Gene ghTLP1b for improving resistance to cotton verticillium wilt and application thereof

Abstract

The application relates to application of a cotton class sweet protein gene GhTLP1b in improving resistance of cotton to verticillium wilt. The application clones a GhTLP1b gene from a disease-resistant material ZZM2, uses an agrobacterium-mediated transformation technology to obtain transgenic cotton strains of gene overexpression and gene silencing of the gene, and proves that the GhTLP1b overexpression strain enhances the resistance of cotton to the verticillium wilt, and is characterized by delayed disease occurrence and reduced disease index; the GhTLP1b silencing strain makes the cotton susceptible, and is characterized by weakened active oxygen burst and reduced lignin and callose accumulation. The application identifies and verifies an important resistance gene of cotton to the verticillium wilt, and provides an effective gene resource for genetic breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a gene GhTLP1b that enhances cotton resistance to Verticillium wilt and its application in improving cotton resistance to Verticillium wilt. Background Technology

[0002] Cotton, as an important economic crop, is frequently affected by various diseases in terms of yield and fiber quality. Among them, Verticillium dahliae, a soil-borne vascular disease, is a serious threat. After infecting the roots, this pathogen blocks the plant's vascular bundles, leading to yellowing, wilting, and leaf drop, and in severe cases, plant death. It typically causes a 10%-30% reduction in cotton yield annually. Currently, there are no highly effective and stable control methods for this disease, and the pathogen's long survival time in the soil and wide host range limit the effectiveness of measures such as crop rotation. Therefore, breeding disease-resistant varieties has become a long-term and effective strategy for controlling this disease.

[0003] Plants have developed complex immune systems through long-term evolution, in which pathogenesis-related proteins (PRs) play a crucial role in disease resistance. Thaumatin-like proteins (TLPs), belonging to the PR-5 family, are widely distributed in plants and have been reported to participate in responses to fungi, bacteria, and abiotic stresses. TLPs typically possess a β-sheet structure and can inhibit pathogen growth by disrupting the pathogen's cell membrane or interacting directly with the pathogen. Several TLP members have been identified in cotton, but their specific functions and mechanisms in Verticillium wilt resistance remain unclear, especially lacking key genes with definite disease resistance effects that can be used for molecular breeding.

[0004] Current cotton Verticillium wilt resistance breeding mainly relies on traditional hybridization and phenotypic selection, which suffers from problems such as long cycles, low efficiency, and complex genetic bases of resistance. Although some resistance-related genes have been reported, key genes with functional verification and significant resistance effects are still few, limiting the development of molecular breeding for resistance. Therefore, identifying cotton endogenous genes with clear resistance functions, elucidating their mechanisms, and developing their applications in resistance breeding are of great significance for improving cotton Verticillium wilt resistance and ensuring production safety. Summary of the Invention

[0005] The purpose of this invention is to provide the cotton Verticillium wilt-resistant material ZZM2 and the application of its carried resistance gene GhTLP1b in enhancing cotton resistance to Verticillium wilt, thus providing effective germplasm resources for breeding upland cotton varieties resistant to Verticillium wilt. This invention identified and cloned a gene, GhTLP1b, that enhances cotton resistance to Verticillium wilt. It was confirmed that GhTLP1b is upregulated after induction by Verticillium dahliae, and overexpression of this gene in cotton enhances cotton's resistance to Verticillium wilt, while silencing the gene increases plant susceptibility.

[0006] To achieve the above objectives, the present invention provides a gene GhTLP1b that regulates resistance to Verticillium wilt in cotton, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides the protein encoded by the gene, the coding region sequence (CDS) of which is shown in SEQ ID NO.2 and the corresponding amino acid sequence is shown in SEQ ID NO.3.

[0008] The present invention also provides the application of the GhTLP1b gene in improving cotton resistance to Verticillium wilt, and the nucleotide sequence of the GhTLP1b gene is shown in SEQ ID NO.1.

[0009] Preferably, the application is to enhance the resistance of cotton to Verticillium wilt by increasing the expression level of the GhTLP1b gene or the protein activity in cotton.

[0010] Preferably, the enhanced resistance of cotton to Verticillium wilt is manifested by delayed onset and reduced disease index.

[0011] Preferably, the application also includes weakening cotton's resistance to Verticillium wilt by silencing or reducing the expression level or protein activity of the GhTLP1b gene in cotton.

[0012] Preferably, the weakening of cotton's resistance to Verticillium wilt is manifested in the following ways: reduced reactive oxygen species emission, reduced lignin accumulation, and reduced callose accumulation.

[0013] The present invention also provides a biomaterial for improving the resistance of cotton to Verticillium wilt, wherein the biomaterial is an overexpression line of the GhTLP1b gene obtained in cotton.

[0014] This invention also provides the application of the GhTLP1b gene in genetic breeding for resistance to Verticillium wilt in cotton.

[0015] The present invention also provides a method for enhancing the resistance of cotton to Verticillium wilt, including the step of increasing the expression level or protein activity of the GhTLP1b gene in the recipient cotton.

