Ghagl8 protein and coding gene in cotton disease resistance and early maturation synergistic improvement

Abstract

The application discloses application of GhAGL8 protein and a coding gene in cotton disease resistance and early maturity synergic improvement and belongs to the technical field of plant genetic engineering. GhAGL8 It is found for the first time that increasing the expression level of the gene can make cotton simultaneously obtain the phenotypes of enhanced disease resistance and enhanced early maturity, and new functional gene resources are provided for synergic genetic improvement of important agronomic traits of cotton. GhAGL8 It is found for the first time that increasing the expression level of the gene can make cotton simultaneously obtain the phenotypes of enhanced disease resistance and enhanced early maturity, and new functional gene resources are provided for synergic genetic improvement of important agronomic traits of cotton.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of a GhAGL8 protein and its encoding gene in the synergistic improvement of cotton disease resistance and early maturity. Background Technology

[0002] Cotton is an important natural fiber crop and economic crop, but existing major cotton-producing areas face two major challenges: first, cotton has a long growing season, making it susceptible to damage from late spring frosts and early autumn frosts; second, continuous cropping exacerbates Verticillium wilt, necessitating the breeding of early-maturing cotton varieties that also possess disease resistance to overcome the industry's predicament. However, there is a typical trade-off between early maturity and disease resistance, resulting in early-maturing varieties in existing cotton germplasm resources generally exhibiting weak disease resistance, while disease-resistant varieties are mostly mid-to-late maturing. Varieties with good synergistic effects of early maturity and disease resistance are extremely scarce in production, severely hindering the healthy development of the cotton industry.

[0003] In traditional breeding, selecting early-maturing materials separately and then introducing disease-resistant genes, or obtaining disease-resistant materials first and then continuously selecting for early-maturing traits, usually requires multiple generations of hybridization, backcrossing, and trait identification. This results in a long breeding cycle, and the problem of simultaneously and stably inheriting early maturity and disease resistance may arise in the offspring population. Even with marker-assisted selection, if the early-maturing and disease-resistant genes used are located at different genetic loci, it is still necessary to aggregate multiple target loci and continuously screen for materials that possess both excellent agronomic traits, further increasing the complexity of the breeding work.

[0004] Therefore, developing functional genes that can simultaneously regulate early maturity and Verticillium wilt resistance in cotton, and elucidating their roles in cotton growth, development, and disease resistance, is of great significance for solving the problem of balancing early maturity and disease resistance in existing cotton breeding, shortening the breeding cycle of superior varieties, and improving the stability of cotton production. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of the GhAGL8 protein and its encoding gene in the synergistic improvement of cotton disease resistance and early maturity. This invention identifies a novel disease resistance and early maturity regulator, GhAGL8, in the cotton genome. Overexpression of GhAGL8 in cotton exhibits significant disease resistance and early maturity traits, effectively solving the problem of the difficulty in simultaneously achieving early maturity and disease resistance in existing cotton breeding practices.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of GhAGL8 protein in the synergistic improvement of disease resistance and early maturity in cotton; wherein the GhAGL8 protein is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0007] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fragment expressed by fusion with the target protein through DNA recombination technology. This tag can be used to improve the expression efficiency of the target protein or to facilitate its detection, tracing, and / or purification. To facilitate the purification, detection, or tracing of the protein described in (A1), a protein tag can be attached to the amino and / or carboxyl termini of the protein. The protein tag includes, but is not limited to, Poly-Arg tags, Poly-His tags, FLAG tags, Strep-tag II tags, or c-Myc tags; wherein, the Poly-Arg tag can be six consecutive arginine residues (RRRRRR), the Poly-His tag can be six consecutive histidine residues (HHHHHH), the FLAG tag can have an amino acid sequence of DYKDDDDK, the Strep-tag II tag can have an amino acid sequence of WSHPQFEK, and the c-Myc tag can have an amino acid sequence of EQKLISEEDL.

