or a transgenic plant tissue containing the expression cassette of (c1), or a transgenic plant tissue containing the recombinant vector of (c2);

[0023] (c6) a transgenic plant organ containing the GbTCP20 gene, or a transgenic plant organ containing the expression cassette of (c1), or a transgenic plant organ containing the recombinant vector of (c2).

[0024] Further, the biological material for silencing, interfering with, or inhibiting the GbTCP20 gene in (b3) is at least one of (d1) to (d4) below:

[0025] (d1) an interfering fragment or a silencing fragment of the GbTCP20 gene;

[0026] (d2) a primer for amplifying the interfering fragment or the silencing fragment of the GbTCP20 gene of (d1);

[0027] (d3) an interfering expression vector or a silencing vector of the GbTCP20 gene;

[0028] (d4) a recombinant microorganism containing the interfering expression vector or the silencing vector of (d3).

[0029] Further, the above-mentioned application is to take the GbTCP20 gene as a target gene, to improve the disease resistance of plants or to breed new plant germplasm with improved disease resistance by overexpressing the GbTCP20 gene through genetic engineering methods; or to reduce the disease resistance of plants or to breed new plant germplasm with reduced disease resistance by inhibiting the expression of the GbTCP20 gene.

[0030] Further, the disease resistance is the Verticillium wilt disease resistance; and the plant is cotton or Arabidopsis, but is not limited thereto.

[0031] In a second aspect, the present application claims to protect a method for improving the Verticillium wilt resistance of cotton, which overexpresses the GbTCP20 gene with the nucleotide sequence shown in SEQ ID NO. 1 in cotton.

[0032] In a third aspect, the present application claims to protect a GbTCP20 gene capable of improving the disease resistance of plants, which has the nucleotide sequence shown in SEQ ID NO. 1.

[0033] In a fourth aspect, the present application claims to protect a protein encoded by the above-mentioned GbTCP20 gene, which has the amino acid sequence shown in SEQ ID NO. 2.

[0034] In a fifth aspect, the present application claims to protect a biological material containing the above-mentioned TCP20 gene or containing an interfering fragment or a silencing fragment of the GbTCP20 gene, which is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

[0035] The application clones a TCP transcription factor protein gene GbTCP20, the cDNA ORF sequence of the gene is shown as SEQ ID NO. 1. The protein encoded by the gene has an amino acid sequence shown as SEQ ID NO. 2. Research finds that inhibiting the expression of the GbTCP20 gene can significantly reduce the resistance of the plant to verticillium wilt. Overexpression of the GbTCP20 gene can significantly improve the resistance of the plant to verticillium wilt. In the specific embodiments of the application, the cotton TCP transcription factor protein gene GbTCP20 is taken as a target gene, and the expression level of the GbTCP20 gene of the plant is inhibited or improved by the method of virus-induced gene silencing (VIGS) or the method of Arabidopsis heterologous overexpression, which can be used for improving the disease resistance of cotton to verticillium wilt or creating new germplasm with disease resistance and other production applications.

[0036] The application has the following advantages:

[0037] (1) The TCP transcription factor protein gene GbTCP20 cloned by the application has not been studied in cotton before. The GbTCP20 gene in cotton is identified for the first time, and the gene is subjected to systematic sequence structure, expression pattern and function analysis, and the important role of GbTCP20 in the resistance of cotton plants to verticillium wilt is determined.

[0038] (2) The tissue expression pattern analysis shows that the gene is expressed to a certain extent in each tissue. The real-time fluorescent quantitative PCR result shows that the expression amount of the gene is significantly up-regulated at 12h, 24h, 72h and 96h after the cotton plant is induced by verticillium wilt compared with that before induction (0h), which indicates that the gene is closely related to the resistance to verticillium wilt.

[0039] (3) The expression of the gene is inhibited by the method of virus-induced gene silencing (VIGS), and the phenotype is studied by taking the genetic standard line of G.barbadense L.acc.Hai7124 as a receptor. The expression analysis of the VIGS material shows that the expression of GbTCP20 is significantly reduced in the strain in which the expression of the gene is inhibited compared with the control, and the plant in which the expression of GbTCP20 is inhibited shows the phenotype of reduced resistance to verticillium wilt, which indicates that the expression level of the gene affects the resistance of cotton to verticillium wilt.

[0040] (4) The expression of GbTCP20 is inhibited, which significantly reduces the resistance of cotton to verticillium wilt. The molecular biology experiment proves that the silencing of GbTCP20 significantly inhibits the accumulation of lignin in the phenylpropanoid synthesis pathway.

[0041] (5) The expression of GbTCP20 is improved, which significantly enhances the resistance of Arabidopsis to verticillium wilt. The application provides new evidence for understanding the function of TCP family members in the resistance of plants to verticillium wilt. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 TCP20 is a feature description of the cotton;

[0043] A, the bHLH domain amino acid sequence alignment of TCP20 in Gossypium barbadense genetic standard line Hai7124 and Arabidopsis thaliana; B, the expression pattern of GbTCP20 in different tissues and organs of Hai7124, and in ovules and fibers at different development stages; C, the expression pattern analysis of GbTCP20 in cotton induced by V. dahliae by real-time fluorescent quantitative PCR (RT-qPCR); D, the subcellular localization of transcription factor GbTCP20 in tobacco leaves. Error bars represent standard deviation (SD). Asterisks represent statistical significance, determined by t-test (**P<0.01).