[0016] Preferably, the enhancement of GhTLP1b gene expression or protein activity is achieved by introducing an expression vector containing the coding region sequence shown in SEQ ID NO. 2 into recipient cotton and overexpressing it.

[0017] Specifically, the method involves enhancing resistance by increasing the expression level of the GhTLP1b gene or its protein activity in recipient cotton. The coding sequence of the GhTLP1b gene is amplified, ligated into the pCAMBIA2300 vector, and transformed into wild-type cotton via Agrobacterium-mediated transformation, resulting in transgenic overexpression cotton material. When the overexpression cotton material and wild-type cotton are inoculated with Verticillium dahliae, the transgenic overexpression cotton material exhibits improved resistance to Verticillium wilt compared to wild-type cotton. This is mainly reflected in a1)-a3): a1) the susceptibility rate of the transgenic overexpression cotton material is lower than that of wild-type cotton; a2) the disease index of the transgenic overexpression cotton material is lower than that of wild-type cotton; a3) the onset of disease in the transgenic overexpression cotton material is later than that in wild-type cotton.

[0018] Compared with traditional technologies, this invention offers the following advantages: This invention is the first to identify the application of GhTLP1b in enhancing cotton resistance to Verticillium wilt. It was found that compared to the JM11 cotton variety, GhTLP1b is highly expressed in the disease-resistant variety ZZM2, enhancing its resistance to Verticillium wilt. Simultaneously, overexpression of this gene in wild-type cotton enhances resistance to Verticillium wilt; while silencing this gene leads to reduced accumulation of reactive oxygen species, lignin, and callose, resulting in increased susceptibility to Verticillium wilt. This gene provides important genetic material and a theoretical basis for elucidating the molecular mechanism of cotton resistance to Verticillium wilt, and has significant research value. Furthermore, the materials and genes provided by this invention can significantly enhance cotton resistance to Verticillium wilt, providing effective genetic resources for cotton disease-resistant breeding and possessing significant practical application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show the embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The figure shows the results of the resistance identification analysis of ZZM2 and JM11 against Verticillium dahliae V991.

[0021] In this table, A represents the phenotype of JM11 and ZZM2 after 21 days of inoculation with Verticillium dahliae; B represents the disease severity of ZZM2 and JM11 after 21 days of treatment; C represents the disease index of ZZM2 and JM11 after 21 days of treatment; and D represents the stem dissection results of ZZM2 and JM11 after 21 days of treatment.

[0022] Figure 2 Figure showing the results of GhTLP1b response to pathogen induction;

[0023] Where A represents the expression level of GhTLP1b in roots and leaves of ZZM2 and JM11; B represents the expression level of GhTLP1b in different tissues (roots, stems and leaves) of ZZM2; and C represents the induced expression of GhTLP1b in ZZM2 after treatment with Verticillium dahliae.

[0024] Figure 3 The figure shows the phenotypic and resistance analysis results after GhTLP1b gene silencing;

[0025] Where A represents the disease incidence 21 days after silencing GhTLP1b in ZZM2; B represents the silencing efficiency of GhTLP1b in roots and leaves; and C represents the disease severity 21 days after inoculation treatment following GhTLP1b silencing.

[0026] Figure 4 The figure shows the phenotypic and resistance analysis results after GhTLP1b gene silencing;

[0027] Among them, A represents the detection of ROS deposition by DAB staining after silencing GhTLP1b; B represents the detection of lignin deposition by phloroglucinol staining in paraffin sections after silencing GhTLP1b; C represents the detection of callus accumulation by aniline blue staining after silencing GhTLP1b; D represents the detection of fungal recovery 21 days after silencing the GhTLP1b gene; and E represents the detection of callus content accumulation after silencing GhTLP1b.

[0028] Figure 5 shows the results of inoculation analysis of GhTLP1b gene overexpression strains.

[0029] Among them, A is the expression level detection of the GhTLP1b overexpression line created using cotton J668 as background material; B is the phenotype and disease index of the overexpression line after inoculation with bacteria; C is the disease index of the overexpression transgenic material after inoculation with Verticillium dahliae for 21 days. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments. Obviously, the described embodiments are only a part of the implementation methods of this application, and not all of the implementation methods. It should be noted that all other embodiments obtained by those skilled in the art based on these embodiments of the present invention are within the protection scope of the present invention.