[0008] In the above application, the synergistic improvement of cotton disease resistance and early maturity specifically refers to: improving cotton's resistance to Verticillium wilt and promoting early maturity by overexpressing GhAGL8 protein.

[0009] Furthermore, the Verticillium wilt is caused by Verticillium dahliae.

[0010] A second aspect of the present invention provides the application of the gene encoding the GhAGL8 protein in the synergistic improvement of cotton disease resistance and early maturity; said encoding gene is GhAGL8 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

[0011] In the above applications, by promoting GhAGL8 Gene expression is used to improve cotton's resistance to Verticillium wilt and promote early maturity.

[0012] Furthermore, the Verticillium wilt is caused by Verticillium dahliae.

[0013] By overexpressing in cotton GhAGL8 Genes that enhance the resistance of cotton materials to pathogen infection, thereby reducing the adverse effects of pathogen infection on the normal growth and development of cotton. Specifically, when the pathogen is *Verticillium dahliae* (… Verticillium dahliae )hour, GhAGL8Cotton with overexpressed gene showed strong resistance to Verticillium wilt, indicating that... GhAGL8 Genes can serve as important genetic regulatory factors to enhance cotton's resistance to Verticillium dahliae, providing new target genes for cotton Verticillium wilt resistance breeding.

[0014] Meanwhile, the present invention discovered GhAGL8 The gene not only plays a role in regulating disease resistance, but also promotes the reproductive growth process of cotton, leading to overexpression. GhAGL8 The cotton genus exhibits an early-maturing phenotype, characterized by earlier flowering and / or earlier maturity. Therefore, compared to single-function genes that only possess disease resistance or only regulate reproductive time, the present invention… GhAGL8 Genes can simultaneously improve disease resistance and early maturity in the same cotton material through the regulation of the same genetic factor.

[0015] Preferred, promoting GhAGL8 The substance used for gene expression is any one of the following: C1) contains GhAGL8 Gene expression cassettes; C2) contains GhAGL8 Recombinant expression vectors of genes, or recombinant expression vectors containing the expression cassette described in C1); C3) contains GhAGL8 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant expression vector described in C2); C4) contains GhAGL8 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains GhAGL8 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains GhAGL8 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

[0016] In this invention, plant expression vectors commonly used in the art can be used to construct a structure containing... GhAGL8 Recombinant gene expression vectors. The type of plant expression vector is not particularly limited, as long as it can carry... GhAGL8 The gene is transcribed and / or expressed in plant cells, plant tissues, and / or plant plants. The plant expression vectors include, but are not limited to, binary plant expression vectors suitable for Agrobacterium-mediated genetic transformation, plasmid expression vectors suitable for plant genetic transformation, and expression vectors suitable for gene gun or microparticle bombardment-mediated plant genetic transformation.

[0017] In some implementations, containing GhAGL8The recombinant gene expression vector is constructed using the following methods: Will GhAGL8 The gene was inserted between the BamHI and SalI sites of the pCambia2300 vector to construct a vector containing... GhAGL8 Recombinant gene expression vectors.

[0018] The beneficial effects of this invention are: This invention identified a transcription factor gene from the cotton genome that has both disease resistance and early maturity regulation functions. GhAGL8 And for the first time, it was discovered that improving GhAGL8 The expression level of this gene can enable cotton to simultaneously acquire phenotypes of enhanced disease resistance and early maturity, providing new functional gene resources for the synergistic genetic improvement of important agronomic traits in cotton.

[0019] The invention discovered GhAGL8 The aforementioned dual regulatory function helps to solve the problem of separate selection and multi-site aggregation of early maturity and disease resistance in traditional cotton breeding. It reduces the breeding steps of multi-generation hybridization, backcrossing and gene aggregation to obtain the dual superior traits of disease resistance and early maturity. It can also reduce the risk of trait segregation or adverse linkage during the genetic aggregation of different target traits, thus providing a new technical approach to shorten the creation cycle of disease-resistant and early-maturing cotton materials. Attached Figure Description

[0020] Figure 1 RT-qPCR for identifying transgenic materials GhAGL8 Level of expression.