[0044] Figure 2 GbTCP20 silencing weakens the resistance of cotton to Verticillium wilt;

[0045] TRV is a tobacco rattle virus (TRV) vector of cotton, comprising pTRV1 and pTRV2. Among them, pTRV1 is an auxiliary vector. The CLA (cloroplastos alterados 1) gene encodes 1-deoxyxylulose 5-phosphate synthase, which is involved in the process of chloroplast development, and pTRV is a public vector. pTRV:CLA is used as a positive control. After the white phenotype appears in the CLA-silenced plants, the silencing efficiency of GbTCP20 gene is detected. Among them, A, the expression level of GbTCP20 gene in GbTCP20-silenced plants and control plants is detected by RT-qPCR; B, compared with the control plants, the disease resistance of GbTCP20-silenced plants is significantly weakened at 11 days, 13 days, 18 days, 20 days and 25 days after inoculation with Verticillium wilt. Error bars represent standard deviation (SD). Asterisks represent statistical significance, determined by t-test (**P<0.01, ***P<0.001).

[0046] Figure 3 GbTCP20-silenced plants are significantly enriched in the phenylpropanoid biosynthesis pathway;

[0047] A, differential expression gene analysis of 0h, 24h and 96h after inoculation of V. dahliae in the control group (TRV:00) and GbTCP20 silenced group (TRV: GbTCP20). B and C, KEGG analysis of differentially expressed genes at 24h (B) and 96h (C) after inoculation of V. dahliae. D, RT-qPCR verification of the expression pattern of differentially expressed genes in the phenylpropanoid synthesis pathway. Error bars represent standard deviation (SD). Asterisks represent statistical significance, determined by t-test (*P<0.01, **P<0.01, ***P<0.001).

[0048] Figure 4 To identify the off-target of homologous genes in the roots of GbTCP20 silenced plants;

[0049] The genes with high homology to GbTCP20 in Gossypium barbadense Hai7124 were obtained by blast through software Seqhunter. Gossypium barbadense is tetraploid, and GbTCP20 has very high similarity in A (GB_A12G1677) and D (GB_D12G1669) subgroups. Among them, A, the phylogenetic tree of homologous genes and GbTCP20 and gene similarity analysis. The asterisk indicates the target gene GbTCP20. B, the expression level of homologous genes in GbTCP20 silenced plants has no significant difference compared with the control plants, and the results show that the silenced fragment of GbTCP20 silenced plants is specific. The data is derived from the transcriptome file of GbTCP20 gene silenced plants and control plants, and the error bars represent the standard deviation (SD) of three biological replicates. Asterisks represent statistical significance, determined by t-test (***P<0.001).

[0050] Figure 5 To inhibit the deposition of lignin by GbTCP20 silencing;

[0051] A, B and C, the interaction of transcription factor GbTCP20 with target element (GGGCCCAC) was verified. The minimal concentration of aureobasidin A to inhibit the growth of pAbAi-target bait strain was 200 ng / mL. The pGADT7-GbTCP20 transformed pAbAi-target was inoculated on SD / -Leu / -UraAbA (200 ng / mL) and cultured at 30°C for 3-5 days, and the growth was normal. The luciferase reporter assay (B) and (C), the fluorescence signal of 35S::GbTCP20 / target-mini35S-LUC was significantly enhanced compared with 35S::GbTCP20 / mutant-mini35S-LUC after co-injection into tobacco leaves, and the LUC / REN value was also increased. D and E, the interaction of transcription factor GbTCP20 with GbCOMT promoter was detected by luciferase reporter assay. The fluorescence signal of 35S::GbTCP20 / GbCOMTpro-LUC was significantly enhanced compared with 35S::GbTCP20 / empty-LUC after co-injection into tobacco leaves, and the LUC / REN value was also increased. F and G, lignin content detection and histochemical staining analysis. The lignin content of stems was determined by acetylation staining (F), and the total lignin content of GbTCP20 silenced plants was significantly lower than that of control plants. Histochemical staining analysis was performed by HCl-mellitene staining (G), and the coloring degree of stems of GbTCP20 silenced plants was significantly reduced compared with control plants. Error bars represent standard deviation (SD) of three biological replicates. Asterisks represent statistical significance determined by t-test (**P<0.01, ***P<0.001).

[0052] Figure 6 The position of the GbCOMT gene promoter cis-acting element is

[0053] Among them, the target element combined by GbTCP20 in the promoter of GbCOMT is located at-383-391 bp upstream of the ATG of the gene.

