TLP1-like gene molecular marker related to verticillium wilt resistance of cotton and application of TLP1-like gene molecular marker

Abstract

The invention discloses a TLP1-like gene molecular marker related to cotton verticillium wilt resistance and application of the TLP1-like gene molecular marker, and belongs to the field of biotechnology application. The promoter of the TLP1 protein gene TLP1-like has two haplotypes, the promoter sequence of the haplotype 1 is SEQ ID NO.5, and the genomic sequence of the haplotype 2 is SEQ ID NO.6. The invention also discloses a method for preparing the TLP1 protein gene TLP1-like promoter of the TLP1 protein gene TLP1-like promoter of the TLP1 protein gene. Compared with the haplotype 1, the haplotype 2 has an SV insertion of 2623 bp at a-899 bp position of an ATG upstream promoter region of a TLP1-like gene, so that the expression quantity of the TLP1-like gene is reduced. An InDel functional marker capable of remarkably distinguishing the haplotype 1 and the haplotype 2 is further developed. Compared with the haplotype 1, the haplotype 2 has a remarkable advantage in the aspect of improving the verticillium wilt resistance of the cotton, and the silent haplotype 1 cotton plant TLP1-like remarkably improves the resistance of the cotton to verticillium dahliae. The haplotype 2 has important effects and application prospects in screening cotton disease-resistant materials and cultivating cotton high-resistance varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology applications and relates to a molecular marker of a TLP1-Like protein gene related to cotton Verticillium wilt function and an application thereof. Background Art

[0002] Cotton (Gossypium spp.) is one of the important economic crops. It is an important strategic material related to my country's national economy and people's livelihood and a raw material for the textile industry. Upland cotton and sea island cotton are the two main allotetraploid cultivated cotton species. The two originated from a common ancestor but were domesticated independently. Upland cotton, also known as fine-staple cotton, has medium fiber quality, strong adaptability, and high yield, accounting for 90% of the world's total cotton production; sea island cotton, also known as long-staple cotton, has excellent fiber quality and high resistance to Verticillium wilt but low yield. Cotton Verticillium wilt (Verticillium wilt) is a soil-borne vascular disease caused by Verticillium dahliae, which can cause cotton leaves to yellow, wilt, and fall off, and even the death of the entire plant. This disease seriously affects the sustainable development of the cotton industry. Therefore, the rapid screening of cotton varieties resistant to Verticillium wilt is an effective means to solve this problem.

[0003] With the development of cotton genome sequencing technology and bioinformatics, some molecular markers linked to cotton Verticillium wilt resistance have been obtained. However, few functional genes for Verticillium wilt resistance in sea island cotton have been identified, and even fewer selection markers can be directly used to assist breeding. Summary of the Invention

[0004] The present invention aims to provide a TLP1-like molecular marker of a TLP1 protein gene related to the Verticillium wilt resistance trait of cotton and its application.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention seeks to protect at least one application of an InDel molecular marker associated with cotton Verticillium wilt resistance or a substance for detecting the InDel molecular marker in the following (a1) to (a5):

[0007] (a1) screening or assisting in screening cotton germplasm resistant to Verticillium wilt, or preparing a product for screening or assisting in screening cotton germplasm resistant to Verticillium wilt;

[0008] (a2) identifying or assisting in identifying the Verticillium wilt resistance trait of cotton, or preparing a product for identifying or assisting in identifying the Verticillium wilt resistance trait of cotton;

[0009] (a3) identifying the promoter haplotype of a cotton TLP1 protein gene TLP1-like, or preparing a product for identifying the promoter haplotype of a cotton TLP1 protein gene TLP1-like;

[0010] (a4) Improving the Verticillium wilt resistance of cotton, or preparing products for improving the Verticillium wilt resistance of cotton;

[0011] (a5) Breeding cotton with high resistance to Verticillium wilt, or preparing products for breeding cotton with high resistance to Verticillium wilt;

[0012] The InDel molecular marker is based on the insertion / deletion of a 2623 bp nucleotide fragment as shown in SEQ ID NO.7 at the -899 bp position of the promoter sequence of the cotton TLP1 protein gene GbTLP1-like as shown in SEQ ID NO.6.

[0013] The InDel molecular marker is based on the nucleotide sequence of the cotton TLP1 protein gene TLP1-like as shown in SEQ ID NO.6, that is, there is an insertion / deletion of a 2623bp nucleotide fragment upstream (-899bp) of the start codon (ATG).

[0014] Furthermore, the promoter of the cotton TLP1 protein gene TLP1-like has two haplotypes, the promoter sequence of haplotype 1 is shown in SEQ ID NO.5, and the promoter sequence of haplotype 2 is shown in SEQ ID NO.6. Compared with haplotype 1, haplotype 2 has a 2623bp nucleotide fragment insertion of a nucleotide sequence as shown in SEQ ID NO.7 at -899bp upstream of the start codon (ATG) of the gene, resulting in reduced expression of the TLP1-like gene; the disease resistance of haplotype 2 plants is better than that of haplotype 1.

[0015] In a second aspect, the present invention seeks protection for a product containing a substance for detecting an InDel molecule marker, and the substance is at least one of the following (b1) to (b5):

[0016] (b1) Screening or assisting in screening products for different cotton varieties resistant to Verticillium wilt;

[0017] (b2) Products that identify or assist in identifying cotton's resistance to Verticillium wilt;

[0018] (b3) Identification of products of cotton TLP1-like promoter haplotypes;

[0019] (b4) Products for improving cotton's resistance to Verticillium wilt;

[0020] (b5) Products for breeding cotton with high Verticillium wilt resistance.

[0021] In a third aspect, the present invention claims protection for a method, which is at least one of the following methods (c1) to (c5):

[0022] (c1) a method for screening or assisting in screening cotton germplasm with high resistance to Verticillium wilt;

[0023] (c2) Methods for identifying or assisting in identifying Verticillium wilt resistance traits in cotton;

[0024] (c3) Method for identifying the haplotype of the promoter of the cotton TLP1-like protein gene;

[0025] (c4) Methods for improving cotton's resistance to Verticillium wilt;

[0026] (c5) Methods for breeding cotton with high Verticillium wilt resistance;

[0027] The method comprises the following steps: using a substance for detecting InDel molecular markers to detect whether a promoter of a TLP1-like protein gene TLP1-like of a cotton germplasm material to be tested contains the InDel molecular marker as claimed in claim 1, thereby identifying the haplotype of the promoter of the cotton TLP1-like protein gene TLP1-like; two haplotypes exist in the promoter region of the cotton TLP1 protein gene TLP1-like, and haplotype 2 has a 2623 bp nucleotide fragment insertion of a nucleotide sequence as shown in SEQ ID NO.7 at a position -899 bp upstream of the start codon ATG of the gene compared to haplotype 1; haplotype 2 has better disease resistance than haplotype 1; and the cotton germplasm material to be tested carrying haplotype 2 is selected as a cotton germplasm with Verticillium wilt resistance, used in cotton breeding, and improved Verticillium wilt resistance of cotton.

[0028] In a specific embodiment of the present invention, PCR amplification is performed using InDel molecular marker primers, and different haplotypes are identified based on the presence or absence of amplified products. Amplified products with a molecular weight of 501 bp using InDel molecular marker primers are haplotype 2, while no amplified products are haplotype 1.