[0021] Figure 2 : GhAGL8-OE Results of early maturity identification in overexpression plants; in the figure, A represents wild type (WT) and... GhAGL8-OE Phenotypes of overexpressing plants (GhAGL8-OE4, GhAGL8-OE27); B represents wild-type (WT) and... GhAGL8-OE Plant height statistics of overexpression plants (GhAGL8-OE4, GhAGL8-OE27); C represents wild type (WT) and GhAGL8-OE Statistics on the number of flowering days of overexpression plants (GhAGL8-OE4, GhAGL8-OE27).

[0022] Figure 3 : GhAGL8-OE Results of disease resistance identification in overexpressing plants; in the figure, A represents wild-type (WT) and... GhAGL8- OE Disease phenotype of overexpressing plants (GhAGL8-OE4, GhAGL8-OE27) after inoculation with Vd991; B represents wild-type (WT) and GhAGL8-OEStatistical analysis of disease index of overexpressing plants (GhAGL8-OE4, GhAGL8-OE27) after inoculation with Vd991. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] As mentioned earlier, there is a typical trade-off between early maturity and disease resistance, resulting in weak overall disease resistance in early-maturing varieties among existing cotton germplasm resources, while disease-resistant varieties are mostly mid-to-late maturing. In production, there is a severe shortage of varieties with good synergy between early maturity and disease resistance.

[0025] GhAGL8 The gene is a known flowering regulatory gene, and its nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence of the GhAGL8 protein is shown in SEQ ID NO.2, as follows: GhAGL8 The nucleotide sequence of the gene: ATGGGGAGGGGTAGGGTTCAGTTGAAGAGAATTGAAAACAAGATCAACAGGCAAGTCACTTTCTCAAAGAGAAGGTCTGGTTTATTGAAGAAAGCCCATGAAATCTCTGTGCTTTGTGATGCTGAAGTTGCTTTGATTGTTTTCTCAACTAAAGGGAAGCTCTTTGAATACTCATCAGATTCTTGCATGGAGAGGATCCTCGAACGGTATGAAAGATATTCTTATGCAGAGAGGCAACTTGCTGCAAATGAAAATGAACGAACTGGTAGTTGGACTTTGGAACATGCAAAACTTAAAGCCAGGATGGAGGTGTTACAAAGAAACCAAAGGCATTATATGGGAGAAGATCTTGAGAATTTGAGTCTTAGAGAGCTTCAGAACTTGGAGCACCAACTTGATTCTGCCCTTAAACACATACGTTCAAGAAAGAATCAGCTCATGTTTGAATCCATTTCCGAGCTTCAGAAAAAGGATAAAGCATTACAAGAGCAGAACAATGTCCTTGCAAAGAAGGTAAAGGAAAAGGAGAAGGAGAAGGAAAAGGAGAAGGAGAAGGAAATGACCCATCAGCCACAACAAAACAATTGCCAAGATTCATCCTCAATGCTTCCACAACCACTGCAGTCCTTGAACATCAGTGACACATATGAAGCAAGGAGCAATGGAAGAGAAGAGGGTAATCCTAGTGCAGCACAACATCGCAACTCCAATGTGCTATTGCCACCGTGGATGATTCCTCGTATTGAGTAA

[0026] Amino acid sequence of GhAGL8 protein: MGRGRVQLKRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSTKGKLFEYSSDSCMERILERYSYAERQLAANENERTGSWTLEHAKLKARMEVLQRNQRHYMGEDLENLSLREL QNLEHQLDSALKHIRSRKNQLMFESISELQKKDKALQEQNNVLAKKVKEKEKEKEKEKEMTHQPQQNNCQDSSSMLPQPLQSLNISDTYEARSNGREEGNPSAAQHRNSNVLLPPWMIPRIE.