[0031] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] SEQ ID NO.1

[0034]

[0035] SEQID NO.2

[0036] >GH_A06G0026

[0037] ATGGCCCAATATACATTTGTTGCTGCTATCTGTGTTCTTATTCTTTCACATTCGTTCATATCAGTAGTGAAAGCCACAACCTTTACAATAGTAAACGAATGCAATTATGTTGTATGGCCAGGGATTCTATCAAACGCAGGTGTTCCAACGCTTCCCACCACTGGTTTCGCTCTCCAAAGCGGCGAAACCAAAGCCATTACTGCACCGGCATCATGGGGTGGTCGGTTCTGGGGTAGAACCCATTGCTCCGAGGATTCCACCGGGAAATTTTCCTGCCTTACCGGTGATTGTGGCTCCGGGAAGGTGGAATGTTCTGGAAACGGGGCTGCTCCGCCCGCGAGTTTAGCTGAGTTCACATTGGACGGTGCTGGCGGCCTTGATTTTTTCGACGTGAGTTTGGTCGACGGTTACAACATCCCTATGTTGGTTGTTCCACAGGGTGGCACGGGACAAAACTGTACCAACACCGGCTGCGTGGTTGACCTTAATGGCTCTTGCCCTTCCGAACTAAAGGTGATGAGCACCGACGGCACAGATGGCGTCGCTTGCAAGAGTGCTTGCGAAGCCTTCGGCGACCCTCAGTACTGTTGCAGCGGCGCGTATGGTACACCCGATACTTGCAAACCGTCTTCGTACTCGGAGGTGTTTAAGACAGCTTGCCCACGCGCATACAGCTATGCCTATGATGATAAAACCAGTACCTTCACATGCGCCAACGCTGATTATACAATTACTTTCTGTCCTTCGCCTAACACTAGCCAAAAATCATCGCAAGAGCAGCAACAAAACACGGAAACGACAACACCACCTCTTATCAACACCGCGATGGTGTATGAAGGTGCATTGGACCAAAATGTAGCATCACCGTCCTCCTGCACCCACGTGTTCGTAAACGTGGGCATCATTATGGCAATTTGGTGGTCGTGGAAACTCTTCTAA

[0038] SEQID NO.3

[0039] >GH_A06G0026

[0040] MAQYTFVAAICVLILSHSFISVVKATTFTIVNECNYVVWPGILSNAGVPPTLPTTGFALQSGETKAITAPASWGGRFWGRTHCSEDSTGKFSCLTGDCGSGKVECSGNGAAPPASLAEFTLDGAGGLDFFDVSLVDGYNIPMLVVPQGGTGQNCTNT GCVVDLNGSCPSELKVMSTDGTDGVACKSACEAFGDPQYCCSGAYGTPDTCKPSSYSEVFKTACPRAYSYAYDDKTSTFTCANADYTITFCPSPNTSQKSSQEQQQNTETTTPPLINTAMVYEGALDQNVASPSSCTHVFVNVGIIMAIWWSWKLF*

[0041] Example 1: Identification of resistance of ZZM2 and JM11 to Verticillium dahliae V991

[0042] 1. Source and Cultivation Conditions of Plant Materials: The plant materials used in the experiment were Zhongzhimian No. 2 (from the National Southern Breeding Research Institute Laboratory of the Chinese Academy of Agricultural Sciences in Sanya) (hereinafter referred to as ZZM2) and Jimian No. 11 (from the National Southern Breeding Research Institute Laboratory of the Chinese Academy of Agricultural Sciences in Sanya) (hereinafter referred to as JM11). 200 seeds from each of these two materials were selected, pre-germinated, and then sown in flowerpots filled with nutrient soil (nutrient soil: vermiculite = 2:1). The pots were covered with plastic wrap to maintain moisture and temperature, and cultured in a greenhouse (28℃, light / dark = 16 h:8 h). 2. Activation and Cultivation of Verticillium wilt: 100 μL of V991 Verticillium dahliae spore suspension stored at -80℃ was inoculated into liquid Czapek medium and cultured with shaking at 25℃ and 180 rpm for 3 to 5 days. The culture product was filtered through double-layered gauze to obtain a spore suspension, which was then thoroughly mixed and diluted 10 times. The five-point sampling method was used to determine the number of spores under an optical microscope using a hemocytometer, and the concentration of the spore suspension was calculated and adjusted to 1×10⁻⁶ using distilled water. 7 3. Verticillium wilt inoculation: Select cotton seedlings with uniform growth and inoculate them with Verticillium wilt V991 using the root soaking method. The specific method is as follows:

[0043] Mock control group: The roots of the seedlings were slightly damaged with a steel ruler, and then 10 mL of distilled water was added to the paper tray;

[0044] Inoculation treatment group: The roots of the seedlings were slightly damaged with a steel ruler, and then 10 mL of a 1×10⁻⁶ solution was added to the paper tray. 7 Prepare a spore suspension of 1 spore per mL and gently shake the paper tray during inoculation to ensure even distribution of the bacterial solution.

[0045] The two groups of seedlings were transplanted into nutrient soil (nutrient soil: vermiculite = 2:1, volume ratio) and placed in an artificial climate culture room at 25℃ with a photoperiod of 16 h light / 8 h darkness.

[0046] Nine to twelve days after inoculation, when cotton seedlings exhibited typical symptoms of Verticillium wilt, the incidence of disease in each treatment group was statistically analyzed. Based on the symptoms such as leaf dehydration, wilting, drying, yellowing, and leaf drop, the disease index was calculated. The Disease Index (DI) was calculated according to the "Technical Specification for Identification of Cotton Verticillium Wilt Resistance" (NY / T2952-2016), classifying the severity of Verticillium wilt into five disease levels: 0, 1, 2, 3, and 4. The formula for calculating the disease index is: Disease Index (DI) = 100 × ∑(Number of diseased leaves at each level × Representative value at each level) / (Total number of leaves surveyed × Highest representative value).