[0029] In the technical solution of the present invention, the substance for detecting the InDel molecular marker is the following (d1) or (d2) or (d3) or (d4):

[0030] (d1) in vitro nucleic acid amplification primers as shown in SEQ ID NO.8 and SEQ ID NO.9 for specifically amplifying the InDel molecular marker;

[0031] (d2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in (d1);

[0032] (d3) a kit containing the in vitro nucleic acid amplification primer described in (d1) or the in vitro nucleic acid amplification reagent described in (d2);

[0033] (d4) A detection instrument containing the in vitro nucleic acid amplification primer (d1), the in vitro nucleic acid amplification reagent (d2) or the kit (d3).

[0034] In the technical solution of the present invention, the cotton Verticillium wilt resistance trait is at least one of a disease phenotype and a disease incidence rate.

[0035] In a fourth aspect, the present invention claims protection for the use of a substance that inhibits the expression of a TLP1-like protein gene TLP1-like or a substance that reduces the activity or content of a protein encoded by the TLP1-like protein gene TLP1-like in the following (e1) or (e2):

[0036] (e1) improving cotton resistance to Verticillium wilt or cultivating new cotton germplasm with improved resistance to Verticillium wilt;

[0037] (e2) Promote disease resistance in target plants.

[0038] The TLP1-like protein gene TLP1-like is a DNA molecule of the following (f1) or (f2) or (f3) or (f4):

[0039] (f1) a DNA molecule whose coding region is shown in SEQ ID NO. 1 or SEQ ID NO. 3;

[0040] (f2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3;

[0041] (f3) a DNA molecule that hybridizes with the DNA molecule defined in (f1) or (f2) under stringent conditions and encodes a TLP1-like protein;

[0042] (f4) A DNA molecule derived from upland cotton or sea island cotton that has at least 98% homology to the DNA molecule defined in (f1) or (f2) and encodes a TLP1-like protein.

[0043] Furthermore, the protein encoded by the TLP1-like protein gene TLP1-like is (g1) or (g2) or (g3):

[0044] (g1) a protein with an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 4;

[0045] (g2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of (g1);

[0046] (g3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4.

[0047] The present invention shows that when the TLP1-like protein gene TLP1-like (TRV: GhTLP1-like) is silenced in upland cotton, compared with the control group (TRV: 00), the disease resistance of the cotton in the silenced group is increased, the browning degree of the stem is reduced, the fungal recovery rate is slowed down, and the fungal invasion amount is reduced.

[0048] Furthermore, the substance that inhibits the expression of the TLP1-like protein gene TLP1-like or reduces the activity or content of the protein encoded by the TLP1-like protein gene TLP1-like is a biomaterial, and the biomaterial is any one of the following hl) to h6):

[0049] (h1) a nucleic acid molecule that silences, inhibits, interferes with, or gene-edits the expression of the TLP1-like protein gene;

[0050] (h2) an expression cassette containing the nucleic acid molecule described in (h1);

[0051] (h3) a recombinant vector containing the nucleic acid molecule described in (h1), or a recombinant vector containing the expression cassette described in (h2);

[0052] (h4) a recombinant microorganism containing the nucleic acid molecule described in (h1), or a recombinant microorganism containing the expression cassette described in (h2), or a recombinant microorganism containing the recombinant vector described in (h3);

[0053] (h5) A gene-edited plant cell line containing the nucleic acid molecule described in (h1), or a gene-edited plant cell line containing the expression cassette described in (h2), or a gene-edited plant cell line containing the recombinant vector described in (h3);

[0054] (h6) gene-edited plant tissue containing the nucleic acid molecule described in (h1), or transgenic plant tissue containing the expression cassette described in (h2), or transgenic plant tissue containing the recombinant vector described in (h3);

[0055] (h7) A gene-edited plant organ containing the nucleic acid molecule described in (h1), or a gene-edited plant organ containing the expression cassette described in (h2), or a gene-edited plant organ containing the recombinant vector described in (h3).

[0056] In the fifth aspect, the present invention requests protection for a method for improving the disease resistance of cotton, taking the TLP1-like protein gene TLP1-like as the target gene, and through genetic engineering methods, silencing, inhibiting, interfering or gene editing the nucleotide sequence of the TLP1-like protein gene TLP1-like shown in SEQID NO.1 and SEQ ID NO.3 in cotton, inhibiting its expression, improving the disease resistance of cotton or cultivating new cotton germplasm with resistance to Verticillium wilt.

[0057] Through transcriptome analysis, the present invention identified a thaumatin-like protein (TLP) gene, TLP1-like, composed of an N-signal peptide (SP) and a THN domain. The gene's ID numbers in G. barbadense L. acc. Hai7124 and G. hirsutum L. acc. TM-1 are GH_A09G1614 and GB_A09G1742, respectively. Normally, this gene is only expressed at low levels in filaments and not in other tissues. However, after induction with Verticillium dahliae, GhTLP1-like expression was dramatically upregulated in the stems of the susceptible cotton variety TM-1, while its expression level in the resistant variety Hai7124 was very low, 54 times weaker than that of TM-1. Sequence alignment of the TLP1-like gene's promoter region revealed a 2623-bp nucleotide sequence insertion upstream of the ATG (-899 bp) in Hai7124 compared to TM-1. Therefore, the TLP1-like promoter sequence differences in TM-1 and Hai7124 were designated haplotype 1 and haplotype 2, respectively. A dual-luciferase reporter assay was used to detect promoter activity, demonstrating that a 2623-bp SV insertion in the promoter region of the TLP1-like gene from Hai7124 significantly impaired reporter gene expression. Furthermore, an InDel (InDel) functional molecular marker targeting this insertion site was developed. InDel marker primers amplified a 501-bp product in promoters containing SV insertions, while no product was detected in promoters without SV insertions. Genotyping of natural populations of Gossypium barbadense and Upland cotton using these InDel primers revealed that the TLP1-like promoter contained two haplotypes in the Gossypium barbadense population and only haplotype 1 in the Upland population. Combined analysis of disease resistance phenotypic data from natural populations revealed that haplotype 2 exhibited significantly lower TLP1-like expression, reduced disease incidence, and enhanced disease resistance compared to haplotype 1. Furthermore, VIGS technology further elucidated the role of TLP1-like in Verticillium wilt resistance. Silencing GhTLP1-like in upland cotton TM-1 significantly enhanced cotton resistance to Verticillium wilt. This suggests that GhTLP1-like negatively regulates cotton disease resistance. Therefore, SV insertion in the promoter, which reduces the expression of the susceptible gene TLP1-like, is beneficial for enhancing cotton disease resistance.

[0058] A functional molecular marker screened by the present invention is used, and functional molecular marker-specific primers are used to detect the difference sites between different haplotypes in the promoter region of the TLP1-like protein gene. The sample carrying the haplotype 2 of the TLP1-like protein gene is selected as the cotton germplasm with high disease resistance. The forward and reverse primer sequences of the functional molecular marker-specific primers are shown in SEQ ID NO.8 and SEQ ID NO.9. The detection includes detection at the gene level. Studies have shown that when the haplotype 2 gene sequence is present in cotton plants, the expression level of the allele or its protein is reduced, which promotes the improvement of cotton disease resistance, indicating that the gene haplotype 2 has an important role and application prospects in improving cotton disease resistance traits and breeding new cotton disease-resistant varieties.

