Application of GhDWARF27 gene in regulating resistance of plants to verticillium wilt and breeding plants resistant to verticillium wilt

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of GhDWARF27 gene in regulation of plant verticillium wilt resistance and breeding of verticillium wilt resistant plants. The application discloses that application of GR24, an artificial synthetic analogue of strigolactone (SLs), or overexpression of a SLs biosynthesis gene GhDWARF27 can improve the resistance of plants to verticillium wilt, and silencing of the GhDWARF27 gene can result in reduction of SLs content in roots of the plants and reduction of the resistance of the plants to verticillium wilt. SLs can activate expression of JA response related genes through JA and ABA biosynthesis and signal pathways, and can enhance the resistance of the plants to verticillium wilt in an ABA dependent manner by enhancing the activity of antioxidant enzymes. The technical scheme and molecular mechanism of SLs for enhancing the resistance of cotton to verticillium wilt will provide a new approach for cotton breeding and prevention of cotton verticillium wilt.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the GhDWARF27 gene in regulating plant resistance to Verticillium wilt and cultivating Verticillium wilt-resistant plants. Background Technology

[0002] Controlling Verticillium wilt in cotton is a challenging problem, not only because the pathogen has a wide host range, but also because its sclerotia are highly resistant and can survive in the soil for many years. Various factors, such as sclerotia density in the soil, temperature, and seeding rate, can affect the severity of Verticillium wilt (Paplomatas et al., 1992). Furthermore, most plants show no symptoms in the early stages of infection, making it difficult to predict the onset and severity of the disease, and consequently, to determine appropriate control measures and timing. Currently available control methods include chemical control, biological control, and agricultural control. Breeding and planting resistant varieties is the most economical and effective method.

[0003] In recent years, a number of cotton varieties resistant to Verticillium wilt and with excellent comprehensive traits, such as Liaomian 10, Liaomian 12, and Zhongmian Institute 35, have been promoted in cotton-growing areas severely affected by Verticillium wilt. These varieties have played an important role in controlling the spread of Verticillium wilt and have significantly reduced yield and economic losses caused by the disease (Wang Hongmei, 2015). However, due to the lack of highly resistant upland cotton resources and the variation of the Verticillium wilt pathogen, breeding cotton varieties resistant to Verticillium wilt is difficult to meet the needs of cotton production. In recent years, advances in the molecular biology of the interaction between cotton and Verticillium wilt pathogen have provided an important foundation for the discovery of Verticillium wilt-related genes. Using VIGS and transgenic technology, genes such as GhGPA (Chen et al., 2021a), GhMAPKKK2 (Li Xiuqing et al., 2022), GhPP2C52 (Li et al., 2023b), and GhMYB33 (Guang et al., 2023) have been identified as playing important roles in cotton's response to Verticillium wilt resistance. Host-induced gene silencing (HIGS) technology has been used to identify several genes that play a key role in the pathogenicity of Verticillium wilt (Ghag and Pathology, 2017). Zhang et al. (2016) first reported that HIGS-induced gene silencing in cotton Verticillium dahliae led to a significant decrease in pathogenicity. The VdH1 knockout mutant of Verticillium dahliae showed delayed onset and reduced severity of disease in cotton plants (Zhang et al., 2016). Inhibition of VdILV2 or VdILV6 in the highly pathogenic Verticillium dahliae Vd991 via HIGS significantly reduced the pathogenicity of Verticillium dahliae (Wei et al., 2020). Silencing the chitin synthase genes VdChs5 and VdChs7 in susceptible cotton varieties significantly enhanced resistance to Verticillium wilt (Chen et al., 2023). Zhang et al. (2023) identified a key gene for resistance to Verticillium wilt, GhRVD1, through a genome-wide association study (GWAS) of 419 upland cotton accessions (Zhang et al., 2023b).

[0004] Therefore, in-depth research into cotton's Verticillium wilt resistance genes can enhance the understanding of the molecular mechanisms of disease resistance. Discovering key resistance genes and applying molecular breeding techniques has become an efficient strategy for controlling Verticillium wilt. Currently, there is no publicly available research in this field regarding the role of the cotton GhDWARF27 gene in cotton's resistance to Verticillium wilt. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the GhDWARF27 gene in regulating plant resistance to Verticillium wilt and in breeding Verticillium wilt-resistant plants. The GhDWARF27 gene can improve plant resistance to Verticillium wilt and provide a technical basis for breeding Verticillium wilt-resistant plants.

[0006] This invention provides the application of strigolactones and / or the GhDWARF27 gene, which regulates the biosynthesis of said strigolactones, in regulating plant resistance to Verticillium wilt and / or cultivating plants resistant to Verticillium wilt, wherein the amino acid sequence encoded by said GhDWARF27 gene is shown in SEQ ID NO.64.

[0007] Preferably, the application includes any one of the following:

[0008] I: The application of positive regulation of the GhDWARF27 gene in improving plant resistance to Verticillium wilt and / or in cultivating Verticillium wilt-resistant plants;

[0009] II: Application of negative regulation of the GhDWARF27 gene in a model for reducing plant resistance to Verticillium wilt;

[0010] III: Application of exogenous application of synthetic analogues of strigolactone in improving plant resistance to Verticillium wilt.

[0011] The present invention also provides the application of biomaterials that positively regulate the GhDWARF27 gene in improving plant resistance to Verticillium wilt and / or cultivating Verticillium wilt-resistant plants, wherein the amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO.64.

[0012] Preferably, the biomaterial includes any one or more of the following:

[0013] 1) Expression cassettes containing the CDS sequence of the GhDWARF27 gene;

[0014] 2) Recombinant expression vectors containing the CDS sequence of the GhDWARF27 gene;

[0015] 3) A recombinant expression vector containing the expression cassette described in 1);

[0016] 4) Engineered bacteria containing the CDS sequence of the GhDWARF27 gene;

[0017] 5) Engineered bacteria containing the expression cassette described in 1);

[0018] 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3);

[0019] The amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO.64.

[0020] The present invention also provides the application of biomaterials that negatively regulate the GhDWARF27 gene in reducing plant resistance to Verticillium wilt and / or in cultivating Verticillium wilt-susceptible plant models, wherein the amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO. 64.

[0021] Preferably, the biomaterial includes any one of the following:

[0022] A) Silencing the nucleic acid molecules of the GhDWARF27 gene;

[0023] B) A TRV2 vector containing the nucleic acid molecule described in A);

[0024] C) A virus-mediated gene silencing system containing the TRV2 vector described in B).

[0025] Preferably, the nucleotide sequence of the nucleic acid molecule described in A) is as shown in SEQ ID NO.65.

[0026] Preferably, the nucleotide sequence of the CDS sequence of the GhDWARF27 gene is shown in SEQ ID NO.63.

[0027] Preferably, the plant includes cotton.

[0028] The present invention also provides a method for cultivating transgenic plants, wherein the transgenic plants include Verticillium wilt-resistant plants or Verticillium wilt-susceptible plant models; the cultivation steps of the Verticillium wilt-resistant plants include: increasing the content of GhDWARF27 protein in the recipient plant or promoting the expression of the GhDWARF27 gene in the recipient plant to obtain the Verticillium wilt-resistant plants;

[0029] The steps for cultivating the Verticillium wilt-susceptible plant model include: reducing the content of GhDWARF27 protein in the recipient plant or silencing the GhDWARF27 gene in the recipient plant to obtain the Verticillium wilt-susceptible plant model.

[0030] Beneficial effects:

[0031] This invention provides the application of the GhDWARF27 gene in regulating plant resistance to Verticillium wilt and / or breeding Verticillium wilt-resistant plants. The amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO. 64. This invention clarifies that exogenous application of the synthetic strigolactone (SL) analog GR24 or overexpression of the SL biosynthetic gene GhDWARF27 can enhance plant resistance to Verticillium wilt. Silencing the GhDWARF27 gene leads to a reduction in SL content in plant roots and decreases plant resistance to Verticillium wilt. SLs activate the expression of JA response-related genes through JA and ABA biosynthesis and signaling pathways, and enhance antioxidant enzyme activity in an ABA-dependent manner, thereby enhancing plant resistance to Verticillium wilt. The technical solution and molecular mechanism of SLs enhancing cotton Verticillium wilt resistance will provide new approaches for cotton Verticillium wilt-resistant breeding and control. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0033] Figure 1 The following are the amino acid sequence analysis results of the GhDWARF27 gene in Example 1:

[0034] Figure 2 This is a phylogenetic tree of DWARF27 in different plant species in Example 1 and the expression pattern of GhDWARF27 in different tissues of cotton;

[0035] Figure 3 The promoter sequence of GhDWARF27 in Example 1;

[0036] Figure 4 Phenotypic diagrams of the positive and negative controls of the VIGS system in Example 1;

[0037] Figure 5 The expression levels of GhDWARF27 and the content of SLs in the VIGS strain in Example 1;

[0038] Figure 6 The symptoms of Verticillium wilt in GR24 pretreated VIGS plants 21 days after inoculation with Verticillium wilt pathogen in Example 1;

[0039] Figure 7 The image shows the trypan blue staining of leaves from GR24-pretreated VIGS plants in Example 1 21 days after inoculation with Verticillium wilt.

[0040] Figure 8 The disease index of GR24 pretreated VIGS plants in Example 1 after inoculation with Verticillium wilt is statistically analyzed;

[0041] Figure 9 The differentially expressed genes after inoculation with Verticillium wilt in Example 1;

[0042] Figure 10 The results of GO enrichment of differentially expressed genes in Example 1;

[0043] Figure 11 The results of KEGG enrichment of differentially expressed genes in Example 1;

[0044] Figure 12 The expression changes of genes involved in SLs biosynthesis in Example 1;

[0045] Figure 13 The effects of GR24 treatment and silencing GhDWARF27 on ABA biosynthesis and signaling pathway gene expression in cotton roots, as shown in Example 1;

[0046] Figure 14 The root enzyme activity of different plants after inoculation with Verticillium wilt in Example 1;

[0047] Figure 15 The results of DAB staining on cotton plant leaves under different treatments in Example 1;

[0048] Figure 16 qRT-PCR analysis of the expression levels of antioxidant enzyme genes in cotton roots under different treatments in Example 1;

[0049] Figure 17 The effect of GR24 treatment and silencing GhDWARF27 on ABA content in cotton roots in Example 1;

[0050] Figure 18 The results of qRT-PCR for the genes involved in ABA biosynthesis and signal transduction pathways in Example 1;

[0051] Figure 19 The effects of GR24 treatment and silencing GhDWARF27 on JA biosynthesis and signaling pathway gene expression in cotton roots, as shown in Example 1;

[0052] Figure 20 The JA content in the roots of cotton treated with GR24 and silenced with GhDWARF27 in Example 1;

[0053] Figure 21 The results of qRT-PCR analysis of genes involved in JA biosynthesis and signal transduction pathways in Example 1 are shown.