[0047] Table 1. Criteria for Classifying the Disease Severity of Cotton Verticillium Wilt

[0048] Disease level standard Level 0 perfectly healthy plants Level 1 A single cotyledon becomes infected Level 2 Two cotyledons or one true leaf are infected. Level 3 The cotyledons and two or more true leaves are affected. Level 4 The leaves withered and died completely.

[0049] 4. Resistance Identification and Analysis of ZZM2 and JM11 to V991: Cotton varieties ZZM2 and JM11 were infected with the deciduous strain V991 and planted under greenhouse conditions. Disease incidence was observed in both varieties 21 days after inoculation. The results are as follows: Figure 1 As shown. Significant differences existed in disease manifestations between the two varieties: JM11-inoculated plants exhibited widespread leaf yellowing, dehydration, and wilting symptoms; in contrast, ZZM2 showed significantly milder symptoms. Further quantitative analysis using the disease index was conducted, and the significance of the differences between the two groups was compared using a t-test. Results ( Figure 1 The results in C) showed that the disease index of JM11 was close to 90%, while that of ZZM2 was less than 50%, with a significant difference between the two (*p<0.05).

[0050] After completing the observation of the disease condition and the statistics of the disease index, the cotton plant stems were dissected to observe the browning of the vascular bundles. The results are as follows: Figure 1 As shown in Figure D, the vascular bundles of JM11 stems exhibit significant browning, while the vascular bundles of most stem segments in ZZM2 remain normal. Based on the above phenotypic and data analysis results, it is indicated that JM11 has significantly lower resistance to Verticillium wilt than ZZM2.

[0051] Example 2: Response of the GhTLP1b gene to Verticillium wilt induction in ZZM2 and JM11.

[0052] 1. Source and Culture Conditions of Plant Materials: The plant materials used in the experiment were Zhongzhimian No. 2 (hereinafter referred to as ZZM2) and Jimian No. 11 (hereinafter referred to as JM11). 200 seeds from each of these two materials were selected, pre-germinated, and then sown in pots filled with nutrient soil (nutrient soil: vermiculite = 2:1). The pots were covered with plastic wrap to maintain moisture and temperature, and cultured in a greenhouse (28℃, light / dark = 16 h:8 h). 2. Activation, Culture, and Inoculation Statistics of Verticillium Wilt: The specific operation method was the same as in Example 1. 3. RNA Extraction and Expression Detection: RNA was extracted from ZZM2 material at 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, and 96 h after inoculation, as well as from the roots and leaves of both ZZM2 and JM11 materials. The trend of gene expression and the dominant sites of the gene were observed. The methods for extracting total RNA from plants and synthesizing cDNA are as follows:

[0053] (1) Total RNA was extracted at different time points using the FastPure Universal Plant Total RNA Isolation Kit. The RNA extraction steps are as follows:

[0054] 1) Place the plant sample in liquid nitrogen and grind it into powder quickly. Transfer it to a centrifuge tube, add 600 μL of Buffer PSL, vortex for 30–60 s immediately, and centrifuge at 12000 rpm for 5 min after complete lysis.

[0055] 2) Take about 500 μL of supernatant and transfer it to FastPure gDNA-Filter Column III. Centrifuge at 12000 rpm for 30 seconds, discard the adsorption column, and collect the filtrate.

[0056] 3) Add 0.5 times the volume of anhydrous ethanol to the filtrate and vortex to mix.

[0057] 4) Transfer the entire mixture to FastPure RNA Column V, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0058] 5) Add 700 μL of Buffer RWA to RNA Column V, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0059] 6) Add 500 μL Buffer RWB, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0060] 7) Repeat step 6 once.

[0061] 8) Place RNA Column V back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to completely remove residual washing solution.

[0062] 9) Transfer RNA Column V to a new 1.5 mL centrifuge tube, add 30–100 μL of RNAase-free ddH2O to the center of the adsorption membrane, let stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 30 s to elute RNA.

[0063] RNA concentration was determined using a micro-volume ultraviolet spectrophotometer (Thermo Scientific, Nanodrop2000). The obtained RNA can be used directly for subsequent experiments or stored at -85°C to -65°C for later use.

[0064] (2) The extracted RNA was used to synthesize cDNA using the Evo M-MLV reverse transcription kit (containing a gDNA removal reagent for qPCR). The reverse transcription steps are as follows.

[0065] Removal of genomic DNA:

[0066] 1) Prepare the reaction solution according to the table below and carry out the genomic DNA removal reaction;

[0067]

[0068] Reaction conditions: 42°C for 2 min

[0069] 4°C

[0070] Reverse transcription reaction

[0071] 2) Prepare the reaction solution according to the table below and carry out the reverse transcription reaction.