[0054] Figure 7 GbTCP20 enhances the resistance of Arabidopsis to Verticillium wilt;

[0055] A, RT-qPCR was used to detect the expression level of GbTCP20 in transgenic Arabidopsis (top); PCR was used to identify the integration of GbTCP20 into the genome of Arabidopsis (bottom). Ubq5 (At3g62250) was used as an internal reference gene. B and C, the disease phenotype and disease investigation of Arabidopsis 12 days after inoculation with Verticillium wilt fungus, the resistance of GbTCP20 overexpression plants (OE4, OE11 and OE19) to Verticillium wilt was significantly increased compared with the control plants (WT). The disease level rate of GbTCP20 overexpression plants and the control plants was significantly lower than the disease index (C). Error bars represent standard deviation (SD). Asterisks represent statistical significance, determined by t-test (*P<0.05, **P<0.01).

[0056] Figure 8 A schematic diagram of GbTCP20 regulating the resistance of plants to Verticillium wilt;

[0057] After being infected by Verticillium wilt, the total amount of the transcription factor GbTCP20 in the plant was significantly increased, which activated the transcription of the GbCOMT gene in the lignin synthesis pathway and further promoted the accumulation of lignin, thereby endowing the plant with resistance to Verticillium wilt. DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0059] Example 1 Planting of plant materials and growth conditions

[0060] The Verticillium wilt resistant variety G.barbadense L.acc.Hai7124, N. benthamiana and Arabidopsis thaliana of Columbia type were used as research objects and were planted in the growth room of Nanjing Agricultural University. The seedlings cultured in the greenhouse (16h:8h, light:dark, 23-25℃) for 1 week were used for VIGS experiments, and the 3-week-old cotton seedlings were used for inoculation with Verticillium wilt fungus. The GbTCP20 transgenic Arabidopsis and wild-type Arabidopsis were cultured in the greenhouse (16h:8h, dark:light, 21-23℃) for 4 weeks for inoculation with Verticillium wilt fungus; the growth conditions of the inoculated Arabidopsis were changed to (16h:8h, light:dark, 21-23℃).

[0061] The leaf-decaying Verticillium wilt strain (V. dahliae, VD8) was cultured on potato dextrose agar (PDA) at 25°C for one week, followed by incubation in Czapek's liquid medium (25°C, 180 rpm) for 3-5 days. Seedlings were inoculated using the root-damage method, with a VD8 spore suspension concentration of 1×10⁻⁶. 7 mL -1 Samples were taken at 0h, 12h, 24h, 72h, and 96h after Hai7124 seedlings were inoculated with VD8; the spore suspension used for inoculating Arabidopsis seedlings with VD8 was 1×10⁻⁶. 6 mL -1 .

[0062] Example 2: RT-qPCR analysis of the cotton GbTCP20 gene

[0063] Table 1: Primers used for amplification

[0064]

[0065] The published transcriptome of sea island cotton reveals that the GbTCP20 gene is expressed in all tissues (e.g., Figure 1 (As shown in B). RT-qPCR was performed on root tissues of the island cotton genetic standard line Hai7124 at 0h, 12h, 24h, 72h, and 96h after inoculation with Verticillium wilt. The results showed that, compared to 0h, the expression of GbTCP20 was significantly upregulated at 12h, 24h, 72h, and 96h (e.g., as shown in B). Figure 1 (as shown in C).

[0066] Example 3: Subcellular localization of GbTCP20 in cotton

[0067] GbTCP20 belongs to the typical class I TCP family and contains Basic helix I loop helix II (bHLH) structure fields (such as...). Figure 1The coding sequence of GbTCP20 (SEQ ID NO. 1, 885 bp) was cloned and inserted into the pBinGFP vector with Kpnl and BamHI as restriction enzyme sites, and a 35S: GbTCP20-GFP vector was constructed by homologous recombination. The 35S: GbTCP20-GFP and P19 were transformed into Agrobacterium tumefaciens GV3101 strain after correct positive clones were selected by colony PCR and sequencing. P19 was used as an auxiliary plasmid, and P19 and 35S: GbTCP20-GFP were co-injected into N. benthamiana leaves to observe the location of GbTCP20. DAPI (4', 6-diamidino-2'-phenylindole) dye was used as a nuclear marker. The fluorescence signal in the tobacco epidermal cells was detected by LSM780 confocal microscopy (Zeiss, Germany) 2-3 days after injection (as shown in FIG. 2D). Figure 1 D as shown in FIG. 2D.

[0068] Table 2: Primers used for amplification

[0069]