[0054] Figure 22 The results of qRT-PCR analysis of JA-related response genes in Example 1;

[0055] Figure 23The effects of GR24 treatment and silencing GhDWARF27 on the expression of SA response genes and SA content in cotton roots, as shown in Example 1;

[0056] Figure 24 For the identification of the T3 generation of GhDWARF27 overexpression transgenic cotton in Example 1;

[0057] Figure 25 The content of SLs in the three GhDWARF27 overexpression transgenic lines in Example 1;

[0058] Figure 26 For the identification of Verticillium wilt resistance in the GhDWARF27 overexpression transgenic line in Example 1;

[0059] Figure 27 The disease index of the three overexpressing transgenic lines at different time points after inoculation in Example 1;

[0060] Figure 28 The leaves of the transgenic lines and control plants in Example 1 after inoculation with Verticillium wilt were stained with DAB and trypan blue. Detailed Implementation

[0061] This invention provides the application of strigolactones and / or the GhDWARF27 gene, which regulates the biosynthesis of said strigolactones, in regulating plant resistance to Verticillium wilt and / or cultivating Verticillium wilt-resistant plants. The amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO. 64. The preferred CDS sequence of the GhDWARF27 gene of this invention is shown in SEQ ID NO. 63.

[0062] The preferred applications described in this invention include any one of the following:

[0063] I: The application of positive regulation of the GhDWARF27 gene in improving plant resistance to Verticillium wilt and / or in cultivating Verticillium wilt-resistant plants;

[0064] II: Application of negative regulation of the GhDWARF27 gene in reducing plant resistance to Verticillium wilt and / or in cultivating Verticillium wilt-susceptible plant models;

[0065] III: Application of exogenous application of synthetic analogues of strigolactone in improving plant resistance to Verticillium wilt.

[0066] The synthetic analogues of strigolactone described in this invention preferably include, but are not limited to, GR24.

[0067] This invention clarifies that strigolactones (SLs) can positively regulate cotton resistance to Verticillium wilt, and that GhDWARF27 plays an important role in SLs biosynthesis. Using upland cotton genome data, bioinformatics analysis identified the full-length CDS of the GhDWARF27 gene as 807 bp, encoding 268 amino acids.

[0068] The present invention also provides the application of biomaterials that positively regulate the GhDWARF27 gene in improving plant resistance to Verticillium wilt and / or cultivating Verticillium wilt-resistant plants, wherein the amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO.64.

[0069] The biomaterials of the present invention preferably include any one or more of the following: 1) an expression cassette containing the CDS sequence of the GhDWARF27 gene; 2) a recombinant expression vector containing the CDS sequence of the GhDWARF27 gene; 3) a recombinant expression vector containing the expression cassette of 1); 4) engineered bacteria containing the CDS sequence of the GhDWARF27 gene; 5) engineered bacteria containing the expression cassette of 1).

[0070] 6) An engineered bacterium containing the recombinant expression vector described in 2) or 3); the amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO. 64. The nucleotide sequence of the CDS sequence of the GhDWARF27 gene in this invention is shown in SEQ ID NO. 63. This invention does not specifically limit the elements in the expression cassette, and any expression cassette containing the CDS sequence of the GhDWARF27 gene is within the scope of protection of this invention. The recombinant expression vector of this invention preferably includes an initial vector and an expression cassette containing the CDS sequence of the GhDWARF27 gene inserted into the initial vector, wherein the initial vector preferably includes a plasmid vector, more preferably pCAMBIA1300. The initial strain in the engineered bacterium of this invention preferably includes Agrobacterium, more preferably Agrobacterium GV3101. This invention does not specifically limit the construction method of the recombinant expression vector and the engineered bacterium, and conventional construction methods for recombinant expression vectors or engineered bacteria in the art can be used.

[0071] The present invention also provides the application of biomaterials that negatively regulate the GhDWARF27 gene in reducing plant resistance to Verticillium wilt and / or in cultivating Verticillium wilt-susceptible plant models, wherein the amino acid sequence encoded by the GhDWARF27 gene is shown in SEQ ID NO. 64.

[0072] The biological material of this invention comprises any one of the following: A) a nucleic acid molecule silencing the GhDWARF27 gene; B) a TRV2 vector containing the nucleic acid molecule of A); C) a virus-mediated gene silencing system containing the TRV2 vector of B). In this invention, the nucleotide sequence of the nucleic acid molecule is preferably as shown in SEQ ID NO. 65, specifically: 5'-GGCTCATTGGACCTTCTGAGGTTCGGGAATCGG AGCTCAACGGAAGAAGAGAAAAGAATGTAGTGTATGTGAAAAAGTGCAGGTTCCTAGAGCAAAGCAACTGTGTAGGAATGTGCATCAACCTATGCAAGATGCCATCTCAAGCCTTTATTAAGGATTCCCTAGGAATGCCACTCAACATGGTTCCAAATTTTGATGATATGAGCTGCGAAATGATTTTCGGTCAGGATCCTCCAACACCAAATGATGATC-3'. The nucleotide sequences of the upstream primer VIGS-GhDWARF27-F and the downstream primer VIGS-GhDWARF27-R for amplifying the nucleic acid molecule described in A) are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The nucleic acid molecule of this invention is inserted between EcoRI and KpnRI of the TRV2 vector. The construction method of the TRV2 vector is not particularly limited; conventional construction methods in the art are acceptable. The virus-mediated gene silencing system of this invention preferably includes an Agrobacterium strain containing a TRV1 vector and an Agrobacterium strain containing the TRV2 vector of the nucleic acid molecule. The ratio of the effective viable counts of the Agrobacterium strain containing the TRV1 vector to the Agrobacterium strain containing the TRV2 vector of the nucleic acid molecule is preferably 1:1. This invention does not particularly limit the construction method of the virus-mediated gene silencing system; conventional construction methods in the art are acceptable.

[0073] In the application of the above-mentioned technical solutions of the present invention, the plant is preferably including but not limited to cotton, and more preferably upland cotton.

[0074] This invention utilizes virus-induced gene silencing (VIGS) technology to obtain silenced GhDWARF27 cotton plants. After inoculation with Verticillium wilt, the content of endogenous SLs in the roots of GhDWARF27 gene-silenced plants was significantly lower than that of wild-type cotton, resulting in a marked decrease in plant resistance to Verticillium wilt. Extensive wilting and yellowing of leaves, severe browning of vascular bundles, and a significant increase in the accumulation of fungal hyphae on stems led to a 53.03% increase in the disease index. Furthermore, after inoculation with Verticillium wilt, GhDWARF27 gene-silenced plants accumulated more H2O2 in their leaves than control plants, exhibiting more severe leaf cell damage. External application of GR24 treatment restored the resistance of TRV:DWARF27 plants to Verticillium wilt. Using genetic engineering techniques, a GhDWARF27 gene overexpression vector was constructed and transformed into the upland cotton standard line TM-1 to obtain a GhDWARF27 overexpression transgenic line. The root SL content of cotton lines overexpressing the GhDWARF27 gene increased by 105.96% compared to the control. Verticillium wilt inoculation results showed that the GhDWARF27 overexpressing transgenic lines exhibited significantly enhanced Verticillium wilt resistance, with the Verticillium wilt symptom index decreasing by 57.14% compared to the control.

[0075] The present invention also provides a method for cultivating transgenic plants, wherein the transgenic plants include Verticillium wilt-resistant plants or Verticillium wilt-susceptible plant models; the cultivation steps of the Verticillium wilt-resistant plants include: increasing the content of GhDWARF27 protein in the recipient plant or promoting the expression of the GhDWARF27 gene in the recipient plant to obtain the Verticillium wilt-resistant plants.

[0076] The steps for cultivating the Verticillium wilt-susceptible plant model include: reducing the content of GhDWARF27 protein in the recipient plant or silencing the GhDWARF27 gene in the recipient plant to obtain the Verticillium wilt-susceptible plant model.

[0077] The method for obtaining the plant resistant to Verticillium wilt according to the present invention preferably includes: introducing the GhDWARF27 gene into the recipient plant, more preferably, transferring the recombinant expression vector described in the above technical solution into the recipient plant to increase the content of GhDWARF27 protein in the recipient plant or promote the expression of the GhDWARF27 gene in the recipient plant.

[0078] The method for silencing the GhDWARF27 gene in recipient plants according to the present invention preferably includes introducing the virus-mediated gene silencing system containing the TRV2 vector described in the above-described technical solution into the recipient plant to achieve the purpose of silencing the GhDWARF27 gene; the TRV2 vector contains a nucleic acid molecule that silences the GhDWARF27 gene; the characteristics of the nucleic acid molecule that silences the GhDWARF27 gene have been defined in the above-described technical solution and will not be repeated here. The present invention does not specifically limit the transgenic methods used in the cultivation of the transgenic plants; conventional transgenic methods and steps in the art can be used.

[0079] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0080] Example 1

[0081] The verification steps for the role of the GhDWARF27 gene in regulating plant resistance to Verticillium wilt are as follows:

[0082] 1. Experimental Materials

[0083] 1.1 Plant materials

[0084] The plant materials used in this embodiment are upland cotton and Arabidopsis thaliana. The upland cotton materials include: Verticillium wilt susceptible varieties Xinluzao 36 (XLZ-36) and Jimian 11 (JM-11), Verticillium wilt resistant varieties Jimian 958 (JM-958) and Zhongzhimian 2 (ZZM-2), genetic standard line TM-1, and transgenic cotton overexpressing GhDWARF27 obtained by cotton embryo tip transformation technology.

[0085] 1.2 Strains and Vectors

[0086] Escherichia coli DH5α and Agrobacterium GV3101 were purchased from Shanghai Weidi Company. The cotton deciduous wilt strain (D) is Verticillium dahliae strain Vd_086 (Vd_086), a known and publicly available strain preserved in our laboratory.