[0072]

[0073] Reaction conditions: 37°C for 15 min

[0074] 85°C for 5 seconds

[0075] 4°C

[0076] The obtained cDNA was stored in a -20°C freezer.

[0077] 4. Analysis of GhTLP1b response to pathogen induction: To clarify the dominant expression sites and tissue expression patterns of GhTLP1b in cotton, total RNA was extracted from the root, stem, and leaf tissues of the parental materials ZZM2 (resistant) and JM11 (susceptible), and the expression level of this gene in different varieties and tissues was detected. Results are as follows: Figure 2 As shown in Figure A, under uninoculated conditions, the expression level of GhTLP1b in all tissues of ZZM2 was significantly higher than that in the corresponding tissue of JM11, indicating that the expression difference of this gene may be related to its resistance.

[0078] Further analysis was conducted on the expression distribution of GhTLP1b in different tissues of ZZM2. For example... Figure 2 As shown in B, the gene is most highly expressed in leaves, followed by roots and stems, indicating that GhTLP1b has a tissue-specific expression pattern in cotton.

[0079] To verify whether GhTLP1b responds to Verticillium wilt infection, RNA was extracted from the roots and leaves of ZZM2 and JM11 samples at 0h, 1h, 3h, 6h, 9h, 12h, 24h, 48h, 72h, and 96h under both inoculated and uninoculated V991 conditions, and the dynamic changes in gene expression were detected. The results are as follows: Figure 2 As shown in Figure C, GhTLP1b expression significantly increased after V991 inoculation, exhibiting a clear trend of induced expression at 9h, 24h, 48h, and 96h; while in the uninoculated control group, there was no significant temporal change in gene expression. Further comparison of expression levels between inoculated and uninoculated samples at the same time points revealed that the expression levels at each time point after inoculation were higher than those in the corresponding uninoculated control group.

[0080] In summary, the expression of GhTLP1b varies among varieties and tissues and is induced by Verticillium dahliae infection, suggesting that it may positively regulate cotton resistance to Verticillium wilt.

[0081] Example 3: Silencing GhTLP1b increases cotton susceptibility to Verticillium wilt.

[0082] 1. Construction of VIGS expression vector: Based on the nucleotide sequence information of the CDS region of the candidate gene for the TM-1 reference genome provided in the CottonFGD database (https: / / cottonfgd.net / ), primers were designed by selecting the target gene-specific region as the reference sequence through BLASTN alignment analysis. The primer design followed the following principle: the amplified fragment length was controlled between 300-500 bp.

[0083] The reference sequence is shown below:

[0084] >GhTLP1b-VIGS_(49-348)

[0085] CATTCGTTCATATCAGTAGTGAAAGCCACAACCTTTACAATAGTAAACGAATGCAATTATGTTGTATGGCCAGGGATTCTATCAAACGCAGGTGTTCCAACGCTTCCCACCACTGGTTTCGCTCTCCAAAGCGGCGAAACCAAAGCCATT ACTGCACCGGCATCATGGGGGTGGTCGGTTCTGGGGTAGAACCCATTGCTCCGAGGATTCCACCGGGAAATTTTCCTGCCTTACCGGTGATTGTGGCTCCGGGAAGGTGGAATGTTCTGGAAACGGGGCTGCTCCGCCCGCGAGTTTAGCT

[0086] The primer sequence is: TRV-GhTLP1b-F: TAAGGTTACCGAATTCCATTCGTTCATATCAGT

[0087] TRV-GhTLP1b-R: GCTCGGTACCGGATCCAGCTAAACTCGCGGGC.

[0088] The purified PCR product was ligated into the TRV:00 plasmid, which had been double-digested with EcoRI and BamHI, using the In-Fusion™ (Clontech) cloning method, and then transformed into DH5α competent cells. The specific ligation reaction mixture consisted of: 1.0 μL Exase II, 2.0 μL 5×Buffer, 100 ng of double-digested TRV:00 plasmid, 10 ng of purified PCR product, and ddH2O to a final volume of 10 μL. The prepared In-Fusion mixture was incubated at 50°C for 12 min. After the reaction, the product was added to the thawed DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then rapidly cooled on ice for 2 min. Subsequently, 200 μL of LB liquid medium was added, and the cells were incubated at 37°C with shaking at 180 rpm for 40 min. 100 μL of the bacterial culture was then evenly spread onto LB agar plates containing 0.1% kanamycin and inverted at 37°C for approximately 12 h. Single colonies were picked and PCR amplified using TRV-F and TRV-R primers for positive detection. Positive clones were sent to a biotechnology company for sequencing verification. After amplifying the correctly sequenced positive single colonies, the bacterial culture was diluted with 25% glycerol and stored at -80℃ for later use. Subsequently, the empty vector (TRV::00), helper vector (TRV-192), positive control vector (TRV::GhPDS), and recombinant vector (TRV::GhTLP1b) were transformed into Agrobacterium GV3101 competent cells, respectively.

[0089] 2. Activation, culture and inoculation statistics of Verticillium wilt pathogen: The specific operation method is the same as in Example 1.