[0070] Example 4: Construction of a cotton pTRV-GbTCP20 vector

[0071] TRV is a cotton tobacco rattle virus vector, which is a public vector. pTRV1 and pTRV2 vectors were used to perform virus-induced gene silencing (VIGS) experiments in cotton. pTRV1 is an auxiliary vector, and a specific fragment (441 bp) of GbTCP20 was inserted into the pTRV2 vector with EcoR1 and Xhol as restriction enzyme sites. A TRV: GbTCP20 vector was constructed by homologous recombination, and the primers were designed by Primer 6 software. The cDNA of the genetic standard line Hai7124 of upland cotton was used as a template for PCR amplification, and 2 μL of 10x Loading buffer was added after the amplification reaction was completed. Agarose gel electrophoresis was performed, and the target band was recovered by cutting the gel. The specific steps are described in the gel recovery kit instruction manual. The pTRV2 vector plasmid was digested, and then the target fragment was placed in a PCR instrument for recombination reaction at 37°C for 30 min. The recombination product was immediately taken out and placed on ice, and then transformed into E. coli competent cells. After plating, it was placed in a 37°C incubator for inverted culture for 12 h. Single colonies were picked from the plate and placed in liquid LB medium containing 700 μL of corresponding antibiotics, and cultured at 37°C and 200 rpm for 5-6 h. The bacterial solution was used as a template for PCR amplification with universal primers on the vector. After gel electrophoresis, the positive bacterial solution was sent to a sequencing company for sequencing. The bacterial solution with correct sequencing sequence was considered as a successful vector construction. The plasmid was extracted and stored at -20°C for subsequent Agrobacterium transformation experiments, and finally Agrobacterium containing TRV: GbTCP20 was obtained.

[0072] Table 3: Primers used for amplification

[0073]

[0074] Target fragment (441 bp) sequence:

[0075] AATCCGATGGCGAGACAATTCAGTGGCTGTTGCAGCAATCGGAACCATCTATCATTGC

[0076] TGCAACTGGAACTGGGACGATTCCCGCTTCAGCTCTGGCGGCTGCTGGAGCCTCTGT

[0077] TTGTGCGCAGGGGAACTCTGTTTCTGCTGGTTTGCATACCAAAATGGGACTGGGGGC

[0078] ATGTACTGGGTCCAAAGATAGGAATAATTGGGCAATGTTGGGTGGTAATTTAGGAAGA

[0079] TCCCAAATCCCAAGTGGGGCATGGTCTTCTAGTAATGGAATTGGATCAGGGCTTGTTC

[0080] AAGTTTCAGAGCAATCCACATCAGCTTCAAATTTTGGGAATGAAAACTCCAATCATAT

[0081] CCACCACAACTATGGGTTCCAGGGGCTTGAATTTCCAAATATGAATATGGGTTTTGTGAGTTTTTCGTCGCTGCTCAACGGTAGTAACCTCCAGG (SEQ ID NO. 3)

[0082] Example 5 Identification of GbTCP20 silenced plants for resistance to Verticillium wilt

[0083] To clarify the role of GbTCP20 in cotton resistance to Verticillium wilt, we used virus-induced gene silencing (VIGS) to analyze the function of cotton resistance to Verticillium wilt. The stored pTRV1 and pTRV2 Agrobacterium cultures were removed from the -80°C freezer and streaked on plates containing Kan and Rif antibiotics and incubated at 28°C for 2 days in an inverted incubator. Single colonies from the plates were picked into 700 μL of liquid LB medium containing the corresponding antibiotics and incubated at 28°C for 24 h at 200 rpm. The cultures were then incubated in 50 mL of liquid LB medium containing Kan and Rif antibiotics and incubated at 28°C overnight at 200 rpm for 12 h, until the OD 600 was about 1.5, and the bacteria were collected by centrifugation at 4000 rpm for 10 min, and the supernatant LB was discarded. Resuspension buffer was added to each tube to a final volume of 10 mL, and the bacteria at the bottom of the tube were resuspended. After resuspending the Agrobacterium, the bacteria were centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The Agrobacterium was resuspended with resuspension buffer and adjusted to an OD 600 of 1.2-1.4. The adjusted bacterial solution was then incubated at 28°C in the dark for 3 h. The Agrobacterium strains containing the pTRV1 and pTRV2 vectors were combined at a ratio of 1:1 and injected into the back of the cotyledons of cotton seedlings. The injection target was the cotyledons of 7-day-old cotton seedlings. TRV:CLA1 (Cloroplastos alterados 1) was used as a positive control, and the cotton plants were injected with TRV:CLA1. When the cotton plants showed a white phenotype (about 2 weeks), the silencing efficiency of GbTCP20 was detected. TRV:00 was used as a negative control. The expression of GbTCP20 in the GbTCP20 silencing group was significantly lower than that in the control plants (e.g., Fig. 2A). Figure 2 The cotton seedlings inoculated with VD8 were analyzed for disease symptoms 11-25 days after inoculation. The disease leaf rate of the GbTCP20 silencing plants was significantly higher than that of the control plants, indicating that GbTCP20 silencing reduced the resistance of cotton to Verticillium wilt (e.g., Fig. 2B). Figure 2

[0084] Example 6 Transcriptome analysis of the roots of GbTCP20 silencing plants and control plants