[0027] For research GhAGL8 The function of genes, this invention constructs a gene containing GhAGL8 Recombinant gene expression vectors were used to obtain overexpression using Agrobacterium-mediated genetic transformation technology. GhAGL8 Transgenic cotton lines expressing the gene. Treatment of transgenic cotton lines with *Verticillium dahliae* revealed overexpression. GhAGL8 The transgenic cotton lines expressing the gene exhibited a clear disease resistance phenotype; simultaneously, overexpression of the gene... GhAGL8 The transgenic cotton lines with the gene flowered earlier and were shorter, exhibiting obvious early-maturing characteristics. Therefore, GhAGL8 Genes have the dual function of regulating cotton disease resistance and early maturity, and can synergistically improve cotton early maturity and disease resistance, thus proposing this invention.

[0028] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0029] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, conventional conditions are generally followed. Where: Verticillium dahliae Vd991 is an existing Verticillium dahliae species. Verticillium dahliae One isolate was described in the journal article (Xu RQ, Wang JN, Chen JY, Dai X F. Analysis of T-DNAInsertional Flanking Sequence and Mutant Phenotypic Characteristics inVerticillium dahliae. Scientia Agricultura Sinica, 2010, 43(3):489–496.).

[0030] Czapek's liquid medium: NaNO3 2g, KCl 0.5g, FeSO4·7H2O 0.02g, MgSO4·7H2O 0.5g, sucrose 30g, K2HPO4 1.31g, distilled water to a final volume of 1000mL.

[0031] Czapek's solid medium: Add 15g of agar to Czapek's liquid medium.

[0032] Example 1: Overexpression GhAGL8 Construction of transgenic cotton lines 1. Construct containing GhAGL8 Recombinant gene expression vectors: Obtain "Zhongmian 24" from the CotonFGD website (https: / / cottonfgd.net / ). GhAGL8 The CDS sequence of the gene (Ghicr24_A05G103300) is shown in SEQ ID NO.1.

[0033] according to GhAGL8 Based on the CDS sequence of the gene, amplification primers were designed for PCR amplification. The sequences of the amplification primers are as follows: GhAGL8 -CDS-F: GAACACGGGGGACgtcgacATGGGGAGGGGTAGGGTT; (SEQ ID NO.3) GhAGL8 -CDS-R:TTTGTAGTCCTCGACCATggatccCTCAATACGAGGAATCATC. (SEQ IDNO.4) The PCR amplification product was inserted between the BamHI and SalI sites of the pCambia2300 vector. Sequencing confirmed that the vector contained the correct sequence. GhAGL8 Gene overexpression vectors are denoted as 35Sp:GhAGL8-3FLAG .

[0034] 2. Agrobacterium-mediated genetic transformation: (1) Seed germination: Prepare about 300 delinted and uncoated “Zhongmian 24” seeds, soak them in 200ml of 75% alcohol for 30s for disinfection, then soak them in 6% hydrogen peroxide for 30min for disinfection, rinse them with sterile water 3-5 times, then add 200ml of MSB culture medium (formula shown in Table 1), and culture them in the dark at 35℃ until the seeds show white (about 18-24h).

[0035] Table 1: Composition of MSB culture medium (2) Agrobacterium activation: The overexpression vector constructed in Example 1 was activated. 35Sp:GhAGL8-3FLAG Transform Agrobacterium. Spread the transformed Agrobacterium onto LB agar plates corresponding to the appropriate resistance and incubate at 28°C for one day to activate it. Spread the activated Agrobacterium onto the plates again the day before infection and incubate overnight at 28°C. Collect the Agrobacterium on the plates with an inoculation loop, suspend and mix it in CAB solution (formulation shown in Table 2), adjust the OD value to 0.9 using a spectrophotometer, and place at room temperature on a shaker at 90 rpm for 2 hours to maintain bacterial activity.