[0087] The vectors used include pTRV1, pTRV2, pTRV2:CLA1, and pCAMBIA1300, all of which are publicly known vectors stored in our laboratory and can be purchased through commercial channels. The cotton overexpression vector pCAMBIA3300 was provided by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0088] 2. Experimental Methods

[0089] 2.1 Planting of Plant Materials

[0090] Cotton seeds were delinted with concentrated sulfuric acid and soaked in water overnight. The seeds were then sown in a soil substrate (peat:vermiculite (v / v) = 2:1) and transferred to a growth chamber for cultivation. Cultivation conditions were: 0.5m high fluorescent lamp stand, relative humidity 68-78%, light intensity approximately 10000 lx, temperature 28℃ / 25℃, and a photoperiod of 16h / 8h light / dark. Arabidopsis seeds were washed with 75% ethanol solution for 90s, rinsed three times with sterile distilled water, sterilized with 2% NaClO for 10min, rinsed three times with sterile distilled water, and then sown either by spot sowing in a soil substrate (peat:vermiculite = 1:1) or broadcast onto 1 / 2 MS solid medium. After vernalization at 4℃ for 48h, they were transferred to a growth chamber for further growth. The cultivation conditions were: 0.5m high fluorescent lamp stand, relative humidity 68-78%, light intensity of about 10000lx, temperature 22℃ / 20℃, and light / dark cycle of 16h / 8h.

[0091] 2.2 Activation and culture of Verticillium wilt

[0092] The *Verticillium dahliae* Vd_086 strain preserved in the laboratory was removed and thawed. After blotting the surface liquid with sterile filter paper, it was placed in the center of PDA medium and incubated upside down at 25°C in the dark for 7–14 days. Highly active mycelia were then transferred to Czapek's medium and cultured with shaking at 25°C for 7 days. Mycelial pellets in the culture medium were filtered through gauze, and spore concentration was calculated using a hemocytometer. Finally, the spore concentration was adjusted to 1 × 10⁻⁶ using sterile distilled water. 7 cells / mL and 5×10 3 Available at 1 / mL. The relevant culture medium formulation is as follows:

[0093] Culture medium formulation:

[0094] PDA medium: 200g potato, 20g glucose, 10g agar, ddH2O to a final volume of 1000mL.

[0095] Czapek's culture medium: Sodium nitrate 2.0g, potassium dihydrogen phosphate 0.7g, dipotassium hydrogen phosphate 0.3g, potassium chloride 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, sucrose 30.0g, Tween 0.5g, ddH2O to a final volume of 1000mL.

[0096] 1 / 2MS medium: MS Basal Salt Mixture 2.17g, Murashige and Skoog Vitamin Powder 0.5mL, Inositol 50mg, Agar 8g, ddH2O to a final volume of 1000ml, pH adjusted to 5.8.

[0097] LB medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, ddH2O to a final volume of 1000mL.

[0098] LBA medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 10g agar, and ddH2O to a final volume of 1000mL.

[0099] YEP culture medium: 10g yeast extract, 10g tryptone, 5g sodium chloride, ddH2O to a final volume of 1000mL.

[0100] YEB medium: 10g yeast extract, 10g tryptone, 5g sodium chloride, 10g agar, and ddH2O to a final volume of 1000mL.

[0101] Transformation Buffer for Arabidopsis thaliana: 25 mL MS macro (20×), 0.5 mL MS or B5 micro (1000×), 5 mL MS organic (200×), 5 mL MS iron salt (200×), 50 g sucrose, 10 μ L 6-BA (1 mg / mL), 400 μ L SILWET-77, bring the volume to 1000 mL with ddH2O, and adjust the pH to 5.8.

[0102] 2.3 Inoculation treatment of cotton seedlings with Verticillium wilt

[0103] Stop watering cotton seedlings 3 days before inoculation. When the cotton seedlings have grown to two true leaves and one central leaf, select seedlings of uniform growth and inoculate them with Verticillium wilt using the root-dip method. During inoculation, gently remove the cotton seedlings from the soil, avoiding root damage as much as possible, and slowly rinse the soil substrate attached to the roots with clean water. Dilute the prepared spore concentration to 1×10⁻⁶. 7 For inoculation, the spores were soaked in a solution of 5 mL / mL for 3 minutes, then replanted in pots, and 5 mL of the spore suspension was added to the soil. In the control group, sterile distilled water was used instead of Verticillium dahliae Vd_086 spore suspension for inoculation of cotton seedlings. After inoculation with Verticillium wilt, the cotton seedlings were transferred to a culture room with the following conditions: 0.5 m high fluorescent lamp stand, relative humidity 68-78%, light intensity approximately 10000 lx, temperature 28℃ / 25℃, and a light / dark cycle of 16 h / 8 h. During this period, all plants were irrigated with an equal volume of sterile distilled water.

[0104] 2.4 DNA and RNA extraction

[0105] 0.1g of fresh cotton root tissue sample was ground into powder using liquid nitrogen and then genomic DNA was extracted using the CTAB method. 0.1g of fresh Arabidopsis thaliana tissue was ground and then genomic DNA was extracted using a high-efficiency plant genomic DNA extraction kit (Tiangen Biotech, DP350). 0.1g of fresh cotton root tissue sample was ground and then total RNA was extracted using the RNAprep pure plant total RNA extraction kit (Tiangen Biotech, DP432). The specific procedures for DNA and RNA extraction are as follows:

[0106] DNA extraction using CTAB method

[0107] 1. Transfer 0.1-0.2g of vigorously growing cotton leaves into a 2mL centrifuge tube, add liquid nitrogen and grind into powder. Then transfer to another 2mL centrifuge tube and add 600μL of pre-chilled CTAB extraction buffer (81.8g sodium chloride, 100mL Tris-HCl, 40mL EDTA, 20g CTAB, 20g PVP, and ddH2O to a final volume of 1000mL). Heat the sample in a 65℃ water bath for 40 minutes, inverting it every 15 minutes.

[0108] 2. Add 700 μL of chloroform:isoamyl alcohol solution in a ratio of 24:1 to the centrifuge tube and centrifuge at 12000 rpm for 10 min.

[0109] 3. After centrifugation, discard the precipitate, add 400 μL of isopropanol to the centrifuge tube, and invert until a precipitate appears.

[0110] 4. Centrifuge at 12000 rpm for 5 min, discard the supernatant, and wash the precipitate with 700 μL of 75% ethanol.

[0111] 5. Centrifuge at 12000 rpm for 5 min, collect the precipitate, let it dry naturally, and then store it in TE buffer.

[0112] Rapid Plant Genomic DNA Extraction System

[0113] 1. Take 100 mg of fresh plant tissue, quickly add it to liquid nitrogen, and grind it thoroughly into powder. Add 400 μL of buffer FP1 and 6 μL of LNase A to the tube, shake for 1 min at room temperature, and let stand for 10 min.

[0114] 2. After standing, add 130 μL of FP2 buffer and shake for 1 min.

[0115] 3. Centrifuge at 12000 rpm for 5 min and retain the supernatant.

[0116] 4. Repeat step 3.

[0117] 5. Add 0.7 times the volume of isopropanol to the supernatant and mix well. Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0118] 6. Add 600 μL of 70% ethanol, centrifuge at 12000 rpm for 2 min, and collect the supernatant. Repeat this step once.

[0119] 7. Invert and let stand at room temperature for 5-10 minutes.

[0120] 8. After the ethanol has completely evaporated, add an appropriate amount of TE elution buffer and heat in a 65°C water bath to dissolve the DNA. The OD of the resulting DNA solution is... 260 / OD 280 The ratio should be between 1.7 and 1.9.

[0121] RNA extraction

[0122] (RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit)

[0123] 1. Homogenization: First, prepare 500 μL of lysis buffer SL. After the material is rapidly ground into powder in liquid nitrogen, add it to the lysis buffer and immediately vortex vigorously to mix it. Let it stand for 15 min.

[0124] 2. Centrifuge at 12000 rpm for 2 min.

[0125] 3. Transfer the supernatant to the CS filter column and centrifuge at 12000 rpm for 2 min.

[0126] 4. Add 0.4 times the volume of anhydrous ethanol to the supernatant and transfer it to the CR3 adsorption column. Centrifuge at 12000 rpm for 15 seconds and discard the waste liquid.

[0127] 5. Add 350 μL of protein removal solution RW1 to the adsorption column, centrifuge at 12000 rpm for 15 s, and remove the waste liquid.

[0128] 6. Add 500 μL of bleaching solution RW (check before use if anhydrous ethanol has been added) to the adsorption column CR3, centrifuge at 12000 rpm for 15 s, and remove the waste liquid.

[0129] 7. Repeat step 9.

[0130] 8. Centrifuge at 12000 rpm for 2 min, add 50 μL of Nase-Free ddH2O to the middle of the adsorption column, and let stand at room temperature for 5 min. Centrifuge at 12000 rpm for 1 min to collect RNA.

[0131] 9. Measure RNA concentration to ensure OD 260 / OD 280Readings between 1.8 and 2.1, with a ratio of 2.0 indicating high-quality RNA.

[0132] 2.5 RNA reverse transcription and real-time quantitative PCR

[0133] The first strand of cDNA was synthesized using the Tiangen FastKing cDNA first-strand synthesis kit. The mixing solution was accurately prepared according to the genomic DNA removal system shown in Table 1, and thorough mixing was ensured.

[0134] Table 1 Genomic DNA Removal System

[0135] Components Usage 5×gDNABuffer 2μL TotalRNA 50ng-2μg Sterile ultrapure water Add to 10μL

[0136] After centrifuging the mixture, incubate it at 42°C for 3 minutes. Then store it on ice. The reverse transcription system is shown in Table 2.

[0137] Table 2 Reverse Transcription System

[0138] reagents volume FastKingRTEnzymeMix 1μL PrimerMix 1μL 10×RTBuffer 2μL Sterile ultrapure water Add to 10μL

[0139] Add this mixture to the reaction solution for the genomic DNA removal step and mix well. Incubate at 42°C for 15 min, then at 95°C for 3 min, and store on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0140] qRT-PCR was performed using the SYBR kit (Yisheng Biotechnology Co., Ltd., China) and the Roche LightCycler 96 real-time PCR instrument. The expression levels of all target genes were determined by 2... -ΔΔCT Methodological calculations were performed, and the cotton GhUBQ7 gene was selected as the internal reference gene for qRT-PCR. All quantitative PCR analyses in this experiment were performed at least three biological replicates. The primer sequences for this invention are shown in Table 3, the qRT-PCR reaction system is shown in Table 4, and the qRT-PCR amplification program is shown in Table 5.