[0059] The development process of the technical solution of the present invention specifically includes the following steps:

[0060] 1. Through transcriptome analysis of Hai7124 and TM-1 stems, a TLP1-like protein gene, TLP1-like, was identified. The basal expression level of this gene in both Hai7124 and TM-1 was extremely low, with almost no expression. However, after induction with Verticillium dahliae, its expression was significantly upregulated in TM-1, with the upregulated expression level approximately 54-fold higher than that in Hai7124. The CDS sequence of GhTLP1-like in upland cotton TM-1 is SEQ ID NO.1, and the encoded amino acid sequence is SEQ ID NO.2. The CDS sequence of GbTLP1-like in sea island cotton Hai7124 is SEQ ID NO.3, and the encoded amino acid sequence is SEQ ID NO.4. Analysis of the promoter sequence of the TLP-like gene revealed that the promoter region of Hai7124 contained a large fragment of 2623 bp, compared to TM-1, with the insertion site located upstream (-899 bp) of the start codon (ATG) of TLP1-like. The promoter sequence of TLP1-like is SEQ ID NO. 5 in TM-1 and SEQ ID NO. 6 in Hai7124.

[0061] 2. Using a dual-luc reporter assay (Dual-luc), we detected the driving activity of two haplotypes of TLP1-like promoters and found that the basal driving activity of the promoters in GbTLP1-like_pro and GhTLP1-like_pro was weak. Inoculation with Verticillium dahliae (V991) enhanced reporter gene expression, demonstrating that it belongs to the Verticillium dahliae-induced expression type. After truncating the GbTLP1-like promoter (removing the SV), the expression of the reporter gene was significantly enhanced compared to the full-length promoter (including the intact SV). This demonstrated that SV insertion directly weakened the driving activity of the promoter. InDel markers were developed for insertion / deletion variations in the TLP1-like promoter, and their InDel primer sequences are SEQ ID NO.8 and SEQ ID NO.9, respectively. Genotype identification was performed on natural populations of sea island cotton and upland cotton using these InDel primers, ultimately obtaining genotyping data for the natural populations. The deletions / insertions were named haplotype 1 and haplotype 2, respectively. The results showed that only one genotype, haplotype 1, exists in natural upland cotton populations. There are two types of haplotypes in the sea island cotton population: haplotype 1 and haplotype 2.

[0062] 3. We randomly selected samples of haplotype 1 and haplotype 2 from a natural population of sea island cotton for disease analysis and TLP1-like expression. The results showed that haplotype 1 was more severely diseased than haplotype 2, and TLP1-like expression was significantly higher in haplotype 1 than in haplotype 2. Therefore, differences in TLP1-like expression caused by SV insertions / deletions in the promoter are associated with plant disease resistance.

[0063] 4. The present invention also completed the disease resistance function identification of TLP1-like using VIGS technology. The results showed that the disease resistance of the TRV:GhTLP1-like silenced group in upland cotton TM-1 was significantly improved compared with TRV:00. The diseased leaf rate, fungal biomass, and fungal recovery of the GhTLP1-like silenced group were all lower than those in the control group. This indicates that reduced TLP1-like expression significantly enhances cotton's resistance to Verticillium wilt.

[0064] The advantages of the present invention are:

[0065] 1. TLP1-like was identified by comparing differentially expressed genes in the stem transcriptomes of Hai7124 and TM-1 after Verticillium wilt inoculation. It is a Verticillium wilt-induced gene. Analysis of gene expression patterns in tissues and organs showed that TLP1-like is only lowly expressed in filaments and is absent in other tissues.

[0066] 2. Sequence alignment of the TLP1-like promoter revealed a large insertion / deletion (ID) upstream of the ATG (-899 bp) in the promoter region between Hai7124 and TM-1. Dual-luc assays were used to test the driving activity of the two promoters. The basal driving activity of the TLP1-like promoter was weak in both Hai7124 and TM-1. Inoculation with V991 bacteria enhanced reporter gene expression in both promoters. Furthermore, reporter gene expression was weaker in the GbTLP1-like promoter (-3790 bp to 0 bp) than in the GbTLP1-like promoter (-1167 bp to 0 bp). However, reporter gene expression was significantly enhanced in the GbTLP1-like promoter (-979 bp to 0 bp) compared to the GbTLP1-like promoter (-3790 bp to 0 bp) after promoter truncation. This confirms that insertions in the TLP1-like promoter region directly affect promoter transcriptional activity and can be used to develop functional InDel markers.

[0067] 3. Utilizing the insertion / deletion site (-899 bp upstream of the ATG) in the TLP1-like promoter region of Hai7124 and TM-1, we developed an inDel functional molecular marker to distinguish between two haplotypes of the TLP1-like promoter. This marker can be used to assist in screening or identifying samples for haplotype 2 of the TLP1-like gene.

[0068] 4. In natural populations of sea island cotton, haplotype 2 showed lower TLP1-like expression and stronger disease resistance than haplotype 1. This suggests that haplotype 2 has a significant advantage over haplotype 1 in improving cotton resistance to Verticillium wilt and could be used to improve cotton disease resistance and breed new disease-resistant cotton varieties.

[0069] 5. The results of the resistance function identification of TLP1-like using VIGS technology showed that compared with the control group (TRV:00), the diseased leaf rate of the silenced group (TRV:GhTLP1-like) was significantly reduced, the fungal biomass was significantly reduced, the fungal recovery was weakened, and the browning degree of the stems was reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 .Analysis of gene expression characteristics of TLP1-like.

[0071] a. Expression pattern analysis of A / D subgroup TLP1-like genes in stems of sea island cotton (Hai7124) and upland cotton (TM-1) following Verticillium wilt (V991) induction. The vertical axis of the bar graph represents gene expression levels, expressed as TPM values ​​derived from transcriptome sequencing. The A / D subgroups of TLP1-like have very low basal expression in stem tissue, but expression is significantly upregulated following inoculation with Verticillium wilt. In TM-1, the expression level of subgroup A TLP1-like after induction is approximately 54-fold higher than that in Hai7124, while the upregulation of subgroup D TLP1-like in TM-1 is approximately 42-fold higher than that in Hai7124. Since the upregulation of TLP1-like in subgroup A is significantly higher than that in subgroup D, it is likely that subgroup A TLP1-like is primarily functional in cotton. b. Domain prediction of TLP1-like. Prediction results indicate that TLP1-like contains a typical THN domain, and that the presence of an N-signal peptide differs between subgroups A and D. c. Subcellular localization of TLP1-like in tobacco epidermal cells. Before plasmolysis, green and red fluorescence overlap. After plasmolysis, green fluorescence localizes to the apoplast. Red fluorescence indicates the cell membrane localization marker, AtPIP2-RFP. D-Mannitol is D-mannitol, used for plasmolysis in tobacco epidermal cells. Error bars represent standard deviation (SD). Asterisks indicate statistical significance determined by t-test (*P < 0.05, ***P < 0.001).

[0072] Figure 2 .Detection of driving activity of TLP1-like promoters of different sizes.