[0090] 3. Method for silencing GhTLP1b: Add 400 μL of the Agrobacterium stock solution to 7 mL of LB liquid medium and incubate overnight at 28°C and 180 rpm for 14 h until the bacterial solution turns orange-yellow. Centrifuge to collect the bacterial cells and discard the supernatant. Alternatively, incubate on LB medium with the corresponding resistance at 28°C and 180 rpm until the bacterial solution turns orange-yellow, then centrifuge and discard the supernatant. Resuspend the bacterial cells in a resuspension solution (10 mM MgCl2, 10 mM ES, 200 μM acetylsylcholine) and adjust the OD600 to 0.8–1.0. Mix the recombinant bacterial solution (or control bacterial solution) with an equal volume of TRV1 bacterial solution and let stand for 2 h. Select cotton seedlings with uniform growth and slowly inject 300 μL of the bacterial solution from the underside of the leaves using a 1 ml sterile syringe. After injection, cover the plants with black plastic film and incubate overnight in the dark. Then, transfer them to a greenhouse at 25°C with a photoperiod of 16 h light / 8 h dark for further cultivation. When the TRV::GhPDS positive control plants showed obvious albino phenotype, samples were taken and the silencing efficiency of the target gene was detected. 4. Silencing effect detection: 10-14 days after treatment, the true leaves of the positive control TRV::GhPDS plants showed obvious albino phenotype. At this time, leaves from the silencing treatment group and the negative control TRV::00 were taken and immediately flash-frozen in liquid nitrogen for subsequent RNA extraction and reverse transcription, or stored at -80℃ for later use. The relative expression level of the target gene was detected by qRT-PCR to evaluate the silencing efficiency.

[0091] Primers are as follows: GhTLP1b-qPCR-F: ATCATCGCAAGAGCAGCAACAA;

[0092] GhTLP1b-qPCR-R:AATGATGCCCACGTTTACGAAC;

[0093] UBQ7-F: AGAGGTCGAGTCTTCGGACA;

[0094] UBQ7-R:GCTTGATCTTCTTGGGCTTG.

[0095] 5. Isolation and culture of pathogens from stem segments: Approximately 21 days after inoculation, collect stems from the hypocotyl or first internode of cotton seedlings and sterilize their surfaces in a clean bench. Immerse the stems in 75% anhydrous ethanol for 2 minutes (with gentle agitation), then rinse 3-4 times with sterile distilled water. Dry the stems on sterile filter paper, then cut them into 2-3 mm segments using a sterile scalpel and arrange them in sections on PDA agar plates. Incubate upside down in a 25°C incubator for 2-4 days, observing and recording mycelial growth.

[0096] 6. Verticillium wilt biomass detection: The first internode of cotton seedlings 21 days after inoculation was collected, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. After grinding the samples in liquid nitrogen, total DNA was extracted using the FastPure Plant DNA Isolation Mini Kit (Vazyme, DC401-01). The DNA template concentration was diluted to 10 ng / μL, and GhUBQ7 (Ghir_A11G011460) was used as an internal control gene. Quantitative detection of fungal biomass within the plant was performed using Verticillium wilt-specific primers.

[0097] qVdP1-F:CATCAGTCTCTCTGTTTATACCAACG-3qVdP2-R:CGATGCGAGCTGTAACTACTACGCAA-3

[0098] 7. Phenotypic observation of stems in VIGS-treated plants: To compare the silencing effect, on day 14 after V991 inoculation, stems of plants from the VIGS-treated group and the negative control (TRV::00) were randomly excised. Three biological replicates were performed for each group. Stem dissection and photographic recording of vascular browning and other phenotypes were conducted. 8. Phenotypic and resistance analysis after GhTLP1b gene silencing: The GhTLP1b gene in ZZM2 was silenced using VIGS technology, and the silenced plants were inoculated with Verticillium wilt pathogen V991 to observe changes in disease resistance phenotype. Approximately 21 days after inoculation, the disease incidence of the negative control TRV::00 and the experimental group TRV::GhTLP1b was compared. The results are as follows: Figure 3 As shown. Figure 3 As shown in Figure A, the negative control plants (TRV::00) exhibited partial yellowing, wilting, and leaf drop; while the TRV::GhTLP1b silent plants showed extensive yellowing, severe wilting, and leaf drop, with significant phenotypic differences.

[0099] Further investigation was conducted to determine gene silencing efficiency and to perform disease index statistics. Figure 3 The B in the figure indicates that GhTLP1b was silenced at an efficiency of 70% in leaves and 60% in roots, proving that the gene was effectively silenced. Figure 3 The C-value in the study showed that the disease index of the silenced plants was significantly higher than that of the negative control, indicating that the resistance of GhTLP1b-silenced plants to Verticillium wilt was significantly reduced. Furthermore, stem segments were collected after inoculation and cultured in PDA medium for 3 days to observe the proportion of diseased stem segments. Figure 4 (D in the text). The results showed that the number of diseased stem segments in the TRV::GhTLP1b treatment group was significantly higher than that in the TRV::00 control group. In conclusion, silencing GhTLP1b can significantly reduce the resistance of ZZM2 to Verticillium wilt, indicating that this gene plays a positive regulatory role in the process of cotton resisting Verticillium dahliae infection.