[0085] ​To further investigate the biological processes by which GbTCP20 regulates plant disease resistance, we performed transcriptome sequencing on roots of GbTCP20-silenced plants and control plants at 0h, 24h, and 96h after inoculation with Verticillium wilt. Sequencing was performed using an Illumina NovaSeq 6000 platform, and the sequences were cutadapted back onto the genome of *G. barbadense* (*G. barbadense* cv. Hai7124(ZJU_v1.1)). Differentially expressed gene analysis (|log2(fc)|≥1 and P-value≤0.05) showed that 11 differentially expressed genes were present between GbTCP20-silenced plants and control plants at 0h, including 7 upregulated genes and 4 downregulated genes; 631 differentially expressed genes were present at 24h, including 410 upregulated genes and 221 downregulated genes; and 1302 differentially expressed genes were present at 96h, including 335 upregulated genes and 967 downregulated genes (e.g., *G. barbadense* cv. Hai7124(ZJU_v1.1)). Figure 3 As shown in A). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of differentially expressed genes at 24h and 96h revealed significant enrichment of the phenylpropane synthesis pathway (e.g., [example data]). Figure 3 (As shown in B-3C). Furthermore, RT-qPCR confirmed that the expression patterns of genes involved in the phenylpropane synthesis pathway (GbPAL, GbC4H, GbpoxN1, GbCOMT, GbF5H, and GbPER11) were consistent with RNA-seq data, meaning that these genes were significantly downregulated in GbTCP20-silenced plants compared to control plants (e.g., ...). Figure 3 (As shown in D). Furthermore, the phylogenetic tree illustrates the evolutionary relationships and similarities between GbTCP20 and its homologs, revealing that the similarity between homologs and GbTCP20 is over 50% (e.g., ...). Figure 4 (As shown in A). This data was used to analyze the off-target silencing rate of homologous genes in GbTCP20-silenced plants. There was no significant difference in the FPKM values ​​of homologous genes between GbTCP20-silenced plants and control plants, indicating that GbTCP20 homologous genes did not exhibit off-target silencing in the VIGS experiment. GbTCP20 exhibits high VIGS fragment specificity and good silencing efficiency (e.g., as shown in A). Figure 4 (as shown in B).

[0086] Table 4: Gene-specific primers

[0087]

[0088]

[0089] Example 7: Yeast One-Hybrid Experiment (YIH)

[0090] To further explore the molecular mechanism of GbTCP20 in regulating plant disease resistance, based on the analysis of the promoters (2000 bp) of the differentially expressed genes related to phenylpropanoid biosynthesis in GbTCP20-silenced and control plants at 24 h and 96 h, it was found that only the promoter (-383 bp to -391 bp) of GbCOMT gene encoding a key enzyme of lignin biosynthesis, caffeic acid-O-methyltransferase, contained the target element GGGCCCAC (as shown in Figure 6 Formula I) of class I TCP transcription factors reported in Arabidopsis.

[0091] The experiment used T4 ligation to construct the vector pAbAi-bait, which was pAbAi-Target and pAbAi-Mutant, respectively. Among them, the SmaI linearized vector pAbAi, the target element (GGGCCCAC) and the mutant element (AAATTTAC) were each set with three tandem repeats. At the same time, the vector pGADT7-GbTCP20 was constructed by homologous recombination, and the enzyme cutting site was EcoR I and BamHI. The pAbAi-bait was transformed into yeast Y1H Gold to obtain the bait yeast, and the minimum inhibitory concentration of aureobasidin A (AbA r ) was tested for the bait yeast. pAbAi-53 was used as a positive control, and its minimum inhibitory concentration was 200 ng / mL. The pAbAi-bait bacteria were inoculated into (SD) / -Ura (AbA r ), and placed at 30°C for 2-3 days. The results showed that the minimum inhibitory concentration of aureobasidin A for pAbAi-Target and pAbAi-Mutant bait bacteria was 200 ng / mL. After pGADT7-GbTCP20 was transformed into pAbAi-target bait bacteria, it was coated into (SD) / -Leu / -Ura AbA (200 ng / mL) at 30°C for 3-5 days to detect the binding of GbTCP20 and the target element. pGADT7-53 transformed pAbAi-53 bait bacteria was used as a positive control, and pGADT7-GbTCP20 transformed pAbAi-mutant bait bacteria was used as a negative control. The results showed that pGADT7-GbTCP20 transformed pAbAi-target bait bacteria could grow normally in (SD) / -Leu / -Ura AbA (200 ng / mL), while pGADT7-GbTCP20 transformed pAbAi-mutant bait bacteria could not grow in (SD) / -Leu / -Ura AbA (200 ng / mL) (as shown in Figure 5 Figure A), indicating that GbTCP20 can bind to the element GGGCCCAC.

[0092] Table 5: Primers required for vector construction

[0093]