[0073] The dual-luciferase reporter assay (Dual-luc) is a molecular biology technique widely used in gene expression regulation research, primarily for detecting promoter activity. a. Dual-luc assays the transcriptional activity of TLP1-like SV insertion / deletion promoters. The box model diagram represents the vector construction. GhTLP1-like_pro is an SV deletion promoter, GbTLP1-like_pro is an SV insertion promoter, and GbTLP1-like_proJ is a truncated version of the SV insertion promoter after SV truncation. The numbers represent the length of the promoter upstream of the TLP1-like start codon (ATG). b. The Luc / Ren value reflects the expression of the luc reporter gene. Reporter gene expression is positively correlated with promoter activity. Results showed that the Luc / Ren values ​​of the three promoters were significantly higher in the bacterial-treated group than in the water-treated group, and the Luc / Ren value of GbTLP1-like_proJ-luc was significantly higher than that of GbTLP1-like_pro-luc. This figure shows that SV insertion leads to a decrease in driver activity in response to Verticillium dahliae. Error bars represent standard deviation (SD). Asterisks indicate statistical significance as determined by t-test (***P < 0.001).

[0074] Figure 3 .Typing analysis of whether SV is inserted in the TLP1-like promoter region in natural populations of sea island cotton and upland cotton.

[0075] a. Primers InDel-F and InDel-R were used to detect SV typing in the TLP1-like promoter region. The figure shows the locations of the SV insertion sites and InDel-F / R sequences in haplotype 1 and haplotype 2 TLP1-like promoters. The SV is missing in the haplotype 1 promoter, resulting in no PCR amplification. The SV insertion is present in the haplotype 2 promoter, resulting in a 501-bp PCR product. Sequencing results showed that the PCR product was consistent with the reference sequence. "ref" represents the publicly available TLP1-like promoter sequence in the Hai7124 genome, and "seq" represents the sequence of the PCR product. b. Statistical analysis of the presence or absence of SV insertions in the TLP1-like promoter in 269 natural populations of Gossypium barbadense and 337 natural populations of Upland cotton. TLP1-like was expressed in two haplotypes in the 269 natural populations of Gossypium barbadense: 218 samples were haplotype 1 and 51 were haplotype 2. In contrast, all 337 samples in the natural population of Upland cotton were haplotype 1. c. Electrophoresis phenotyping of 269 natural populations of Gossypium barbadense and 337 natural populations of Gossypium hirsutum using genetic markers for the presence or absence of SV insertions in the TLP1-like promoter. The first lane of the agarose gel is a DNA marker. The target PCR amplification product is 501 bp in length. Haplotype 1 shows no bands, while haplotype 2 shows bands. The red numbers represent the accession numbers of the natural populations.

[0076] Figure 4 .Comparative analysis of disease resistance of different haplotypes in natural populations of sea island cotton with and without SV insertion in the TLP1-like promoter region.

[0077] a. SV identification of 24 accessions of haplotype 1 from a natural population of sea island cotton and analysis of disease progression 35 days after inoculation with Verticillium dahliae. b. SV identification of 24 accessions of haplotype 2 from a natural population of sea island cotton and analysis of disease progression after inoculation with Verticillium dahliae. c. Analysis of diseased leaf percentage in accessions of haplotype 1 and haplotype 2. The horizontal axis represents the number of days after inoculation, and the vertical axis represents the diseased leaf percentage. d. RT-qPCR analysis of TLP1-like expression in accessions of haplotype 1 and haplotype 2. The results showed that TLP1-like expression was significantly higher in haplotype 1 than in haplotype 2. The diseased leaf percentage of cotton plants in haplotype 2 was significantly lower than that in haplotype 1 15, 20, and 35 days after inoculation. This indicates that haplotype 2 plants experienced less severe disease than haplotype 1 plants. Error bars represent standard deviations (SD). Asterisks indicate statistical significance, determined by t-test (*P<0.05, **P<0.01).

[0078] Figure 5 .Functional identification of cotton TLP1-like involved in Verticillium wilt resistance.

[0079] a. RT-qPCR analysis of the silencing efficiency of GhTLP1-like in TM-1. GhTLP1-like expression was essentially absent after water inoculation. Fourteen days after inoculation, GhTLP1-like expression in the GhTLP1-like silencing group was significantly lower than in the TRV:00 control group, indicating effective GhTLP1-like silencing. b.c. Phenotypic and disease analysis of GhTLP1-like silencing and control TRV:00 plants. The diseased leaf rate in GhTLP1-like silencing plants was significantly lower than in the control group, indicating that GhTLP1-like silencing enhances cotton resistance to Verticillium wilt. d. Determination of fungal recovery and fungal biomass in stem tissues of GhTLP1-like silencing and control TRV:00 plants 14 days after inoculation. Fungal recovery was slower and mycelial biomass was lower in the GhTLP1-like silencing group compared to the control group. f. Comparison of fungal colonization in the GhTLP1-like silencing and control TRV:00 plants by stem section. The stem browning of plants in the GhTLP1-like silencing group was less severe than that in the control group. Error bars represent standard deviation (SD). Asterisks indicate statistical significance, determined by t-test (**P < 0.01, ***P < 0.001). DETAILED DESCRIPTION

[0080] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0082] Example 1: Identification of differentially expressed TLP1-like genes between Hai7124 and TM-1 after Verticillium wilt inoculation by comparative transcriptomics

[0083] Hai7124 and TM-1 are representative cotton accessions that are resistant to and susceptible to Verticillium wilt, respectively. Transcriptome sequencing was performed on these two accessions after watering and inoculation with Verticillium dahliae. Sampling was performed 13 days after inoculation, when the first true leaf became diseased, for Hai7124 and 9 days for TM-1. Sampling was performed on the stem, with three biological replicates for each location. Cotton was inoculated with Verticillium dahliae V991 using the root wounding method at the 'two leaves, one heart' stage, at a spore concentration of 1×10 7 mL -1 Transcriptome sequencing was performed on the Illumina NovaSeq6000 platform, and sequences were mapped to the upland cotton (Gossypium hirsutum L.acc.TM-1 (ZJU_v1.1)) genome using Cutadapt. Differentially expressed genes were analyzed (|log2(fc)| ≥ 1 and P-value ≤ 0.05). Analysis of differentially expressed genes revealed a gene, TLP1-like, that was significantly differentially expressed in stems after inoculation with Verticillium dahliae. Tissue expression patterns of cotton genes were obtained from the public resource database NCBI, available at http: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA802699. The domain structure of the TLP1-like gene was visualized using the online website Smart, available at https: / / smart.embl.de / . For subcellular localization, a commercial pBinGFP vector was selected, with restriction enzyme sites KpnI and BamHl. The 35S::TLP1-like-GFP vector was constructed using homologous recombination. The amplification primers are shown in Table 1. AtPIP2A is a cell membrane marker, fused with the red fluorescent protein RFP, which exhibits red fluorescence. D-Mannitol is D-mannitol, with a concentration of 0.8M, which is used for plasmolysis of tobacco cells. After plasmolysis, the extracellular protein is localized between the cell membrane and the cell wall. The fluorescence signal in tobacco epidermal cells 2-3 days after injection was detected using an LSM780 confocal microscope (Zeiss, Germany). The experimental results are shown in Figure 1. Figure 1 shown.