[0100] Example 4: Silencing GhTLP1b to inhibit reactive oxygen species (ROS) bursts

[0101] 1. Materials and treatment: Cotton plants of the negative control TRV::00 and the experimental group TRV::GhTLP1b were collected at 0h, 1h, 3h, 6h, 9h, 12h and 24h after inoculation with Verticillium dahliae V991.

[0102] 2. DAB Method for Detecting ROS Accumulation: The plant tissue reactive oxygen species (DAB) assay kit was used for staining. Collected leaves were gently washed with tap water, blotted dry with filter paper, and then immersed in DAB staining working solution under a negative pressure of 0.1 MPa for 30 min, followed by staining at room temperature in the dark for 4-12 h. Once the positive areas turned dark brown, the samples were removed, rinsed 3-5 times with distilled water, and then immersed in tissue destaining solution in a 70-80℃ water bath for 20-40 min to remove background color. After destaining, the samples were rinsed with distilled water, soaked in preservation solution, and then photographed for observation.

[0103] 3. Analysis of ROS accumulation after DAB staining: Results are as follows Figure 4 As shown in Figure A, at 1 h, 6 h, and 9 h after V991 infection, the peroxide accumulation in TRV::GhTLP1b-silenced plants was significantly lower than that in the TRV::00 control group. The results indicate that silencing the GhTLP1b gene disrupts the release of reactive oxygen species in the early stages of pathogen infection, thereby reducing the cotton's disease resistance.

[0104] Example 5: Silencing GhTLP1b reduces lignin deposition

[0105] 1. Material preparation: On the 7th day after inoculation with V991, about 3cm of stems were cut from TRV::00 and TRV::GhTLP1b plants, with 3 biological replicates for each group.

[0106] 2. Paraffin Sectioning and Phloroglucinol Staining: Stem segments were fixed with 70% FAA fixative, then embedded in paraffin and sectioned (75-90 μm thick). The sections were stained with 3% phloroglucinol ethanol solution for 10 min, followed by development with 18% hydrochloric acid for approximately 30 s, and then observed under a stereomicroscope on a glass slide. Lignin and phloroglucinol form a brick-red or purplish-red product under acidic conditions.

[0107] 3. Analysis of lignin deposition in paraffin sections: such as... Figure 4 As shown in Figure B, the vascular bundles of the stem segments of TRV::GhTLP1b-silenced plants showed a significantly lighter red staining than the TRV::00 control group, indicating a significant reduction in lignin deposition. This result suggests that silencing GhTLP1b disrupts the lignin metabolic pathway, leading to impaired cell wall reinforcement and decreased disease resistance.

[0108] Example 6: Silencing GhTLP1b reduces callosity accumulation

[0109] 1. Materials and treatment: 72 h after inoculation with V991, leaves of TRV::00 and TRV::GhTLP1b plants were collected for callose staining and content determination.

[0110] 2. Aniline blue staining observation: After leaf samples were fixed with AAF fixative and dehydrated with a series of ethanol solutions, they were stained with callose staining working solution (aniline blue method) in the dark for 1 hour. After mounting, the samples were observed under a fluorescence microscope. Callose showed bright blue or bright green fluorescence under ultraviolet excitation.

[0111] 3. Quantitative detection by fluorescence method: Weigh approximately 0.1g of leaf sample, decolorize with ethanol, add extract and homogenize, incubate at 80℃ for 20min, centrifuge and collect the supernatant. React the supernatant and a series of callosine standards with staining reagents, incubate at 50℃ until the blue color fades, and measure the fluorescence intensity at an excitation wavelength of 400nm and an emission wavelength of 500nm. Calculate the callosine content based on the standard curve.

[0112] 4. Analysis of callosity staining results: such as Figure 4 As shown in C, the callose fluorescence signal in the leaves of TRV::GhTLP1b-silenced plants was significantly weaker than that in the control group. Quantitative results ( Figure 4 The results (E) showed that the callose content in the TRV::00 group was approximately 30 mg / g fresh weight, while it decreased to approximately 24 mg / g in the TRV::GhTLP1b group. This indicates that silencing GhTLP1b inhibited callose deposition at the site of infection, weakening the plant's physical defense barrier.

[0113] Example 7: Overexpression of GhTLP1b gene enhances cotton resistance to Verticillium wilt.

[0114] 1. Creation and Identification of Overexpression Lines: The constructed GhTLP1b overexpression vector was transformed into cotton variety J668 via Agrobacterium-mediated transformation. Positive T0 generation plants were obtained through resistance screening and PCR identification. After propagation, DNA was extracted from the T1 generation plants using the CTAB method for PCR verification, yielding stable overexpression lines GhTLP1b-OE1, GhTLP1b-OE2, and GhTLP1b-OE3. The CTAB DNA extraction steps are as follows:

[0115] Take 100-200 mg of fresh plant tissue (such as young cotton leaves) and grind it rapidly into a fine powder using liquid nitrogen.