[0094] Example 8 Dual-LUC reporter assay

[0095] In this experiment, Dual-LUC technology was used to test the binding ability of GbTCP20 to GGGCCCAC. The 35S::GbTCP20 vector was constructed using homologous recombination, with BamH I and Sac I as the enzyme cutting sites. The ptarget-mini35S-LUC and pmutant-mini35S-LUC vectors were constructed using T4 ligation, with Kpn I and Hind III as the enzyme cutting sites. The mini35S promoter is a minimal promoter of the CAMV 35S, which drives the expression of the LUC reporter gene. The mini35S sequence was inserted into pGreenII0800-LUC to produce the pGreenII0800-LUC(mini35S) vector, with BamH 1 and Hind I as the restriction enzyme sites. Nicotiana benthamiana was transformed with 35S::GbTCP20 / ptarget-mini35S-LUC, 35S::GbTCP20 / pmutant-mini35S-LUC was co-transformed into Nicotiana benthamiana as a negative control, and 35S::LUC was transformed into Nicotiana benthamiana as a positive control. Each experiment was set up with three biological replicates. The activity of firefly luciferase (LUC) in transiently transformed Nicotiana benthamiana leaves was analyzed using a Tanon 5200 CCD imaging system. At the same time, the activities of firefly luciferase (LUC) and Renilla luciferase (REN) were determined using a dual-luciferase assay kit, and the LUC / REN ratio was used to indicate the activity of the promoter. The Dual-LUC results showed that the tobacco leaves injected with 35S::GbTCP20 / pmutant-mini35S-LUC emitted very weak fluorescent signals, while the tobacco leaves injected with 35S::LUC and 35S::GbTCP20 / ptarget-mini35S-LUC emitted strong fluorescence (as shown in Figure 5 As shown in Figure 8A, the LUC / REN value of 35S::GbTCP20 / ptarget-mini35S-LUC was significantly higher than that of 35S::GbTCP20 / pmutant-mini35S-LUC (as shown in Figure 8B), indicating that GbTCP20 can bind to the element GGGCCCAC. Figure 5 As shown in Figure 8A, the LUC / REN value of 35S::GbTCP20 / ptarget-mini35S-LUC was significantly higher than that of 35S::GbTCP20 / pmutant-mini35S-LUC (as shown in Figure 8B), indicating that GbTCP20 can bind to the element GGGCCCAC.

[0096] The candidate target gene GbCOMT promoter (-1000bp) was constructed into pGreenll0800-LUC with BamH I and Hind III as the enzyme cutting sites. 35S::GbTCP20 was the effector. N. benthamiana transformed with 35S::GbTCP20 / GbCOMTpro-LUC, 35S::GbTCP20 / empty-LUC co-transformed N. benthamiana was the negative control, and 35S::LUC transformed N. benthamiana was the positive control. Three biological replicates were set for each experiment. The Dual-LUC results showed that the tobacco leaves injected with 35S::GbTCP20 / empty-LUC emitted very weak fluorescence signals, while the tobacco leaves injected with 35S::LUC and 35S::GbTCP20 / GbCOMTpro-LUC both emitted strong fluorescence (as shown in Figure 5 Fig. 3B). The LUC / REN value of 35S::GbTCP20 / GbCOMTpro-LUC was significantly higher than that of 35S::GbTCP20 / empty-LUC (as shown in Figure 5 Fig. 3C), indicating that GbTCP20 can bind to the GbCOMT promoter.

[0097] Table 6: Primers required for vector construction

[0098]

[0099] Example 9 Determination of lignin content in GbTCP20 silenced plants and control plants

[0100] The lignin content in the stems of GbTCP20 silenced plants and control plants was determined by acetylation method. The samples were the stems (1 cm above the cotyledon node) of Hai7124 inoculated with VD8 / H2O for 10 days. First, the samples were dried in a 60°C oven for 4-6 h, then ground into fine powder in a mortar, and 500 μL of reagent solution 1 and 20 μL of perchloric acid solution were added in sequence in a 10 mL glass test tube, which was sealed with sealing film and placed in a 80°C water bath for 40 min, and shaken every 10 min. After taking out, it was naturally cooled. Second, 500 μL of reagent solution 2 was added and mixed well. Third, 20 μL of supernatant was taken from the mixture into a new 10 mL centrifuge tube, and 980 μL of glacial acetic acid was added. Finally, 200 μL of reaction solution was taken into a 96-well enzyme-labeled plate, and each experimental tube was set with three technical replicates. The absorbance value A at 280 nm was determined by an enzyme-labeled instrument. A blank tube and a determination tube were recorded respectively. The entire experiment was performed in a fume hood. Among them, ΔA = A determination tube - A blank tube.

[0101] The lignin standard curve was y = 0.02776x + 0.0068, R2= 0.9889;

[0102] Lignin (mg / g dry weight) = (ΔA-0.0068) ÷ 0.02776 × Vtotal × 10-3 ÷ W × T = 0.0735 × (ΔA-0.0068) ÷ W × T (Vtotal: total volume of reaction: 1.02 mL; W: sample mass, g; T: dilution factor).

[0103] The analysis of the determination results found that the lignin content of the control plants 10 days after inoculation with VD8 was significantly higher than that 10 days after inoculation with H2O; it was shown that the invasion of G. giganate would promote the accumulation of lignin in cotton stems. However, the lignin content of GbTCP20-silenced plants 10 days after inoculation with bacteria and water had no significant difference (as shown in Figure 5 F), indicating that GbTCP20 gene silencing inhibited the accumulation of cotton lignin under the condition of inoculation with bacteria.