[0084] Table 1: Primers used for amplification

[0085]

[0086]

[0087] Example 2 Luciferase reporter assay to determine the transcriptional activity of the SV haplotype in the TLP1-like promoter region

[0088] To test the transcriptional activity of SV haplotypes in the TLP1-like promoter region, a dual-luciferase reporter assay (Dual-Luc) was used to investigate the effect of SV on promoter activity. We selected the commercially available pGreen0800II-luc vector, which contains BamH I and Hind III restriction enzyme sites. Recombination primers were designed based on the cotton reference genome published by Zhejiang University (ZJU) at NCBI, and DNA from Hai7124 and TM-1, respectively, was used as templates to clone the TLP1-like promoter. Homologous recombination was used to construct the TLP1-like_pro-luc vector, containing GhTLP1-like_pro-luc, GbTLP1-like_pro-luc, and GbTLP1-like_proJ-luc. J represents the promoter with SV truncated. The forward and reverse primer sequences involved are shown in Table 2. Three strains of Agrobacterium transformed with promoter vectors were injected into Nicotiana benthamiana. After injection, they were first cultured in the dark (1 day), then switched to normal culture (1 day). The injection site was then inoculated with V991 and water, respectively. After 12 hours of normal culture, Luc / Ren determination was performed. Three biological replicates were set for each experiment. The experimental results are shown in Figure 2. Figure 2 shown.

[0089] Table 2: Primers used for amplification

[0090]

[0091] Note: The primer pair used for TLP1-like_pro-luc vector construction can be used for both GhTLP1-like_pro-luc and GbTLP1-like_pro-luc construction.

[0092] Example 3 Development of functional markers for SV in the TLP1-like promoter region and typing of natural populations of sea island cotton and upland cotton

[0093] Based on the presence of a 2623-bp SV insertion / deletion (SID) in the promoter region of the two TLP1-like haplotypes, located at -899 bp upstream of the ATG, an InDel (InDel) functional molecular marker was developed. The forward and reverse primer sequences are shown in SEQ ID NOs. 8 and 9. The PCR reaction procedure was as follows: initial denaturation at 95°C for 3 minutes; 30 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 20 seconds; and a final extension at 72°C for 5 minutes. Amplified products were analyzed by agarose gel electrophoresis. These primers can effectively distinguish haplotypes in G. truncatum and G. truncatum. The SV insertion in the promoter produces a 501-bp PCR amplification product (shown in SEQ ID NO. 10). No SV insertion results in a PCR amplification of the promoter without a SV insertion. "ref" represents the promoter sequence in the ZJU genomic database, and "seq" represents the sequence of the PCR product.

[0094] The InDel forward and reverse primers were used to perform genotyping of the InDel loci in a natural population of upland cotton containing 337 materials and a natural population of sea island cotton containing 269 materials. Figure 3 shown.

[0095] Example 4 Functional marker detection TLP1-like gene promoter region SV presence or absence material and disease resistance analysis

[0096] Twenty-four accessions of each of two haplotypes of sea island cotton were randomly selected and grown in the plant growth chamber of Nanjing Agricultural University under a 16-hour:8-hour light:dark cycle, 23-25°C. At the two-leaf, one-heart stage, cotton plants were inoculated with Verticillium dahliae (V991), a defoliating Verticillium wilt strain, using the root wounding method. The plants were cultured on potato dextrose agar (PDA) at 25°C for one week and then placed in Czapek's liquid medium (25°C, 180 rpm) for 3-5 days. The spore suspension concentration was 1×10 7 mL -1 RNA was extracted from the stems 20 days after inoculation, and the expression of TLP1-like was identified using RT-qPCR technology. At the same time, the leaf wilting rate of cotton plants 15 days, 20 days and 35 days after inoculation was photographed and counted. The experimental results are as follows Figure 4 shown.

[0097] Example 5 Analysis of the Function of Upland Cotton GhTLP1-like Gene in Verticillium Wilt Resistance

[0098] To elucidate the role of GhTLP1-like in cotton resistance to Verticillium wilt, we selected the publicly available vector TRV (tobaccorattle virus). Virus-induced gene silencing (VIGS) experiments were conducted in upland cotton TM-1 using the pTRV1 and pTRV2 vectors. pTRV1 is a helper vector. A GhTLP1-like specific fragment (249 bp, as shown in SEQ ID NO. 11) was inserted into the pTRV2 vector, which has restriction enzyme sites EcoR1 and Xhol. The TRV:GhTLP1-like vector was constructed using homologous recombination. Primers were designed using Primer 6 software; forward and reverse primer sequences are shown in Table 3. After constructing the vector and confirming its correctness through sequencing, it was transformed into Agrobacterium tumefaciens GV3101. Transformants were selected on LB solid medium supplemented with Kan and Rif antibiotics. The optimal growth temperature is 28°C. To prepare the Agrobacterium injection solution, the cells were cultured in LB liquid medium containing Kan and Rif antibiotics at 28°C, shaking at 200 rpm. The final OD600 of the Agrobacterium injection solution was adjusted to 1.2-1.4 and allowed to rest in the dark for 3 hours. Agrobacterium strains containing the pTRV1 and pTRV2 vectors were combined in a 1:1 ratio and injected into the undersides of the cotyledons of 7-day-old cotton seedlings. Two to three weeks after injection, during the "two leaves, one new" period, the plants were treated with bacteria and water, respectively. Since TLP1-like is a gene that is inducible by Verticillium wilt, the silencing efficiency of GhTLP1-like was assessed 14 days after inoculation with bacteria and water. Phenotypic observations and disease assessments were conducted on cotton seedlings 15 and 20 days after inoculation with V991. Furthermore, fungal recovery experiments, fungal biomass determination, and stem dissection experiments were performed to further characterize the functional resistance / susceptibility of GhTLP1-like to Verticillium wilt. The fungal resuscitation experiment is to disinfect the surface of the cotton stems by washing with 75% alcohol for 5 minutes, and then rinsing with sterile water 3 times. Repeat the disinfection step 3 times. Then cut into small sections and place them on PDA culture medium, and culture them upright in a constant temperature box at 25°C for 3 days. The fungal biomass determination is to use qPCR to measure the copy number of fungal specific genes (ITS, 18S rRNA) to estimate the biomass. The qPCR template is the DNA of the cotton stems inoculated 14 days ago. The sequences of the fungal specific gene primers and the cotton internal reference gene histone 3 (AF024716) primers are shown in Table 3. The cross-section of the stem was taken using a stereo microscope (Olympus MVX10). The experimental results are shown in Table 3. Figure 5 shown.