[0116] Transfer the powder to 2 mL of preheated CTAB extraction buffer (2% CTAB, 100 mmol / L Tris-HCl pH 8.0, 20 mmol / L EDTA pH 8.0, 1.4 mol / L NaCl, 1% PVP-40, with 0.2% β-mercaptoethanol added before use) and vortex immediately to mix.

[0117] Incubate the mixture in a 65°C water bath for 60 minutes, gently inverting and mixing every 10-15 minutes during this period.

[0118] After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1, v / v), and gently invert for 15 min to form an emulsion. Centrifuge at 12,000 ×g for 15 min at 4 °C, and carefully transfer the upper aqueous phase to a new centrifuge tube.

[0119] Add 0.7 times the volume of pre-cooled isopropanol to the supernatant, gently invert to mix, and incubate at -20°C for 30 min to precipitate DNA. Centrifuge at 12,000 ×g for 15 min at 4°C and discard the supernatant.

[0120] Wash the precipitate twice with 70% ethanol, centrifuging at 8,000 ×g for 5 min each time at 4°C, and discard the ethanol.

[0121] Air dry at room temperature until the ethanol has completely evaporated (about 10 min), then add 50-100 μL of TE buffer (10 mmol / L Tris-HCl pH 8.0, 1 mmol / L EDTA pH 8.0) or sterile ultrapure water to dissolve the DNA.

[0122] The purity was assessed by measuring the A260 / A280 ratio (ideally 1.8-2.0) and the A260 / A230 ratio (ideally >2.0) using a micro-spectrophotometer. DNA integrity was then checked by 1% agarose gel electrophoresis, observing a clear main band with no significant degradation. DNA samples were stored at -80°C for later use.

[0123] 2. Material sources and culture conditions

[0124] The control group in this experiment was J668 (from the National Southern Breeding Research Institute Laboratory of the Chinese Academy of Agricultural Sciences in Sanya), and the experimental group consisted of three GhTLP1b overexpression lines: GhTLP1b-OE1, GhTLP1b-OE2, and GhTLP1b-OE3. Both materials were first pre-germinated, and then seeds showing white sprouts and exhibiting uniform germination were sown in pots filled with nutrient soil (nutrient soil: vermiculite = 2:1), covered with plastic wrap to maintain moisture and temperature, and cultured in a greenhouse (28℃, light / dark = 16 h:8 h). At the "two leaves and one bud" stage, the plants were inoculated with *Verticillium dahliae* V991. 3. Activation, culture, and inoculation statistics of *Verticillium wilt*: Wild-type J668 and the three overexpression lines were cultured to the "two leaves and one bud" stage and inoculated with V991 using the method described in Example 1. Disease incidence was observed 21 days after inoculation. 4. Analysis of inoculation results of overexpression lines: (The text continues with further details on inoculation results for overexpression lines.) Figure 5 As shown in Figure B, after inoculation, the leaves of wild-type J668 plants generally turned yellow, wilted, and fell off, while the three overexpression lines remained healthy and showed no obvious symptoms of disease. Disease index statistics further indicated that the overexpression lines had significantly higher disease resistance than the wild type (…). Figure 5 (A). This result demonstrates that overexpression of GhTLP1b can significantly enhance cotton's resistance to Verticillium wilt.

[0125] In summary, the present invention demonstrates that GhTLP1b plays a crucial role in cotton's resistance to Verticillium dahliae infection by positively regulating defense responses such as reactive oxygen species (ROS) eruption, lignin deposition, and callose accumulation. Molecular breeding using this gene can provide important genetic resources and technical support for the selection of cotton varieties resistant to Verticillium wilt.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the GhTLP1b gene in improving cotton resistance to Verticillium wilt, characterized in that, The protein coding region sequence of the GhTLP1b gene is shown in SEQ ID NO.

2. The application is to enhance the resistance of cotton to Verticillium wilt by increasing the expression level or protein activity of the GhTLP1b gene in cotton, which is caused by Verticillium dahliae.

2. The application as described in claim 1, characterized in that, The enhanced resistance of cotton to Verticillium wilt is manifested by a decrease in the disease index.

3. The application as described in claim 1, characterized in that, The amino acid sequence of the GhTLP1b gene protein is shown in SEQ ID NO.

3.

4. The application of the GhTLP1b gene in the genetic breeding of cotton resistance to Verticillium wilt, characterized in that, The protein coding region sequence of the GhTLP1b gene is shown in SEQ ID NO.

2. The application is to enhance the resistance of cotton to Verticillium wilt by increasing the expression level or protein activity of the GhTLP1b gene in cotton, which is caused by Verticillium dahliae.

5. A method for enhancing cotton's resistance to Verticillium wilt, characterized in that, The method includes steps to increase the expression level or protein activity of the GhTLP1b gene in recipient cotton, the protein coding region sequence of which is shown in SEQ ID NO.2, and the Verticillium wilt is caused by Verticillium dahliae.

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