[0036] Table 2: Composition of CAB solution (3) Infection and co-culture: Wash the seeds with sterile water once to reveal the white leaves, select healthy germinating seeds, hold the seeds, cut open the seed coat to expose the cotyledons, expose and peel off the stem tips, put about 300 stem tips peeled within 2 hours into CAB one by one, then discard CAB, add 15ml of the prepared Agrobacterium bacterial solution, sonicate at 40KHz for 40s at room temperature, shake on a shaker at 90 rpm for 50min at room temperature, place the bacterial solution on sterile filter paper, and air dry on a clean bench for 10min.

[0037] (4) Screening culture and shoot induction: The shoot tips were cultured at 23℃ in the dark for 3-4 days. The co-cultured shoot tip embryonic root ends were inserted into the screening medium (formula shown in Table 3) and cultured for 3 days. Each culture dish contained about 50 shoot tips. The temperature was kept constant at 35±1℃, and a photoperiod control mode of 16 hours light / 8 hours dark was used. Then, the photoperiod was kept constant, and the culture temperature was adjusted to 25℃ and cultured for another 12 days. The roots of the explants were cut off, and the temperature and photoperiod were kept constant and cultured for another 15 days. This step was then repeated once.

[0038] Table 3: Composition of the screening culture medium (5) Rooting culture and transplanting: The resistant green shoots are cultured at 25℃ and under a light-dark cycle of 16h / 8h for 20 days until they take root. The green seedlings are then taken out and placed in a triangular conical bottle containing tap water to harden them for 1-2 weeks. Finally, the regenerated seedlings are moved to the greenhouse until the seeds are harvested.

[0039] (6) PCR positive seedling detection: Total RNA was extracted from cotton transgenic lines using Plant RNA Kit (OMEGA), and cDNA was obtained by reverse transcription using a reverse transcription kit (TransGen).

[0040] design GhAGL8 RT-qPCR primers for genes in transgenic cotton lines GhAGL8The relative expression levels of genes were detected. The specific sequences of the RT-qPCR primers are as follows: RT-qPCR-F:ACCACTGCAGTCCTTGAACA; (SEQ ID NO.5) RT-qPCR-R:CGGTGGCAATAGCACATTGG. (SEQ ID NO.6) The results are as follows Figure 1 As shown, in the transgenic cotton lines (GhAGL8-OE4, GhAGL8-OE27) GhAGL8 The relative expression level of the gene was significantly increased, proving that the present invention has successfully constructed an overexpression gene. GhAGL8 Transgenic cotton lines containing genes.

[0041] Example 2: Overexpression GhAGL8 Identification of early maturity traits in transgenic cotton lines using genetically modified genes 1. Test method: overexpression GhAGL8 The transgenic cotton lines (GhAGL8-OE4, GhAGL8-OE27) and “Zhongmian 24” (WT) were planted in the greenhouse of Anyang Cotton Research Institute in Henan Province and cultured under the same conditions. The phenotypes of the transgenic materials and the recipient plant (“Zhongmian 24”) were then observed regularly.

[0042] The planting day was taken as the first day, and the first flower bloom was taken as the criterion. The time from the planting day to the first flower bloom was taken as the flowering days. The flowering days of transgenic plants and recipient materials were counted.

[0043] The plant height of the transgenic cotton lines and recipient plants was measured 50 days after planting.

[0044] 2. Test Results: The results are as follows Figure 2 As shown, overexpression GhAGL8 The transgenic cotton lines of the gene have a significantly earlier flowering period and a significantly reduced plant height than "Zhongmian 24", exhibiting obvious early maturity traits.

[0045] Example 3: Overexpression GhAGL8 Identification of disease resistance phenotypes in transgenic cotton lines 1. Test method: overexpression GhAGL8The transgenic cotton lines (GhAGL8-OE4, GhAGL8-OE27) and "Zhongmian 24" (WT) were sown in vermiculite and placed in a long-day (LD) culture chamber (16h light / 8h dark) at a temperature of 25°C and a humidity of 60%. Twenty days after planting, they were inoculated with a suspension of Verticillium dahliae spores. The preparation method of the Verticillium dahliae spore suspension is as follows: S1: First, in a sterilized laminar flow hood, use a sterilized 1 mL pipette tip to pick up 2 mm of *Verticillium dahliae* Vd991 from Czapek's solid medium. 2 The samples were inoculated into Czapek's liquid medium for expansion culture.