[0099] Table 3: Primers used for amplification

[0100]

[0101] Taken together, these results indicate that a TLP1-like gene, a TLP1-like protein, was identified using comparative transcriptomics methods and was significantly differentially expressed between the disease-resistant accession Hai7124 and the susceptible accession TM-1. Gene expression pattern analysis revealed that TLP1-like was only lowly expressed in silks and not in other organs. However, it was significantly upregulated after Verticillium wilt induction, with the upregulation rate in Hai7124 lower than that in TM-1. This confirms that this gene is a Verticillium wilt-inducible gene. Genomic structural analysis revealed a 2623-bp insertion in the promoter region of Hai7124, upstream of the ATG (-899 bp). Dual-luc assays revealed that SV inhibited the upregulation of TLP1-like expression after Verticillium wilt induction. The differential sequences in the TLP1-like promoter region between TM-1 and Hai7124 were designated haplotype 1 and haplotype 2, respectively. Haplotype 2 exhibited a significant advantage over haplotype 1 in enhancing Verticillium wilt resistance. Silencing GhTLP1-like in upland cotton resulted in significantly stronger disease resistance than control plants. Low expression of GhTLP1-like has important applications and potential in improving cotton disease resistance and breeding new disease-resistant cotton varieties.

[0102] SEQ ID NO. 1 (CDS sequence of GhTLP1-like from upland cotton (GH_A09G1614))

[0103] ATGATGAAGCTTAATTTGCTCTTTGGTCTTACATTGGCCATCCTCATCTCAGGGGCTCAAATGGCGACCTTCACCGTAAAAAACAACTGTCAGTACCCTATCTGGCCGGCAACCCTTGTCGGTGATCCTAATTTGCCACAATTGCCCAAGACTGGGTTCCAGTTAGACCCTCAAGCACAAGATTCCCTTAACGTTCCTAGCCCATGGAAAGGCAGGCTCTGGGCTCGAACACAATGCTCAACCTCAGGCGGAAGGTTCACTTGTGCCACTGCTGACTGTGGGTCCGACCAGATAGCTTGCAATGGAAAAGGTGGAGCCCCTCCGGCAACCCTGGCCGAGTTCACTATTGCAGCTAACGGAGGACAAGATTACTATGATGTCAGCCTTGTTGATGGTTTTAACTTGCCGCTTTCGATAGCCCCGCAGCGCGGCTCAGGTCCAAACTGCACCAGCACTAGCTGTGCGGCGAATGTGAACTCAGCTTGCCCACCAAACTTTGTTGTCAAAGGATCCGATGGGAATACCATAGGTTGCAAAAGCGCATGTGCAGCTTTGAATGAGCCTCAGTATTGTTGCACAGGTCAATATGGTTCACCCGAGACTTGTAAACCCACGGATTACTCAAAGAAATTCAAGGATCAGTGTCCTGAAGCTTATAGTTATGCTTATGATGATCCAACTAGCACTTTCTCCTGCACTGGTGGGCCTAGTTACCTTATCACTTTCTGTCCATGA

[0104] SEQ ID NO.2 (Amino acid sequence of the protein GhTLP1-like from Gossypium hirsutum (GH_A09G1614))

[0105] MMKLNLLFGLTLAILISGAQMATFTVKNNCQYPIWPATLVGDPNLPQLPKTGFQLDPQAQDSLNVPSPWKGRLWARTQCSTSGGRFTCATADCGSDQIACNGKGGAPPATLAEFTIAANGGQDYYDVSLVDGFNLPLSIAPQRGSGPNCTSTSCAANVNSACPPNFVVKGSDGNTIGCKSACAALNEPQYCCTGQYGSPETCKPTDYSKKFKDQCPEAYSYAYDDPTSTFSCTGGPSYLITFCP*

[0106] SEQ ID NO.3 (CDS sequence of GbTLP1-like in Gossypium barbadense (GB_A09G1742))

[0107] ATGATGAAGCTTAATTTGCTCTTTGGTCTTACATTGGCCATCCTCATCTCAGGGGCTCAAATGGCGACCTTCACCGTAAAAAACAACTGTCAGTACCCTATCTGGCCGGCAACCCTTGTCGGTGATCCTAATTTGCCACAATTGCCCAAGACTGGGTTCCAGTTAGACCCTCAAGCACAAGATTCCCTTAACGTTCCTAGCCCATGGAAAGGCAGGCTCTGGGCTCGAACACAATGCTCAACCTCAGGCGGAAGGTTCACTTGTGCCACTGCTGACTGTGGGTCCGACCAGATAGCTTGCAATGGAAAAGGTGGAGCCCCTCCGGCAACCCTGGCCGAGTTCACTATTGCAGCTAACGGAGGACAAGATTACTATGATGTCAGCCTTGTTGATGGTTTTAACTTGCCGCTTTCGATAGCCCCGCTTTCGATAGCCCCGCAGCGCGGCTCAGGTCCAAACTGCACCAGCACTAGCTGTGCGGCGAATGTGAACTCAGCTTGCCCACCAAACTTTGTTGTCAAAGGATCCGATGGGAATACCATAGGTTGCAAAAGCGCATGTGCAGCTTTGAATGAGCCTCAGTATTGTTGCACAGGTCAATATGGTTCACCCGAGACTTGTAAACCCACGGATTACTCAAAGAAATTCAAGGATCAGTGTCCTGAAGCTTATAGTTATGCTTATGATGATCCAACTAGCACTTTCTCCTGCACTGGTGGGCCTAGTTACCTTATCACTTTCTGTCCATGA

[0108] SEQ ID NO.4 (Amino acid sequence of the protein of Gossypium barbadense GbTLP1-like (GB_A09G1742))

[0109] MMKLNLLFGLTLAILISGAQMATFTVKNNCQYPIWPATLVGDPNLPQLPKTGFQLDPQAQDSLNVPSPWKGRLWARTQCSTSGGRFTCATADCGSDQIACNGKGGAPPATLAEFTIAANGGQDYYDVSLVDGFNLPLSIAPLSIAPQRGSGPNCTSTSCAANVNSACPPNFVVKGSDGNTIGCKSACAALNEPQYCCTGQYGSPETCKPTDYSKKFKDQCPEAYSYAYDDPTSTFSCTGGPSYLITFCP*

[0110] SEQ ID NO.5 (Promoter sequence of GhTLP1-like from Gossypium hirsutum (GH_A09G1614))

[0111] (-1137bp)

[0112] ATTTTGTCACTAAGGTGCAAATTGTGAAAACAAATTGCAGTCTTTTATTCCAATAGCAACAGGAGCACATTAAAGTCAAAACACATAGACATTAAATTCCTAGTTGTTAAAGTAAAAGTTCAGAGTGGTTATGTACTTATAAAGCATATTAGGACAAACTCCATGATTGCGGATCTGCTTACTAATGAACTACACCTAAGGTTTTATAAGAGCACATTGCTCATATGGGTGTAATGTCATTTAAAGGATATTTGGTTTTAGTGGGAGTTTGTATTTTTAAATGCTTTTATGTTATAGATACATTTTCAGTTATTTCAGTTTAAAAATAATAAGTTTATTTTCTGCAGAAATAAAGTTTATTTTGTTTATCCACACTCTGATTTTGGTAATGTTTGATCTCACTAAGGAGGACCAGTTGGAAATAGACACGTTTAGATCATATTGCATGTAATTTCCATGCTACACATCCATACTTGATC(-899bp)

[0113] (no SV)(-898bp)