[0141] Table 3 Primer sequences used in Example 1

[0142]

[0143]

[0144] Table 4 qRT-PCR reaction system

[0145] Components Volume (μL) Final concentration SYBRMasterMix 10 1× Upstream primer (10 μM) 0.4 0.2μM Downstream primer (10 μM) 0.4 0.2μM Template DNA 1 - Sterile ultrapure water 8.2 -

[0146] Table 5 qRT-PCR amplification program

[0147]

[0148] 2.6 Searching and identification of DWARF27 in different plant genomes

[0149] The DWARF27 gene for different plant species, including cacao (Theobroma cacao), grape (Vitisvinifera), soybean (Glycine max), poplar (Populus trichocarpa), Selaginella moellendorffii, Physcomitrellapatens, Zostera marina, European spruce (Picea abies), sago pine (Pinus taeda Linn), rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and foxtail (Setaria italica), can be downloaded from the Phytozome website (http: / / www.phytozome.net). Genome data for upland cotton (G. hirsutum), Gossypium raimondii, and Gossypium arboreum can also be downloaded. The gene AtDWARF27 (AT1G03055), which is related to strigolactone synthesis and was previously reported on the TAIR website (http: / / www.arabidopsis.org), was used as a probe in a local cotton genome database for BLAST analysis. The preliminary sequence obtained was then used as a candidate sequence for further analysis.

[0150] 2.7 Sequence Analysis

[0151] Protein sequences of upland cotton, Arabidopsis thaliana, rice, cocoa, and grape were retrieved from the NCBI database and aligned using the online multiple sequence DNAMAN software to generate corresponding graphs. Phylogenetic trees were constructed using MEGA5 software (Tamura et al., 2011) using the neighbor-joining (NJ) method. The reliability of the phylogenetic trees was assessed using the bootstrap method, with a repeatability of 1000. Nucleotide sequences from the start codon ATG to 2000 bp upstream of the upland cotton gene were extracted from the Cottongene database (https: / / www.cottongen.org / ), and cis-acting elements were predicted using the PlantCARE database.

[0152] 2.8 Transcriptome Sequencing

[0153] Cotton seedlings TM-1 were treated with sterile distilled water (control group) or 20 μM strigolactone (rac-GR24, molecular formula C). 17 H 14 Pretreatment was performed using O5. Cotton seedlings pretreated for 7 days were inoculated with Verticillium wilt. Root samples were taken from GR24 pretreated and distilled water control seedlings at 0, 6, 12, and 24 h post-inoculation for transcriptome sequencing. Three biological replicates were performed. At 24 hpi, three root biological replicates were collected from each of the TRV:DWARF27 and TRV:00 cotton seedlings JM-958 for transcriptome analysis. Transcriptome sequencing was performed by Beijing Novogene (Tianjin, China). Transcriptome analysis methods followed those of Li et al. (2019a). The ggplot2 (Wickham, 2016) R package was used for GO and KEGG enrichment bubble plots. The upland cotton TM-1 genome was used as a reference genome (Zhang et al., 2015).

[0154] 2.9 Construction of Virus-Mediated Gene Silenting (VIGS) System

[0155] Primers were designed using Premier 6.0 software. Using cDNA from *Cotton Upland TM-1* as a template, the GhDWARF27 gene fragment was amplified using primers containing restriction endonuclease sites. The high-fidelity enzyme KOD One was used. TM PCR Master Mix-Blue (TOYOBO) was used for amplification. The reaction system is shown in Table 6, and the PCR reaction procedure is shown in Table 7.

[0156] Table 6 Reaction System

[0157] Components Volume (μL) <![CDATA[KODOne TM PCRMasterMix-Blue]]> 25.0 Upstream primer (10 μM) 1.5 Downstream primer (10 μM) 1.5 cDNA 2.5 <![CDATA[Add ddH2O to the final volume]]> 50.0

[0158] Table 7 PCR reaction procedure

[0159]

[0160] The amplified PCR products were recovered using a DNA gel extraction kit (Jinsha Biotechnology, Beijing). The specific experimental method is as follows:

[0161] DNA gel recovery:

[0162] 1. Under a 365nm long-wave ultraviolet lamp, quickly remove the target DNA band with a clean blade, trying to avoid including non-target DNA bands to minimize the gel volume (note that the operation should be quick to reduce DNA damage due to prolonged ultraviolet irradiation).

[0163] 2. Transfer the cut gel block containing the target DNA to a 2mL centrifuge tube and add 500μL of Buffer GN (if the gel block is too large, add Buffer GN until the solution is pale yellow).

[0164] 3. Heat at 65℃ for 10-15 minutes until the gel is completely melted.

[0165] 4. Transfer the molten solution into the adsorption column, centrifuge at 12000 rpm for 1 min, and then discard the waste liquid.

[0166] 5. Before use, check that the appropriate volume of anhydrous ethanol has been added to Buffer W1. Add 600 μL of Buffer W1 to the adsorption column and centrifuge at 12,000 rpm for 1 min.

[0167] 6. Repeat step 5.

[0168] 7. After discarding the solution in the collection tube, centrifuge at 12000 rpm for 2 min.

[0169] 8. Remove the adsorption column and place it in a clean 1.5 mL centrifuge tube. Incubate at room temperature for 2 min. Add 35–50 μL of preheated Elution Buffer (60–65 °C), incubate for 2 min, and then centrifuge at 12,000 rpm for 2 min. If more DNA is needed, the collected eluent can be added back to the adsorption column and centrifuged for another 2 min to maximize DNA recovery.

[0170] The PCR product was cloned into the pTRV2 vector using the homologous recombination method, with EcoRI and KpnRI selected as double restriction sites. The double restriction system is shown in Table 8, and the homologous recombination system is shown in Table 9.

[0171] Table 8 Double enzyme digestion system

[0172] Components volume 10×QuickCutBuffer 1μL DNA 1μg Restriction endonuclease 1 1μL Restriction endonuclease 2 1μL <![CDATA[ddH2O]]> to 10μL

[0173] Table 9 Homologous Recombination System

[0174] Components Recombination reaction / μL Linearized carrier X Insert fragment Y 5×CEⅡBuffer 4 Exnase II 2 <![CDATA[ddH2O]]> to20

[0175] The cotton CLA1 gene was used as a positive control for VIGS and was obtained by constructing the TRV2 vector using primers: GhCLAl-F: 5'-attctgtgagtaaggtt accgaattcATTCCACAACATCGATGATTTAG-3' (SEQ ID NO.61); GhCLAl-R: 5'-gcctcgagacgcgtgagctcggtaccGTAC CATGATGAGTAGATTGCAC-3' (SEQ ID NO.62). Each construct was independently transformed into *E. coli* DH5α cells and validated by plasmid DNA sequencing. The sequenced-validated vectors were then transformed into *Agrobacterium* strain GV3101 by heat shock, validated by colony PCR, and stored at -80°C until use. The specific transformation methods for *E. coli* DH5α and *Agrobacterium* GV3101 competent cells are as follows:

[0176] Transformed competent cells DH5α:

[0177] 1. Thaw DH5α competent cells on ice for 8-10 minutes.

[0178] 2. Add 10 μL of the recombinant product to 100 μL of competent cells, then gently tap the bottom of the tube to ensure thorough mixing. Incubate on ice for 30 min.

[0179] 3. Heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 2 minutes.

[0180] 4. Add 900 μL of LB medium without any antibiotics and incubate at 37°C with shaking (200-250 rpm) for 1 hour (competent cells do not need to be handled on ice at this time).

[0181] 5. Centrifuge at 5000 rpm for 5 min and discard the supernatant. Resuspend the bacteria and spread them on plates containing kanamycin resistance.

[0182] 6. Incubate at 37℃ for 12-16 hours. Once colonies with a diameter of 1 mm are visible, pick bacteria onto LB medium containing the appropriate antibiotic, incubate at 37℃ with shaking at 200 rpm, and then send for sequencing. Select the correct sequencing vector for the next step of the experiment.

[0183] Transformation of competent Agrobacterium GV3101:

[0184] 1. Remove the competent Agrobacterium cells from the -80℃ ultra-low temperature freezer. When they have thawed to a mixture of ice and water at room temperature, quickly insert them into ice for storage.

[0185] 2. Add 0.5 μg of plasmid DNA to 50 μL of competent cells and gently tap the bottom of the tube to mix thoroughly. Then, place the mixture on ice, in liquid nitrogen, in a 37°C water bath, and in an ice bath for 5 min in sequence.

[0186] 3. Add 700 μL of antibiotic-free culture medium and incubate at 28°C with shaking for 2-3 hours.

[0187] 4. After shaking culture, centrifuge at 6000 rpm for 1 min, and retain approximately 100 μL of supernatant to spread onto YEP plates containing the appropriate antibiotic. Invert the plates and incubate at 28°C for 2-3 days (the centrifugation step can be omitted, as the bacteria on the plate will become mushy after centrifugation. Usually, after shaking culture, simply aspirate 60 μL and spread it directly onto a plate containing the appropriate antibiotic).

[0188] 5. After picking and shaking culture, perform bacterial PCR verification. When the bacterial culture becomes turbid, preserve the bacteria (mix with 30% glycerol at a 1:1 ratio and place in a -80℃ refrigerator).

[0189] Agrobacterium tumefaciens culture with the desired carrier was added to antibiotic selection liquid medium containing rifampin and kanamycin and incubated overnight at 28°C. Transformed Agrobacterium cells were collected and resuspended in infiltration medium (10 mM MgCl2, 10 mM MES, and 200 mM acetylsalicylic acid), and then OD200 was calculated. 600 The value was adjusted to 1. After standing in the dark at 28℃ for 2-3 hours, Agrobacterium strains containing pTRV1 and pTRV2, pTRV1 and pTRV2:CLA1, and pTRV1 and pTRV2:DWARF27 vectors were mixed at a 1:1 ratio. After mixing thoroughly by inverting, the Agrobacterium suspension was injected into the cotyledons of 7-day-old TM-1 seedlings using a sterile needle. After culturing in the dark for 24 hours, the cotton seedlings were transferred to a growth chamber at 25℃ and subjected to a 16-hour light / 8-hour dark cycle. The pTRV2 vector was used as a negative control. pTRV2:CLA1 was used as a positive control to verify the silencing effect of the VIGS system. At least 32 cotton seedlings were used each time, and all VIGS experiments were performed in triplicate.