[0104] Example 10: Lignin histochemical staining analysis of GbTCP20-silenced plants and control plants

[0105] The lignin histochemical staining analysis of GbTCP20-silenced plants and control plants was performed by using the phloroglucinol-hydrochloric acid method, and the samples were still the stems of Hai7124 10 days after inoculation with VD8. The stems of Hai7124 were cut 1 cm above the cotyledon node, and the cut materials were placed on glass slides, 50 μL of lignin acid solution was added, and after 2-3 min, an equal amount of phloroglucinol staining solution was added, and then the materials were observed under an optical microscope (10x). The coloring degree of the primary xylem of the stems of the two treatment groups inoculated with VD8 was observed, and it was found that the coloring range of the primary xylem of the GbTCP20-silenced plants was shallower than that of the control plants (as shown in Figure 5 G).

[0106] Example 11: Analysis of the resistance of GbTCP20-overexpressing Arabidopsis to Verticillium wilt

[0107] To explore the relationship between GbTCP20 overexpression and the resistance of plants to Verticillium wilt, we constructed a GbTCP20 overexpression vector driven by the 35S promoter (pBI121 vector) with BamH I and Sac I as the enzyme cutting sites. The wild-type Col-0 Arabidopsis (WT) was transformed by using the ‘floral dip’ method mediated by Agrobacterium tumefaciens to obtain transgenic Arabidopsis. The harvested seeds of T0 generation were subjected to antibiotic screening and PCR molecular identification, and until T3 generation, the genomes and transcriptomes of 8 independent GbTCP20 overexpression transgenic lines (OE) were identified, and finally, 3 overexpression lines (OE4, OE11 and OE19) with higher transcription levels were selected according to the relative expression levels for the resistance analysis of Verticillium wilt (as shown in Figure 7A). Disease severity investigation was conducted from 0 (healthy plant) to 4 (dead plant). Disease index (DI) was calculated by the formula: DI = [(∑ number of leaves at each level x each level representative value) / (total number of leaves investigated x highest level representative value)] x 100. Wild type (WT) showed more severe wilting than 3 OE lines 12 days after VD8 inoculation (as shown in Fig. 1 Figure 7 B). Meanwhile, disease severity investigation and disease index calculation were conducted on plants 12 days after VD8 inoculation. The results showed that wild type (WT) reached 59% disease index 12 days after VD8 inoculation, which was 13%, 21% and 16% higher than OE4, OE11 and OE19, respectively (as shown in Fig. 2 Figure 7 C). It indicated that GbTCP20 overexpression significantly improved the resistance of Arabidopsis to Verticillium wilt.

[0108] Table 7: Primers for vector construction and positive plant identification

[0109]

[0110] Verticillium wilt poses a significant threat to global crop industries. Transcriptional regulation is a key step in controlling the expression of resistance genes. TCP transcription factors are involved in the regulation of immune responses, including plant hormone signaling, activation of systemic acquired resistance (SAR), and interaction with pathogen effectors. AtTCP20 has been reported to act as a node regulator of jasmonic acid (JA) and salicylic acid (SA) signaling pathways, and can bind to effectors secreted by bacteria (Ralstonia pseudosolanacearum) and fungi (Golovinomyces orontii). Cotton is an important economic crop and a source of textile fibers and seed oil. However, TCP proteins in cotton have only been reported to be associated with developmental regulation, such as fiber development, cell wall thickening, and leaf branching. The role of TCP proteins in the defense of cotton against Verticillium wilt remains unclear. In this study, we identified a Gossypium barbadense transcription factor, GbTCP20, which positively regulates the resistance of cotton to Verticillium wilt (as shown in Fig. 3 Figure 1). GbTCP20 silencing will reduce the resistance of cotton to Verticillium wilt; overexpression of GbTCP20 in Arabidopsis will enhance the resistance of plants to Verticillium wilt. In addition, lignin is an aromatic heteropolymer mainly existing in the secondary cell wall of vascular plants. Lignin plays a key role in water transport, mechanical support and defense against plant pathogens. Physiological and biochemical experimental evidence shows that after the plant responds to pathogen invasion and suffers induced stress, the expression level of genes related to lignin synthesis increases significantly, enzyme activity increases, and ultimately leads to lignin deposition. There are obvious differences in the rate and total amount of lignin synthesis and deposition between resistant and susceptible cotton varieties. At present, the transcriptional regulatory network of lignin synthesis has basically been perfected. The transcription factors (TFs) related to lignin synthesis mainly include MYB, WRKY, NAC, AP2 / ERF and BEL1-like families. Among them, MYB TFs account for the largest proportion and play an important role in regulating lignin biosynthesis. However, there are relatively few reports about TCP proteins participating in the regulation of lignin biosynthesis. There is only one report about the indirect regulation of transcription factor TCP4 on lignin synthesis in Arabidopsis, that is, AtTCP4 promotes the biosynthesis of secondary cell walls such as lignin and cellulose by regulating the expression of VND7. VND7 belongs to the NAC transcription factor family. In this study, we prove that GbTCP20 confers resistance to Verticillium wilt in cotton by directly activating the expression of GbCOMT (caffeic acid-O-methyltransferase), a key enzyme gene for lignin synthesis. And studies have shown that silencing GhCOMT will reduce the content of lignin and weaken the resistance of cotton to Verticillium wilt. The research of the present application provides a new regulatory network and a new perspective for understanding the TCP resistance mechanism of cotton to Verticillium wilt.