[0114] TATGTCATTTGGTTGTGTTAATATATGTGACCATGGATGGATTTAATTACGATATATGTAACGAAAGTCAACTTGGTTCTATGTTAACATAATTAATGGACGAGATTGTTCAGAATACCTTTTGGATATGATAGTAAAATTTTGAGCTCATAAGGTTATATAATGGCATGAATTATAAAGTAATTAGTATATATATGAGGTCCAAGTGGGAGATTGTTGGAAAATTTGGACATCACATATATAATAAGGATAATTACATGTTATTTTTTACTATCACGATAGGTTAGCCCAAATTAAAAGTGATCTAATTTGGTTAGAATTTCTTGTGTGCTAATTTGGAAGATCAAATCCCCAAGTCCCAGAAAGTTTCAAGGAATCCATGAATTCAGGTACGCTTCCGCATCTAGTTTTGTTCTTGATTTATTATTAATGATTTGACATGACAGATCCTAGTTTATTAAATTATATTGTAGATTTATTTTATAAATTCTAACATAAGGAGGAAAAAGTATTGCAGTAACTCCCAATCCAAATAACCTTAGGATACAGTGCTTGTTTCCATTGGAATATAACACCAAAAATAGATACACAAATCAAACACCTGTGCTGTCCGCACACTTTAAAAAACAAAAGGTTTACGTAATTTGATCAAATATATACTGTTGCAAGTCAAGGAAATGTTTGACGGTTAAGTCAATCAATCTGAATAATTTGTCTTTTTTGTTTATTTCATGCTGAAAAGTCTGGTGATTAATGAAATAATTTGGTTTTGCGAGGACCTTTACTGCAAAACATTAAAAACGTGAATGCTATATATACCACCTTGCTAGGTACCACAACTTCACCATTCATTCCAATTTCCAAAGCAATTTAATCAGTCGATTGTTTAAGCAAAGTA ATG

[0115] SEQ ID NO.6 (Promoter sequence of GbTLP1L haplotype 2 in Gossypium barbadense (GB_A09G1742))

[0116] (-4000bp)

[0117] ATTTTGTCACTAAGGTGCAAATTGTGAAAACAAATTGCAGTCTTTTATTCCAATAGCAACAGGAGCACATTAAAGTCAAAACACATAGACATTAAATTCCTAGTTGTTAAAGTAAAAGTTCAGAGTGGTTATGTACTTATAAAGCATATTAGGACAAACTCCATGATTGCGGATCTGCTTACTAATGAACTACACCTAAGGTTTTATAAGAGCACATTGCTCATATGGGTGTAATGTCATTTAAAGGATATTTGGTTTTAGTGGGAGTTTGTATTTTTAAATGCTTTTATGTTATAGATACATTTTCAGTTATTTCAGTTTAAAAATAATAAGTTTATTTTCTGCAGAAATAAAGTTTATTTTGTTTATCCACACTCTGATTTTGGTAATGTTTGATCTCACTAAGGAGGACCAGTTGGAAATAGACACGTTTAGATCATATTGCATGTAATTTCCATGCTACACATCCATACTTGATC(-3522bp)

[0118] SV(-3521bp)

[0119]

[0120] (-898bp)

[0121] TATGTCATTTGGTTGTGTTAATATATGTGACCATGGATGGATTTAATTACGATATATGTAACGAAAGTCAACTTGGTTCTATGTTAACATAATTAATGGACGAGATTGTTCAGAATACCTTTTGGATATGATAGTAAAATTTTGAGCTCATAAGGTTATATAATGGCATGAATTATAAAGTAATTAGTATATATATGAGGTCCAAGTGGGAGATTGTTGGAAAATTTGGACATCACATATATAATAAGGATAATTACATGTTATTTTTTACTATCACGATAGGTTAGCCCAAATTAAAAGTGATCTAATTTGGTTAGAATTTCTTGTGTGCTAATTTGGAAGATCAAATTCCCCAAGTCCCAGAAAGTTTCAAGGAATCCATAAATTCAGGTACGCTTCCGCATCTAGTTTTGTTCTTGATTTATTATTAATGATTTGACATGACAGATC CTAGTTTATTAAGTTATATTGTAGATTTATTTTATAAATTCTAACATAAGGAGGAAAAAGTATTGCAGTAACTCCCAATCCAAATAACCTTAGGATACAGTGCTTGTTTCCATTGGAATATAACACCAAAAATAGATACACAAATCAAACACCTGTGCTGTCCGCACACTTTAAAAAACAAAAGGTTTACGTAATTTGATCAAATATATACTGTTGCAAGTCAAGGAAATGTTTGACGGTTAAGTCAATCAATCTGAATAATTTGTCTTTTTTGTTTATTTCATGCTGAAAAGTCTGGTGATTAATGAAATAATTTGGTTTTGCGAGGACCTTTACTGCAAAACATTAAAAACGTGAATGCTATATATACCACCTTGCTAGGTACCACAACTTCACCATTCATTCCAATTTCCAAAGCAATTTAATCAGTCGATTGTTTAAGCAAAGTA ATG

[0122] SEQ ID NO.7(GbTLP1-like_promoter_SV(2623bp))

[0123]

[0124] SEQ ID NO.8 (Forward primer sequence for TLP1-like promoter InDel site molecular marker for genotyping)

[0125] CTCCATGATTGCGGATCTGCTT

[0126] SEQ ID NO.9 (Reverse primer sequence for TLP1-like promoter InDel site molecular marker for genotyping)

[0127] ATGGGAGCAGGTGGAGGAGA

[0128] SEQ ID NO. 10 (PCR product (501 bp) sequence)

[0129] CTCCATGATTGCGGATCTGCTTACTAATGAACTACACCTAAGGTTTTATAAGAGCACATTGCTCATATGGGTGTAATGTCATTTAAAGGATATTTGGTTTTAGTGGGAGTTTGTATTTTAAATGCTTTTATGTTATAGATACATTTTCAGTTATTTCAGTTTAAAAATAATAAGTTTATTTTCTGCAGAAATAAAGTTTATTTTGTTTATCCACACTCTGATTTTGGTAATGTTTGATCTCACTAAGGA GGACCAGTTGGAAATAGACACGTTTAGATCATATTGCATGTAATTTCCATGCTACACATCCATACTTGATCTATGTTGTTGCAGGCCCATTTGCCCGGGCCCAAGAAAGAAATAAAAGGCCCAGCACCCTGGCCCAACAGCCAAGCCCACATCAGACTAGGGTTTCAGAAACTGAAACCCTAGTATCCCACTTGCCGCTGCTCTCTGTCAGCCGCACGTCCCATCGCCATCTCTCCTCCACCTGCTCCCAT

[0130] SEQ ID NO.11 (GhTLP1-like target fragment (249 bp) sequence)

[0131] GCTGTGCGGCGAATGTGAACTCAGCTTGCCCACCAAACTTTGTTGTCAAAGGATCCGATGGGAATACCATAGGTTGCAAAAGCGCATGTGCAGCTTTGAATGAGCCTCAGTATTGTTGCACAGGTCAATATGGTTCACCCGAGACTTGTAAACCCACGGATTACTCAAAGAAATTCAAGGATCAGTGTCCTGAAGCTTATAGTTATGCTTATGATGATCCAACTAGCACTTTCTCCTGCACTGGTGGGC。

Claims

1. Use of an InDel molecular marker associated with cotton Verticillium wilt resistance or a substance for detecting the InDel molecular marker in at least one of the following (a1) to (a5): (a1) screening or assisting in screening cotton germplasm resistant to Verticillium wilt, or preparing a product for screening or assisting in screening cotton germplasm resistant to Verticillium wilt; (a2) identifying or assisting in identifying the Verticillium wilt resistance trait of cotton, or preparing a product for identifying or assisting in identifying the Verticillium wilt resistance trait of cotton; (a3) identifying the promoter haplotype of a cotton TLP1 protein gene TLP1-like, or preparing a product for identifying the promoter haplotype of a cotton TLP1 protein gene TLP1-like; (a4) Improving the Verticillium wilt resistance of cotton, or preparing products for improving the Verticillium wilt resistance of cotton; (a5) Breeding cotton with high resistance to Verticillium wilt, or preparing products for breeding cotton with high resistance to Verticillium wilt; The InDel molecular marker is based on the insertion / deletion of a 2623 bp nucleotide fragment as shown in SEQ ID NO.7 at the -899 bp position of the promoter sequence of the cotton TLP1 protein gene GbTLP1-like as shown in SEQ ID NO.