[0046] S2: Place the inoculated bacteria in a shaker at 25℃ and set the rotation speed to 150 r / min for 5 days.

[0047] S3: Clean the container needed for filtration, filter the bacterial solution in the Czapek liquid culture medium through four layers of gauze to remove mycelia and obtain Verticillium dahliae spore solution.

[0048] S4: Use a pipette to draw a small amount of spore solution and drop it onto the grid of a hemocytometer. Cover with a coverslip, absorb excess liquid with absorbent paper, and observe under a regular optical microscope to determine the spore concentration in the spore solution. Wait until the spore concentration reaches 1×10⁻⁶. 7 When the concentration of *Verticillium dahliae* spores is 1 / mL, the suspension of *Verticillium dahliae* spores is ready.

[0049] An equal volume of Czapek's liquid culture medium without Vd991 was used as a control (CK).

[0050] The severity of disease was observed 21 days after inoculation and classified into five grades: Grade 0, Grade 1, Grade 2, Grade 3, and Grade 4. The specific criteria for each grade are as follows: Grade 0: No yellowing of leaves; the entire plant is healthy. Grade 1: Less than 25% of the leaf area is yellowed; the plant appears relatively healthy overall. Grade 2: Approximately 25%-65% of the leaf area is yellowed; the plant is in an unhealthy state. Grade 3: Approximately 65%-85% of the leaf area is withered and wilted; the entire plant is wilted. Grade 4: The entire cotton plant is completely wilted and dead.

[0051] The disease severity index (%) is calculated according to the following formula based on the severity level of the disease.

[0052] DI (Disease Index) = (Σ (number of each disease level) × (number of each plant) / (highest disease level (4) × total number of plants)) × 100.

[0053] 2. Test Results: The results are as follows Figure 3As shown, overexpression GhAGL8 After inoculation with Verticillium dahliae spore suspension, the disease index of transgenic cotton lines containing the gene was significantly lower than that of the wild-type "Zhongmian 24". This demonstrates overexpression. GhAGL8 Genes can enhance cotton’s resistance to Verticillium dahliae.

[0054] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of GhAGL8 protein in cotton disease resistance and early maturation synergistic improvement, characterized in that, The GhAGL8 protein is the protein shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. Use according to claim 1, characterized in that, The specific method of improving cotton disease resistance and early maturity is as follows: by overexpressing GhAGL8 protein, cotton's resistance to Verticillium wilt is improved and early maturity is promoted.

3. Use according to claim 2, characterized in that, Verticillium wilt is caused by Verticillium dahliae.

4. The application of the gene encoding the GhAGL8 protein in the synergistic improvement of cotton disease resistance and early maturity, characterized in that... The coding gene is GhAGL8 The gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, By promoting GhAGL8 increasing cotton resistance to verticillium wilt and promoting early maturity of cotton by promoting 6. Use according to claim 5, characterized in that, Verticillium wilt is caused by Verticillium dahliae.

7. The application according to claim 5, characterized in that, promote GhAGL8 the substance that promotes gene expression is any one of the following: C1) contains GhAGL8 Gene expression cassettes; C2) contains GhAGL8 Recombinant expression vectors of genes, or recombinant expression vectors containing the expression cassette described in C1); C3) contains GhAGL8 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant expression vector described in C2); C4) contains GhAGL8 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains GhAGL8 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains GhAGL8 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

8. The application according to claim 7, characterized in that, contain GhAGL8 The recombinant gene expression vector is constructed using the following methods: Will GhAGL8 The gene was inserted between the BamHI and SalI sites of the pCambia2300 vector to construct a vector containing... GhAGL8 Recombinant gene expression vectors.

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