[0190] 2.10 Obtaining transgenic cotton with overexpression of GhDWARF27

[0191] The transgenic cotton was obtained by WIMI Biotechnology (Jiangsu) Co., Ltd. using the embryo tip transformation system. The vector used was the pCAMBIA3300 vector provided by WIMI. An overexpression vector was constructed using the linearized vector backbone and the target fragment (primers for amplifying the target fragment were OE-GhDWARF27-F and OE-GhDWARF27-R, Table 3) through homologous recombination, and transformed into *E. coli* DH5α. The correctly sequenced vector was transformed into *Agrobacterium* EHA105 and identified. Cotton embryo tip material was soaked in 50 mL of *Agrobacterium* suspension for 1 day and incubated at room temperature for 30 min for infection. The explants were then placed in a co-culture medium and incubated at 23°C in the dark for 3 days. Afterward, the embryos were transferred to resting medium and cultured under light at 28°C for 7 days, then transferred to resistance selection medium for 6 weeks, and then transferred to elongation medium for 3-6 weeks under light at 28°C and 5000 lx until rooting. After hardening off, cotton plants were transplanted into seedling trays. Genetic testing was performed on T0 plants to verify their positivity. The obtained positive plants were propagated and identified in greenhouses at Zhejiang University's Huajiachi Campus and Hainan Research Institute to obtain positive T3 plants for subsequent experiments. Table 3 provides the primers used for vector construction and PCR screening.

[0192] 2.11 Phenotypic observation and disease index statistics after inoculation

[0193] Resistance to Verticillium wilt in cotton was investigated using the criteria outlined in Xu et al., 2012: Grade 0: no obvious signs of wilting or yellowing; Grade 1: one or two cotyledons withered or fell off; Grade 2: one true leaf withered or fell off; Grade 3: two true leaves withered or fell off; Grade 4: all true leaves fell off or the plant died. Symptoms of the disease caused by Verticillium wilt were recorded 3–33 days after inoculation. The disease index was calculated using the following formula: Disease Index = [(ΣDisease Grade × Number of Diseased Plants) / (Total Number of Plants Assessed × 4)] × 100. Each treatment group had at least 32 plants, and all experiments were repeated three times.

[0194] Resistance to Verticillium wilt in Arabidopsis thaliana was investigated using the criteria outlined in (Song et al., 2021), classifying disease incidence into five grades: Grade 0: healthy plants, no symptoms; Grade 1: less than 25% of leaves infected; Grade 2: 25%-50% of leaves infected; Grade 3: 50%-75% of leaves infected; Grade 4: more than 75% of leaves infected. Symptoms of Verticillium wilt were recorded 21 days after inoculation, and the disease index was calculated. The disease index was calculated using the formula described above. Each treatment had at least 16 T3 plants, with three replicates.

[0195] 2.12 Recovery culture of Verticillium wilt

[0196] Samples were randomly selected from cotton plants inoculated with Verticillium wilt 21 days prior. Stem segments from the top 1 cm of the cotyledonary node were used as material for Verticillium wilt recovery culture. The obtained material was sterilized by immersion in 70% ethanol for 30 seconds, followed by rinsing three times with sterile distilled water. Then, it was soaked in 5% NaClO for 20 minutes and rinsed 5-6 times with sterile water. The cotton stem segments were transversely cut into uniform pieces using a sterile blade and placed on PDA medium. After incubation at 25℃ for 7 days, mycelial growth was observed, data were recorded, and photographs were taken.

[0197] 2.13 DAB staining

[0198] Weigh out DAB powder and dissolve it in sterile distilled water to prepare a 1 mg / mL solution, adjusting the pH to 3.8. For staining, immerse the leaves in the staining solution and store at 28°C in the dark for 8-12 hours. Then, boil the leaves in 95% ethanol for 20 minutes and in 50% ethanol for 10 minutes to remove chlorophyll. Finally, soak the leaves in 70% glycerol, observe under a microscope, and photograph the results.

[0199] 2.14 Trypan blue staining

[0200] The leaves to be stained were immersed in the staining solution (10 mL 85% lactic acid, 10 g water-saturated phenol, 10 mL glycerol, 10 mL ddH2O and 10 mg trypan blue dye), and the staining solution containing the leaves was placed in a boiling water bath for 5 min. After naturally cooling to room temperature, the staining solution was discarded, and the leaves were decolorized with 2.5 g / mL chloral hydrate. The leaves were observed under a microscope and photographed for record-keeping.

[0201] 2.15 Assay of antioxidant enzyme activity

[0202] Peroxidase (POD) kit, superoxide dismutase (SOD) assay kit (WST-8 method), ascorbate peroxidase (APX) kit, catalase (CAT) kit (UV absorption method), and glutathione S-transferase (GST) kit were all purchased from Suzhou Keming Biotechnology Co., Ltd. The activity of each enzyme was determined according to the manufacturer's instructions. All experiments were performed in triplicate.

[0203] 2.16 Determination of Plant Hormone Content

[0204] Determination of ABA and JA content: Weigh approximately 0.2 g of sample, add 1 mL of 80% acetonitrile extract, extract overnight at 4°C, centrifuge at 10000 rpm for 10 min, extract the residue with 0.5 mL of 80% acetonitrile for 2 h, centrifuge, remove the supernatant, combine the two supernatants, blow with nitrogen at 40°C until no organic phase is present, add 0.5 mL of petroleum ether for extraction and decolorization three times, discard the upper ether phase, add 60 μL of citric acid to adjust the pH to 2.8, extract with ethyl acetate three times, combine the organic phases, blow with nitrogen to dryness, vortex with 0.5 mL of methanol to dissolve, filter with a syringe filter, and store at -20°C for testing.

[0205] Determination of strigolactone (SLs) content: 0.5 g of frozen root sample was ground in liquid nitrogen using a mortar and pestle, transferred to a 10 mL centrifuge tube, and 2 mL of 30% acetone aqueous solution was added. The mixture was vortexed thoroughly and centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant was discarded. The remaining insoluble residue was re-extracted with 2 mL of 60% acetone aqueous solution, centrifuged at 12000 rpm for 5 min at 4 °C, and the supernatant was collected and stored at -20 °C for analysis. The test solution was concentrated to dryness using a nitrogen blower, reconstituted with 200 μL of 50% methanol aqueous solution, and centrifuged by vortexing. The supernatant was then used for subsequent determination by high-performance liquid chromatography-mass spectrometry (HPLC-MS).

[0206] 3. Analysis of Experimental Results

[0207] 3.1 Identification and analysis of cotton SLs synthetic genes

[0208] 3.1.1 Sequence Analysis

[0209] Using the key Arabidopsis thaliana SLs synthesis gene AtDWARF27 (AT1G03055) as a probe, the genome data of upland cotton was retrieved, and the cotton GhDWARF27 gene was obtained. Sequence analysis revealed that the full-length CDS of the GhDWARF27 gene is 807 bp, encoding 268 amino acids. BLAST sequence analysis showed that GhDWARF27 shares sequence homology with DWARF27 proteins from other species, with 76.12% similarity to cocoa TcDWARF27, 59.33% similarity to grape VvDWARF27, 53.01% similarity to rice OsDWARF27, and 46.84% similarity to Arabidopsis thaliana AtDWARF27. Figure 1 As shown, the blue-covered sequences are the same amino acids.

[0210] The CDS and amino acid sequences of the GhDWAWRF27 gene are shown in SEQ ID NO. 63 and SEQ ID NO. 64, respectively:

[0211] Full length of CDS: 5'-ATGGAAGCCAAGGTAGTTTTACAAAGCAGGACCCCGACGGGGACGTCATCGCGGGGAGT AAATAAACAGCGATGCTCTCCGGTTCGGGCGGTGCTAGCGAGGCCGGCTGAAAGCGTAGTCGGAAGTGGAACGAAGGAGAGGTTGAGGTTGAAGTTAAAGCCTGCAGATAGCAAACGTGAGGTAGCACAGGACAGCAGTAGTGTCCATAATGATAACTGGTTTGATCTATGGGCTATTAATTATCTATCCCAGAGTTTACAAGCTGCAACAGGGGTAAAGAGCATGTTGAGTGGGTATGAGAGCTTGGTAGAGACAACAGCAATGATGTCCAAGAAATTTAATACAAAAACGCAACAGGAGCTTGTCATTCAAGTTCTTGACACCGCCATCCCCAAGCTCATTCTAAACATGATAAAAACGTTGTTACCCCAATGTCAATTCACAAGGGAATATTTCGCCGCCTTCACCACTGTGTTTTTTGCTTGGCTCATTGGACCTTCTGAGGTTCGGGAATCGGAGCTCAACGGAAGAAGAGAAAAGAATGTAGTGTATGTGAAAAAGTGCAGGTTCCTAGAGCAAAGCAACTGTGTAGGAATGTGCATCAACCTATGCAAGATGCCATCTCAAGCCTTTATTAAGGATTCCCTAGGAATGCCACTCAACATGGTTCCAAATTTTGATGATATGAGCTGCGAAATGATTTTCGGTCAGGATCCTCCAACACCAAATGATGATCCGGTGCTCAAGCAGCCTTGCTACAAATTATGTAGGGCAAACCAACAGCACACAGTGAAGAGCTCAGGCTAA-3'.

[0212] Amino acid sequence: MEAKVVLQSRTPTGTSSRGVNKQRCSPVRAVLARPAESVVGSGTKERLRLKPADSKREVA QDSSSVHNDNWFDLWAINYLSQSLQAATGVKSMLSGYESLVETTAMMSKKFNTKTQQELVIQVLDTAIPKLILN MIKTLLPQCQFTREYFAAFTTVFFAWLIGPSEVRESELNGRREKNVVYVKKCRFLEQSNCVGMCINLCKMPSQA FIKDSLGMPLNMVPNFDDMSCEMIFGQDPPTPNDDPVLKQPCYKLCRANQQHTVKSSG.

[0213] 3.1.2 Phylogenetic and gene expression pattern analysis of GhDWARF27

[0214] Phylogenetic analysis of DWARF27 homologous protein sequences from different species revealed that different types of DWARF27 clustered independently without overlap, indicating that DWARF27 is ubiquitous in the plant kingdom and is relatively conserved during evolution. Figure 2 To further validate the tissue expression pattern of GhDWAR F27, RNA-Seq data from various cotton tissues were downloaded from a public database (see Hu Y, Chen J, Fang L, et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton[J]. Nature Genetics, 2019, 51(4):739-748.) for tissue expression pattern analysis. The results showed that GhDWAR F27 was highly expressed in cotton roots and leaves, followed by stems, and expressed at lower levels in petals and sepals. Copies of subgroup A were also expressed in ovules and fibers. Figure 2 (B)

[0215] exist Figure 2Figure A represents the phylogenetic tree of GhDWARF27 and its homologs from other plant species. Dicotyledons include: Tc, *Theobroma cacao*; Vv, *Vitis vinifera*; Ga, *G. arcoretum*; Gr, *G. raimondii*; Ptr, *Populus trichocarpa*; Gh, *G. hirsutum*; Gm, *Glycine max*; At, *Arabidopsis thaliana*. Moss includes: Pp, *Physcomitrella patens*. Ferns include: Sm, *Selaginella moellendorffii*. Algae include: Zm, *Zostera marina*. Monocotyledons include: Os, Oryza sativa (rice); Bd, Brachypodium distachyon (sorghum); Sb, Sorghum bicolor (sorghum); Si, Setaria italica (foxtail grass). Gymnosperms include: Pa, Picea abies (European spruce); Pta, Pinus taeda Linn (sago palm). B represents the expression level of GhDWARF27 in different cotton tissues (roots, stems, leaves, petals, sepals, ovules, and fibers).