6.

2. The use according to claim 1, characterized in that The promoter of the cotton TLP1 protein gene TLP1-like has two haplotypes. The promoter sequence of haplotype 1 is shown in SEQ ID NO.5, and the promoter sequence of haplotype 2 is shown in SEQ ID NO.

6. Compared with haplotype 1, haplotype 2 has a 2623bp nucleotide fragment insertion of a nucleotide sequence as shown in SEQ ID NO.7 at -899bp upstream of the ATG promoter region, resulting in reduced expression of the TLP1-like gene; haplotype 2 plants have better disease resistance than haplotype 1.

3. A product, characterized in that The product contains the substance for detecting InDel molecular markers as described in claim 1, and is at least one of the following (b1) to (b5): (b1) Screening or assisting in screening products of different cotton varieties resistant to Verticillium wilt; (b2) Products that identify or assist in identifying cotton's resistance to Verticillium wilt; (b3) Identification of products of cotton TLP1-like promoter haplotypes; (b4) Products for improving cotton's resistance to Verticillium wilt; (b5) Products for breeding cotton with high Verticillium wilt resistance.

4. A method, characterized in that The method is at least one of the following methods (c1) to (c5): (c1) a method for screening or assisting in screening cotton germplasm with high resistance to Verticillium wilt; (c2) Methods for identifying or assisting in identifying Verticillium wilt resistance traits in cotton; (c3) Methods for identifying TLP1-like promoter haplotypes in cotton TLP1 proteins; (c4) Methods for improving cotton's resistance to Verticillium wilt; (c5) Methods for breeding cotton with high Verticillium wilt resistance; The method comprises the following steps: using a substance for detecting InDel molecular markers to detect whether a promoter of a TLP1-like protein gene TLP1-like of a cotton germplasm material to be tested contains the InDel molecular marker as claimed in claim 1, thereby identifying the haplotype of the promoter of the cotton TLP1-like protein gene TLP1-like; two haplotypes exist in the promoter region of the cotton TLP1 protein gene TLP1-like, and haplotype 2 has a 2623 bp nucleotide fragment insertion of a nucleotide sequence such as that shown in SEQ ID NO.7 at -899 bp in the promoter region upstream of the ATG compared to haplotype 1; haplotype 2 has better disease resistance than haplotype 1; and the cotton germplasm material to be tested carrying haplotype 2 is selected as a cotton germplasm with Verticillium wilt resistance, used for cotton breeding, and improved Verticillium wilt resistance of cotton.

5. The use according to claim 1 or 2, the product according to claim 3, the method according to claim 4, characterized in that: The substance for detecting the InDel molecule marker is the following (d1) or (d2) or (d3) or (d4): (d1) in vitro nucleic acid amplification primers as shown in SEQ ID NO.8 and SEQ ID NO.9 for specifically amplifying the InDel molecular marker; (d2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in (d1); (d3) A kit containing the in vitro nucleic acid amplification primers described in (d1) or the in vitro nucleic acid amplification reagents described in (d2); (d4) A detection instrument containing the in vitro nucleic acid amplification primers described in (d1), the in vitro nucleic acid amplification reagents described in (d2) or the kit described in (d3).

6. The use according to claim 1 or 2, the product according to claim 3, the method according to claim 4, characterized in that: The cotton Verticillium wilt resistance trait is at least one of a disease phenotype and a disease incidence rate.

7. Use of a substance that inhibits the expression of a TLP1-like protein gene or a substance that reduces the activity or content of a protein encoded by the TLP1-like protein gene in the following (e1) or (e2): (e1) improving cotton resistance to Verticillium wilt or cultivating new cotton germplasm with improved resistance to Verticillium wilt; (e2) promoting disease resistance in target plants; The TLP1-like protein gene TLP1-like is a DNA molecule of the following (f1) or (f2) or (f3) or (f4): (f1) a DNA molecule whose coding region is shown in SEQ ID NO. 1 or SEQ ID NO. 3; (f2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3; (f3) A DNA molecule that hybridizes with the DNA molecule defined by (f1) or (f2) under stringent conditions and encodes a TLP1-like protein; (f4) A DNA molecule derived from upland cotton or sea island cotton and has 98% homology with the DNA molecule defined by (f1) or (f2) and encodes a TLP1-like protein.

8. The use according to claim 7, characterized in that The protein encoded by the TLP1-like protein gene TLP1-like is (g1) or (g2) or (g3): (g1) a protein with an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 4; (g2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of (g1); (g3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO.

4.

9. The substance that inhibits the expression of the TLP1-like protein gene TLP1-like or the substance that reduces the activity or content of the protein encoded by the TLP1-like protein gene TLP1-like is a biomaterial, and the biomaterial is any one of the following h1) to h6): (h1) a nucleic acid molecule that silences, inhibits, interferes with, or gene-edits the expression of the TLP1-like protein gene; (h2) an expression cassette containing the nucleic acid molecule of (h1); (h3) a recombinant vector containing the nucleic acid molecule described in (h1), or a recombinant vector containing the expression cassette described in (h2); (h4) a recombinant microorganism containing the nucleic acid molecule described in (h1), or a recombinant microorganism containing the expression cassette described in (h2), or a recombinant microorganism containing the recombinant vector described in (h3); (h5) A gene-edited plant cell line containing the nucleic acid molecule described in (h1), or a gene-edited plant cell line containing the expression cassette described in (h2), or a gene-edited plant cell line containing the recombinant vector described in (h3); (h6) gene-edited plant tissue containing the nucleic acid molecule described in (h1), or transgenic plant tissue containing the expression cassette described in (h2), or transgenic plant tissue containing the recombinant vector described in (h3); (h7) A gene-edited plant organ containing the nucleic acid molecule described in (h1), or a gene-edited plant organ containing the expression cassette described in (h2), or a gene-edited plant organ containing the recombinant vector described in (h3).

10. A method for improving disease resistance of cotton, characterized by: Taking the TLP1-like protein gene TLP1-like as the target gene, through genetic engineering methods, the expression of the TLP1-like protein gene TLP1-like shown in SEQ ID NO.1 or SEQ ID NO.3 is silenced, inhibited, interfered or gene-edited in cotton, thereby inhibiting its expression and creating a new cotton germplasm with improved resistance to Verticillium wilt.

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