[0111] SEQUENCE LISTING

[0112] SEQ ID NO. 1 (cDNA ORF sequence of TCP20 in the genome of Gossypium hirsutum L. H7124)

[0113] ATGGAACCTAAGGGCAACGGCAAGGGCTCAAATCATCATCCACAAGAGGTGCCCACC

[0114] TGCTTGACTCCTCAGAAAGCAGAGAACAATAAACCTGCAGAAATAAAAAACTTGCA

[0115] AATCATGATTGCGAGCAAAGATGACAACAAGAAGCAACTAGCTCCCAAGAGAAGCT

[0116] CAAACAAAGACAAGCACAAGAAAGTAGATGGCAGAGGTAGAAGAATAAGGATGCCT

[0117] GCTCTGTGCGCCGCCAGGATTTTCCAATTGACCCGAGAATTGGGTCACAAATCCGATG

[0118] GCGAGACAATTCAGTGGCTGTTGCAGCAATCGGAACCATCTATCATTGCTGCAACTGG

[0119] AACTGGGACGATTCCCGCTTCAGCTCTGGCGGCTGCTGGAGCCTCTGTTTGTGCGCA

[0120] GGGGAACTCTGTTTCTGCTGGTTTGCATACCAAAATGGGACTGGGGGCATGTACTGG

[0121] GTCCAAAGATAGGAATAATTGGGCAATGTTGGGTGGTAATTTAGGAAGATCCCAAATC

[0122] CCAAGTGGGGCATGGTCTTCTAGTAATGGAATTGGATCAGGGCTTGTTCAAGTTTCAG

[0123] AGCAATCCACATCAGCTTCAAATTTTGGGAATGAAAACTCCAATCATATCCACCACAA

[0124] CTATGGGTTCCAGGGGCTTGAATTTCCAAATATGAATATGGGTTTTGTGAGTTTTTCGT

[0125] CGCTGCTCAACGGTAGTAACCTCCAGGTTCCAGGTTTGGAGCTTGGGCTTTCACAGG

[0126] ATCCGCATTTTGGAGTGTCTAATTCCCAAGCTTTTAGCCACTTTTACCAGCAGATTGGG

[0127] CAGCAGCGAGGTGGTGTACGTCCCTTGAATCAGCAGCAGATTGTTGCTGATAAGGAT

[0128] AACTCCCAGGGATCCAAGCAGTAG SEQ ID NO. 2 (amino acid sequence of TCP20 in the genome of Gossypium hirsutum H7124)

[0129] MEPKGNGKGSNHHPQEVPTCLTPQKAENNKPAEIKNLQIMIASKDDNKKQLAPKRSSNK

[0130] DKHKKVDGRGRRIRMPALCAARIFQLTRELGHKSDGETIQWLLQQSEPSIIAATGTGTIPA

[0131] SALAAAGASVCAQGNSVSAGLHTKMGLGACTGSKDRNNWAMLGGNLGRSQIPSGAWS

[0132] SSNGIGSGLVQVSEQSTSASNFGNENSNHIHHNYGFQGLEFPNMNMGFVSFSSLLNGSNL

[0133] QVPGLELGLSQDPHFGVSNSQAFSHFYQQIGQQRGGVRPLNQQQIVADKDNSQGSKQ.

Claims

1. A nucleotide sequence as set forth in SEQ ID NO. 1 GbTCP20 Use of the gene in improving the Verticillium wilt disease resistance of cotton or Arabidopsis or breeding new germplasm of cotton or Arabidopsis with improved Verticillium wilt disease resistance.

2. A recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing a nucleotide sequence as shown in SEQ ID NO. 1 GbTCP20 The use of the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing a nucleotide sequence as shown in SEQ ID NO. 1 in improving the resistance of cotton or Arabidopsis to Verticillium wilt or breeding new germplasm of cotton or Arabidopsis with improved resistance to Verticillium wilt.

3. The nucleotide sequence as shown in SEQ ID NO. 1 GbTCP20 The application of the protein GbTCP20 encoded by the gene in improving the Verticillium wilt resistance of cotton or Arabidopsis or cultivating new germplasm of cotton or Arabidopsis with improved Verticillium wilt resistance.

4. Use according to claim 1 or 2, characterized in that, The application discloses a method for improving the resistance of cotton or Arabidopsis to verticillium wilt by overexpressing a target gene. GbTCP20 The application discloses a method for improving the resistance of cotton or Arabidopsis to verticillium wilt by overexpressing a target gene. GbTCP20 The application discloses a method for improving the resistance of cotton or Arabidopsis to verticillium wilt by overexpressing a target gene.

5. A method of increasing resistance to Verticillium wilt in cotton, comprising, overexpressing a nucleotide sequence as set forth in SEQ ID NO. 1 GbTCP20 GbTCP20 gene.

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