[0216] 3.1.3 Promoter sequence analysis of GhDWARF27

[0217] The promoter sequence of the GhDWARF27 gene was cloned from the DNA of cotton leaf tissue. The promoter sequence upstream of ATG, 1874 bp, was obtained. Figure 3 , Figure 3The start codon is underlined. Cis-regulatory element analysis of the promoter sequence was performed using Plantcare online software. The results showed that the promoter region contains various cis-regulatory elements involved in stress, development, and hormone responses (Table 10). Among the stress-related response elements were the low-temperature stress response element (LTR) and the anaerobic response element (ARE). The promoter region also contained various hormone response elements, including the abscisic acid response element (ABRE), the auxin response element (TGA-element), the methyl jasmonate response element (CGTCA-motif, TGACG-motif), the gibberellin response element (GARE-motif), and the salicylic acid response element (TCA-element). These results indicate that GhDWARF27 plays an important role in plant hormone responses and coping with different environmental stresses.

[0218] Table 10 GhDWARF27 Promoter Cission Actuators

[0219] name sequence Function ABRE CACGTG Abscisic acid response (ABA-responsiveness) ARE AAACCA Anaerobic reaction CGTCA-motif CGTCA Methyl jasmonic acid response (MeJA-responsiveness) GARE-motif TCTGTTG Gibberellin-responsive element GC-motif CCCCCG Anoxic-specific inducibility LTR CCGAAA Low-temperature responsiveness TCA-element CCATCTTTTT Salicylic acid response (SA-responsiveness) TGA-element AACGAC Auxin-responsive element TGACG-motif TGACG Methyl jasmonic acid response (MeJA-responsiveness)

[0220] 3.2 Silencing GhDWARF27 reduces the resistance of cotton plants to Verticillium wilt.

[0221] To further investigate the effects of endogenous gene silencing agents (SLs) on cotton Verticillium wilt resistance, this invention used the Verticillium wilt-resistant cotton variety JM-958 as material and employed virus-mediated gene silencing (VIGS) technology to silence GhDWARF27 to study its disease resistance function. When cotton seedlings reached the stage where both cotyledons were fully expanded, Agrobacterium tumefaciens strains TRV:00, TRV:CLA1, and TRV:DWARF27 were injected into the abaxial surface of the cotyledons. Negative and positive controls were TRV:00 and TRV:CLA1 plants, respectively. Fourteen days after injection, the albinism phenotype of the true leaves in TRV:CLA1 plants demonstrated that the VIGS system successfully suppressed the expression of the target gene. Figure 4 ).

[0222] Fourteen days after injecting Agrobacterium carrying the target gene GhDWARF27 fragment into the cotyledons of cotton seedlings... Figure 5 The results in study A showed that the expression level of TRV:DWARF27 was significantly lower than that of TRV:00, indicating that the GhDWARF27 gene had been successfully silenced. Measurements of endogenous stem cell line (SLs) content in the TRV:00 and TRV:DWARF27-silenced lines showed that the SL content in the TRV:DWARF27 root system was significantly reduced after GhDWARF27 silencing. Figure 5 (B)

[0223] Figure 5Table A shows the qRT-PCR analysis results of the root system GhDWARF27 of TRV:00 and TRV:DWARF27. GhUBQ7 was used as an internal reference gene. Table B shows the content of SLs in the roots of TRV:00 and TRV:DWARF27. A t-test was used; *P<0.05, **P<0.01. Data are presented as mean ± standard deviation, n=3.

[0224] When cotton seedlings reached the stage of two true leaves and one central leaf, TRV:00 and TRV:DWARF27 silenced plants were inoculated with Verticillium wilt. Results showed that the resistance of JM-958 to Verticillium wilt was weakened by the silencing of GhDWARF27. Extensive yellowing and wilting of leaves were observed in TRV:DWARF27 plants, while the negative control plants (TRV:00) showed milder Verticillium wilt symptoms. Stem dissection results showed that the degree of vascular bundle browning in TRV:DWARF27 plants was significantly higher than that in TRV:00 plants. Fungal recovery experiments also showed that the amount of fungal mycelium produced on the stems of the TRV:DWARF27 group was greater than that of the TRV:00 group. Figure 6 ); Figure 6 In section A, VIGS strain was pretreated with distilled water (control) and 20 μM GR24 at 21 dpi to show Verticillium wilt symptoms. Bar = 1 cm. Section B shows browning of the vascular bundles in the stems of cotton seedling A. Photographs were taken at 21 dpi. Bar = 2 mm. Section C shows the fungal recovery experiment on the stems of cotton seedling A. Stems from plants at 21 dpi were used. Bar = 1 cm.

[0225] Trypan blue staining revealed that the staining area of ​​the TRV:DWARF27 strain was larger than that of the TRV:00 strain, and silencing GhDWARF27 resulted in greater cell damage in cotton leaves. Figure 7 (where Bar = 1 cm). Disease index statistics showed that at 21 dpi, the disease indices of TRV:DWARF27 and TRV:00 strains were 50.5 and 33, respectively, with a highly significant difference. Figure 8 The data are presented as mean ± standard deviation (n = 32). These results indicate that inhibiting the endogenous SLs synthesis gene GhDWARF27 reduces cotton's resistance to Verticillium wilt.

[0226] To further verify the role of exogenous GR24 in Verticillium wilt resistance, the gene-silenced plants TRV:00 and TRV:DWARF27 used in the Verticillium wilt infection experiment were treated with distilled water and 20 μM GR24. Specifically, the TRV:00+GR24 group and the TRV:DWARF27+GR24 group were added before inoculation with Verticillium wilt to observe whether exogenous GR24 could alleviate the resistance of the GR24-silenced plants to Verticillium wilt. The results after inoculation with Verticillium wilt showed that 20 μM GR24 treatment enhanced the resistance of TRV:00 and TRV:DWARF27 plants to Verticillium wilt, specifically manifested in reduced leaf yellowing and wilting, reduced browning of stem vascular bundles, and reduced fungal accumulation in the stems. Figure 6 Trypan blue staining results showed that GR24 pretreatment alleviated the degree of leaf damage to some extent. Figure 7 At 21 dpi, the disease index of TRV:DWARF27 was significantly higher than that of TRV:00, while the disease index of the TRV:DWARF27+GR24 group was lower than that of TRV:DWARF27, but still higher than that of the TRV:00 group. These results indicate that external application of GR24 alleviated the reduced resistance to Verticillium wilt in silenced GhDWARF27 plants to some extent.

[0227] In conclusion, silencing GhDWARF27 reduced the resistance of cotton seedlings to Verticillium wilt, and external application of GR24 could alleviate the symptoms of Verticillium wilt in cotton to some extent.

[0228] 3.3 Transcriptome Analysis

[0229] To investigate the molecular mechanism by which SLs enhance cotton resistance to Verticillium wilt, root samples from TM-1 plants were collected at four different time points (0, 6, 12, and 24 hpi) after inoculation with Verticillium wilt using cotton seedlings pretreated with GR24 or distilled water. Transcriptome analysis was performed. Simultaneously, root transcriptome data from JM-958 TRV:00 and TRV:DWARF27 plants were compared 24 h after Verticillium wilt inoculation to understand the changes in cotton resistance to Verticillium wilt after downregulation of GhDWARF27 expression. The significance thresholds for differentially expressed genes (DEGs) were |log2FoldChange| > 1 and P-value < 0.05.

[0230] At each time point, the experimental groups treated with GR24 were compared with the control group treated with distilled water to identify differentially expressed genes. Specifically, four comparison groups were established: an H2O control group (H2O_0h, H2O_6h, H2O_12h, H2O_24h) ​​and a GR24 experimental group (GR24_0h, GR24_6h, GR24_12h, GR24_24h). VIGS silencing lines were divided into a control group (TRV:00) and an experimental group (TRV:DWARF27). After sequencing quality control, high-quality cleanbases of 85.03Gb and 79.79Gb were obtained in the H2O and GR24 groups, respectively, and high-quality cleanbases of 20.6Gb and 20.78Gb were obtained in the TRV:00 and TRV:DWARF27 groups, respectively. The Q30 base percentage of each sample was not less than 92.45% (Tables 11-12). The alignment rates of the upland cotton TM-1 reference genome in the H2O library were 96.39%-97.94%, and in the GR24 library, they were 96.73%-98.03%. The alignment rates in the TRV:00 library were 96.12%-96.75%, and in the TRV:DWARF27 library, they were 94.91%-96.46%. These results indicate that the transcriptome sequencing results are good and meet the requirements for subsequent experiments.

[0231] Table 11 Transcriptome sequencing results

[0232]

[0233]

[0234] Table 12 Basic Information of Matched Reads

[0235]

[0236]

[0237] Differential gene expression analysis at 0, 6, 12, and 24 hpi enriched a total of 2785 upregulated genes and 2417 downregulated genes, of which 665 genes were upregulated at 24 hpi. The highest number of differentially expressed genes were found at 0 hpi and 24 hpi, with 1413 and 1256 genes specifically expressed, respectively. Between TRV:00 and TRV:DWARF27, 1891 differentially expressed genes were identified, of which 1147 were upregulated and 744 were downregulated in TRV:DWARF27 compared to TRV:00. Figure 9 ); Figure 9A represents differentially expressed genes in plants treated with H2O and 20 μM GR24 at 0, 6, 12, and 24 hpi. B is a Venn diagram showing the differences in differentially expressed genes between plants treated with H2O and 20 μM GR24 at 0, 6, 12, and 24 hpi. Numbers indicate overlapping and non-overlapping differentially expressed genes between groups. C is a volcano plot of differentially expressed genes between TRV:00 and TRV:DWARF27 plants at 24 hpi.

[0238] To understand the function of these differentially expressed genes, this invention performed GO enrichment analysis on the upregulated genes in the roots of exogenous GR24 group plants and the downregulated genes in TRV:DWARF27 groups. The results are as follows: Figure 10 As shown, Go enrichment analysis was performed on differentially regulated genes (0, 6, 12, 24 hpi) in the GR24 group compared to the H2O group and differentially regulated genes (24 hpi) in the TRV:DWARF27 group compared to the TRV:00 group. Color depth is based on -log. 10 (P-value), where the size of the circle represents the number of genes.

[0239] Depend on Figure 10 It can be seen that differentially expressed genes are involved in multiple biological processes. Compared with the H2O control group, the GR24 group showed significant enrichment in the peroxidation system and hormone system, especially in the upregulated genes at 24 hpi. These genes were highly enriched in responses to JA and ABA, hormone-mediated signaling pathways, hormone biosynthesis pathways, and oxidative stress responses. Furthermore, these genes were also highly enriched in downregulated genes in the GhDWARF27 silent plants. The GO analysis results suggest that exogenous application of GR24 may enhance cotton resistance to Verticillium wilt through plant hormone pathways.

[0240] KEGG enrichment analysis of differentially expressed genes showed that the upregulated differentially expressed genes in the exogenous GR24 group compared to the control group were mainly enriched in pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis, ascorbic acid and aldehyde metabolism. However, after the GhDWARF27 gene was silenced, the downregulated differentially expressed genes in the TRV:DWARF27 group compared to the TRV:00 group were enriched in phenylpropanoid biosynthesis and terpene and polyketide metabolism. Based on the above analysis, it was found that most differentially expressed genes are mainly related to phenylpropanoid biosynthesis and plant hormone signal transduction. Figure 11 ), Figure 11 KEGG enrichment analysis was performed on differentially regulated genes (0, 6, 12, 24 hpi) in the exogenous GR24 group compared to the H2O group and differentially regulated genes (24 hpi) in the TRV:DWARF27 group compared to the TRV:00 group. Color depth was based on logarithmic values. 10(P-value), where the size of the circle represents the number of genes. As expected, genes involved in the SL biosynthetic pathway were upregulated by GR24 pretreatment and suppressed by GhDWARF27 silencing. Figure 12 ); Figure 12 The expression changes of genes involved in SLs biosynthesis induced by exogenous application of GR24 at 0, 6, 12, and 24 hpi or by silencing GhDWARF27 at 24 hpi were investigated. These results indicate that the SLs-mediated immune response to Verticillium wilt in cotton is associated with other hormones, particularly ABA and JA.

[0241] 3.4 Exogenous GR24 enhances Verticillium wilt resistance through ABA synthesis and signaling pathways

[0242] 3.4.1 Effects of exogenous GR24 and silencing GhDWARF27 on ABA synthesis and signal transduction in cotton

[0243] Based on GO and KEGG enrichment results, this invention analyzed in detail the effects of exogenous application of GR24 and silencing of GhDWARF27 on ABA and JA biosynthesis and signal transduction in cotton plants. This invention analyzed the changes in gene expression levels related to ABA biosynthesis and signal transduction pathways in transcriptome data to examine changes in gene expression levels during inoculation with Verticillium wilt in cotton seedlings. De novo ABA synthesis is achieved through a multi-step enzymatic reaction. The carotenoid zeaxanthin, as the starting material for ABA biosynthesis, is catalyzed by zeaxanthin epoxidase (ZEP) to produce violaxanthin. Violaxanthin can be further catalyzed by neoxanthin synthase (NSY) to neoxanthin. Subsequently, it is converted to xanthoxin under the action of 9-cis-epoxycarotenoid dioxygenase (NCED). Finally, flavonoids undergo a two-step oxidation process in the cytoplasm using xanthoxin dehydrogenase (ABA2) and abscisic-aldehyde oxidase (AAO3) to generate ABA. Figure 13 (Chen et al., 2020). In the resting state, ABA receptors PYR / PYLs / RCARs in the cytoplasm exist as dimers. When coupled with ABA, they bind to phosphatase PP2Cs in monomeric form. After the inhibition of protein kinase SnRK2s by PP2Cs is relieved, transcription factors, including ABF, can be phosphorylated, thereby activating downstream ABA-responsive genes ( ). Figure 13(Seo et al., 2002). Changes in the expression levels of genes related to ABA biosynthesis and signal transduction pathways in the GR24 treatment group and the H2O control group are shown in Figure B. Figure 13 The heatmap on the left shows the expression changes of related genes in the TRV:DWARF27 and TRV:00 groups, while the right side represents the changes in gene expression. According to transcriptomic data, although some genes showed no change or significant upregulation at certain time points, the expression of most genes encoding ABA biosynthetic enzymes (ZEP, NCEDs, ABA2, and AAO3) and genes in the ABA signaling pathway (PYR, PP2C, SnRK2s, and ABF) changed under exogenous GR24 application, particularly at 6 hpi and 24 hpi. This indicates that exogenous GR24 not only activates ABA biosynthesis but also ABA signaling. In contrast, the expression levels of these genes related to ABA biosynthesis and signaling pathways were significantly reduced in the TRV:DWARF27 group. Figure 13 ); Figure 13 Heatmap A shows the expression changes of ABA biosynthetic pathway genes in the GR24-treated and GhDWARF27-silenced groups at 0, 6, 12, and 24 hpi. Heatmap B shows the expression changes of ABA signaling pathway genes in the GR24-treated and GhDWARF27-silenced groups at 0, 6, 12, and 24 hpi. Different colors in the heatmaps represent log2 fold change values. Fold change is the FPKM ratio between the GR24-treated and H2O-treated samples (represented by four consecutive squares of 0, 6, 12, and 24); and the FPKM ratio between the TRV:DWARF27 group and the TRV:00 group (represented by a single square of 24).

[0244] 3.4.2 Effects of external application of GR24 and silencing of GhDWARF27 on the activity of antioxidant enzymes in cotton plants

[0245] Excessive ROS accumulation caused by highly pathogenic Verticillium dahliae is a major cause of yellowing and necrosis in cotton leaves. ABA, as an important stress response hormone, can enhance the antioxidant capacity of plants to resist stress by eliminating ROS bursts caused by harmful pathogen attacks (Turkan et al., 2017; Qiu et al., 2021; Wang et al., 2021). Therefore, this invention compared the activities of SOD, POD, CAT, APX, and GST enzymes in the roots of cotton seedlings treated with 20 μM GR24 or distilled water at 0, 12, and 24 hpi, as well as the related enzyme activities of VIGS-silenced plants TRV:00 and TRV:DWARF27 at 24 hpi. Figure 14As shown, the activities of SOD, POD, CAT, APX, and GST in the roots of plants treated with H2O or 20 μM GR24 (left side of the dashed line) at 0, 12, and 24 hpi, and the activities of TRV:00 and TRV:DWARF27 plants at 24 hpi (right side of the dashed line). A t-test was used; *P<0.05, **P<0.01, ns = no significant difference. Data are presented as mean ± standard deviation, n=3.

[0246] The results showed that the activities of SOD, POD, CAT, APX, and GST antioxidant enzymes in the exogenous 20 μM GR24 treatment group were generally higher than those in the distilled water control group at all three time points. Specifically, at 24 hpi, exogenous GR24 application increased the activities of the five antioxidant enzymes in the plant roots by 27.3%, 21.8%, 35.8%, 67.9%, and 15.8%, respectively, compared with the distilled water control group. However, the SOD, POD, CAT, APX, and GST enzyme activities in TRV:DWARF27 plants showed the opposite trend.

[0247] After infection with Verticillium wilt, DAB staining results showed that the leaves of the control group were more deeply stained than those of the 20 μM GR24 group, indicating that exogenous application of GR24 could reduce ROS accumulation caused by Verticillium wilt infection. Furthermore, the staining area of ​​the TRV:DWARF27 plant was larger than that of the TRV:00 plant, indicating that silencing GhDWARF27 resulted in greater ROS accumulation in the leaves and reduced resistance of cotton seedlings to Verticillium wilt. Comparison between the TRV:DWARF27+GR24 and TRV:DWARF27 groups revealed that exogenous application of GR24 could alleviate ROS accumulation caused by GhDWARF27 gene silencing to a certain extent, restoring resistance to Verticillium wilt. Figure 15 ),exist Figure 15 The image of the medium green leaf was taken before DAB staining, Bar = 1 cm.

[0248] 3.4.3 Effects of external application of GR24 and silencing of GhDWARF27 on the expression levels of antioxidant enzyme genes in cotton plants

[0249] like Figure 16 The results showed that the expression levels of SOD, POD, CAT, APX and GST antioxidant enzyme genes in the exogenous 20μM GR24 treatment group were significantly higher than those in the distilled water control group, while the expression levels of related antioxidant enzyme genes in the TRV:DWARF27 group were significantly lower than those in the TRV:00 group.

[0250] 3.4.4 Effects of external application of GR24 and silencing of GhDWARF27 on ABA content in cotton plant roots

[0251] The ABA content in the roots of cotton plants treated with external GR24 and silent GhDWARF27 was measured 24 hours after inoculation with Verticillium wilt. The results are shown in […]. Figure 17 ; Figure 17 In the table, A represents the ABA content in the roots of cotton seedlings treated with H2O or 20 μM GR24 for 24 hpi. B represents the ABA content in the roots of TRV:00 and TRV:DWARF27 plants. A t-test was used; *P<0.05, **P<0.01. Data are presented as mean ± standard deviation, n=3. It can be seen that the ABA content in the roots of cotton seedlings treated with exogenous GR24 was significantly higher than that in the distilled water control, while the ABA content in the roots of TRV:DWARF27 plants was significantly lower than that in TRV:00 plants. qRT-PCR results further validated the consistency between the relative expression levels of the transcriptome samples and the quantitative analysis results. Figure 18 ); Figure 18 The area to the left of the dashed line represents the roots of plants treated with H2O or 20 μM GR24; the area to the right of the dashed line represents the roots of TRV:00 and TRV:DWARF27 plants. A t-test was used, with *P < 0.05, **P < 0.01, and ns = no statistical significance. Data are presented as mean ± standard deviation, n = 3. The results of this invention indicate that exogenous application of GR24 increased the expression of ABA biosynthesis genes and root ABA content, enhanced the activity of antioxidant enzymes, thereby reducing the accumulation of excess ROS and improving cotton's resistance to Verticillium wilt. Silencing GhDWARF27, however, had the opposite effect.

[0252] 3.5 Exogenous GR24 enhances Verticillium wilt resistance through JA synthesis and signaling pathways

[0253] 3.5.1 Effects of exogenous GR24 and silencing GhDWARF27 on JA synthesis and signal transduction in cotton

[0254] Transcriptome data on JA biosynthesis and signal transduction pathways were analyzed to describe changes in the expression levels of root-related genes after inoculation with Verticillium wilt. JA and its structurally similar metabolites are produced via the oxidative lipid biosynthesis pathway (Hettenhausen et al., 2013). α-linolenic acid is a precursor in the synthesis of jasmonic acid-based plant hormones. α-linolenic acid is oxidized by lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) to form OPDA. Subsequently, OPD A is carried to the oxidosome and oxidized by 12-oxo-phytodienoic reductase (OPR3) to produce JA. Figure 19(Fragoso et al., 2014). In the signal transduction pathway, the key regulator of the JA signaling system, the receptor protein COIl (corona ine insensitive 1), binds to the transcriptional repressor protein JAZ. When JA-Ile binds to COI1, the JAZ repressor protein degrades and releases transcription factors such as MYC2, thereby activating the expression of downstream related response genes (Li et al., 2021b). Transcriptome data analysis revealed that although some genes did not change or were not significantly upregulated at certain time points, exogenous GR24 treatment led to the upregulation of most JA synthesis genes, including GhLOXs, GhAOS, GhAOCs, and GhOPR3, while the expression of these synthesis genes in TRV:DWARF27 was significantly downregulated. Figure 19 The expression levels of JA signaling pathway genes GhC OI1, GhJAZ, and GhMYC2 were upregulated by exogenous GR24, but downregulated in TRV:DWARF27. Figure 19 (B) In Figure 19 In the heatmap, A represents the expression of JA biosynthetic pathway genes in the GR24-treated and GhDWARF27-silenced groups at 0, 6, 12, and 24 hpi. B represents the expression of JA signaling pathway genes in the GR24-treated and GhDWARF27-silenced groups at 0, 6, 12, and 24 hpi. Different colors in the heatmap represent log2 fold change values, where fold change is the FPKM ratio between the GR24-treated and H2O-treated samples (represented by four consecutive squares of 0, 6, 12, and 24); and the FPKM ratio between the TRV:DWARF27 group and the TRV:00 group (represented by a single square of 24).

[0255] 3.5.2 Effects of external application of GR24 and silencing of GhDWARF27 on JA content in cotton plant roots

[0256] The results of the determination of JA content in the roots of each plant with externally applied GR24 and silenced GhDWARF27 are shown in the figure. Figure 20 ;exist Figure 20 In the table, A represents the JA content in the roots of plants inoculated with Verticillium wilt after 24 hours of treatment with H2O or 20 μM GR24. B represents the JA content in the roots of TRV:00 and TRV:DWARF27 plants. A t-test was used; *P<0.05, **P<0.01. Data are presented as mean ± standard deviation, n=3.

[0257] Depend on Figure 20 It can be seen that the JA content in the roots of cotton increased by 51.94% when GR24 was applied externally, while silencing GhDWARF27 reduced the JA content in the roots by 110%, both of which were highly significant.

[0258] 3.5.3 qRT-PCR validation of exogenous GR24 and silenced GhDWARF27 on JA pathway genes

[0259] To validate the transcriptome expression data of genes involved in JA biosynthesis and signaling pathways, the relative expression levels of each gene in the transcriptome samples were analyzed using qRT-PCR. The results are shown below. Figure 21 ; Figure 21 Plants to the left of the dashed line are those treated with H2O or 20 μM GR24; plants to the right of the dashed line are TRV:00 and TRV:DWARF27 plants. A t-test was used, with *P < 0.05 and **P < 0.01. Data are presented as mean ± standard deviation, n = 3.

[0260] Depend on Figure 21 It can be seen that the qRT-PCR results are highly consistent with the transcriptome data. Furthermore, the expression levels of JA response-related genes GhPR3, GhPR4, GhPDF1.2, and GhVSP2 were significantly upregulated in exogenous GR24 treatment, while they were significantly downregulated in GhDWARF27 silenced plants. Figure 22 );exist Figure 22 Plants treated with H2O or 20 μM GR24 are located to the left of the dashed line; plants treated with TRV:00 and TRV:DWARF27 are located to the right of the dashed line. A t-test was used, with *P < 0.05 and **P < 0.01. Data are presented as mean ± standard deviation, n = 3. It is evident that exogenous GR24 enhances cotton resistance to Verticillium wilt, possibly because GR24 activates JA biosynthesis and signal transduction pathways in cotton roots, increasing root JA content and enhancing the expression of downstream response-related genes GhPR3, GhPR4, GhPDF1.2, and GhVSP2, thereby enhancing cotton plant resistance to Verticillium wilt.

[0261] 3.5.4 qRT-PCR validation of SA pathway genes by exogenous GR24 and silencing GhDWARF27

[0262] The qRT-PCR results of SA content and SA response genes in the roots of cotton plants treated with GR24 and silenced with GhDWARF27 showed that the SA content in the roots of cotton seedlings did not change significantly after exogenous GR24 treatment and GhDWARF27 silencing. Quantitative results of SA-related response genes showed that only the GhPR1 gene expression was downregulated with the silencing of GhDWARF27, while other genes did not show significant changes after exogenous GR24 treatment and GhDWARF27 silencing. Figure 23 ); Figure 23In Figure A, the SA content in the roots of plants inoculated with Verticillium wilt after 24 hours of treatment with H2O or 20 μM GR24 is shown. Figure B shows the SA content in the roots of TRV:00 and TRV:DWARF27 plants. Figure C shows the qRT-PCR analysis of the SA response gene; plants to the left of the dashed line are treated with H2O or 20 μM GR24, and plants to the right of the dashed line are TRV:00 and TRV:DWARF27 plants. A t-test was used; *P < 0.05, **P < 0.01, ns = no significant difference. Data are presented as mean ± standard deviation, n = 3.

[0263] 3.6 Results of resistance identification of Verticillium wilt in GhDWARF27 overexpression transgenic cotton

[0264] 3.6.1 Identification of GhDWARF27 overexpression transgenic cotton

[0265] To further investigate the function of GhDWARF27 in cotton resistance to Verticillium wilt, this invention constructed an overexpression vector of the GhDWARF27 gene and transformed it into cotton plants TM-1, obtaining multiple independent transformants. PCR amplification of genomic DNA from the leaves of the overexpressing plants was performed using primers specific to the 35S promoter. Figure 24 No amplification product was found in the wild-type line shown in Figure A. The GhDWARF27 overexpression transgenic line amplified a band of approximately 850 bp. RNA was extracted from the root tissue of the transgenic lines, and the expression level of the GhDWARF27 gene was detected by qRT-PCR. The expression level of the GhDWARF27 gene in the transgenic lines was more than 100 times higher than that in the control. The three transgenic lines with the highest relative expression levels (L56, L58, and L68) were 1137, 1557, and 582 times higher than the wild-type, respectively. (See Figure A). Figure 24 B. In Figure 24 In the table, A represents the transgenic cotton lines overexpressing GhDWARF27 identified by PCR. B represents the relative expression level of GhDWARF27. A t-test was used; *P<0.05, **P<0.01. Data are presented as mean ± standard deviation, n=3.

[0266] 3.6.2 SLs content in GhDWARF27 overexpression transgenic cotton

[0267] The SLs content in cotton roots was measured using wild-type and the three transgenic lines with the highest relative expression levels. The SLs content in the roots of the overexpression materials was 105.96%, 68.05%, and 40.61% higher than that of the wild-type, respectively, with highly significant differences. Figure 25 ); Figure 25 The t-test was used, and *P<0.05; **P<0.01. Data are presented as mean ± standard deviation, n=3.

[0268] 3.6.3 Verticillium wilt resistance in GhDWARF27 overexpression transgenic cotton

[0269] Inoculation of T3 transgenic cotton plants with three stable high-expression transformants (L56, L58, and L68) with Verticillium wilt pathogen yielded the following results: Figure 26 As shown, Figure 26 In Figure A, the symptoms of Verticillium wilt in WT (TM-1) and transgenic plants (L58, L68, and L56) at 21 dpi. Bar = 1 cm. In Figure B, the degree of browning of the vascular bundles in the stems of WT and transgenic plants at 21 dpi. Bar = 2 mm. In Figure C, the fungal recovery results in the stems of WT and transgenic plants at 21 dpi. Bar = 2 mm. Figure 26 The results showed that the GhDWARF27 overexpression transgenic line had stronger resistance to Verticillium wilt, and the yellowing, wilting and leaf drop of seedling leaves were significantly lower than those of the control group. At 21 dpi, the degree of browning of the vascular bundles in the stem was lower, and there was less accumulation of fungal hyphae in the stem.

[0270] Disease index statistics showed that the incidence of disease in the GhDWARF27 overexpression material was lower than that in the wild-type material throughout the entire disease cycle. Specifically, the disease indices at 21 dpi for the wild-type and the three transgenic lines (L56, L58, and L68) were 70, 43, 30, and 41.25, respectively, with statistically significant differences. Figure 27 ); Figure 27 The data are the mean ± standard deviation, n = 32.

[0271] Furthermore, DAB and trypan blue staining results also indicated that the overexpression transgenic lines had less ROS accumulation in their leaves and less leaf cell damage than the control lines. Figure 28 ); Figure 28 Image A shows DAB staining of leaves from WT and transgenic plants at 21 dpi. Bar = 1 cm. Image B shows trypan blue staining of leaves from WT and transgenic plants inoculated with Verticillium wilt at 21 dpi. Bar = 1 cm.

[0272] The above results indicate that the expression level of GhDWARF27 in cotton is positively correlated with cotton's resistance to Verticillium wilt. This result is consistent with the resistance identification results of GR24 and VIGS silencing treatments, suggesting that SLs play a positive role in resisting cotton Verticillium wilt, whether applied externally or by increasing the content in the plant.

[0273] References:

[0274] Paplomatas EJ,Bassett DM,Broome JC,et al.Incidence ofVerticilliumwilt and yield losses ofcotton cultivars(Gossypium hirsutum)based on soilinoculum density of Verticillium dahliae[J].Phytopathology,1992,82(12):1417-1420.

[0275] Wang Hongmei. Occurrence and damage of cotton wilt and Verticillium wilt in China and the effectiveness of disease-resistant breeding [J]. Chinese Agricultural Science Bulletin, 2015, 31(15):124-130.

[0276] Chen B, Zhang Y, Yang J, et al. The G-proteinαsubunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation [J]. The Crop Journal, 2021a, 9(4): 823-833.

[0277] Li Xiuqing, Wang Qian, Hu Ziyao, et al. Functional analysis of GhMAPKKK2 gene in cotton resistance to Verticillium wilt [J]. Cotton Science, 2022, 34(1):1-11.

[0278] Li Y, Zhang

[0279] Guang H,Xiaoyang G,Zhian W,et al.The cotton MYB33 gene is a hub generegulating the trade-offbetween plant growth and defense inVerticilliumdahliae infection[J].Journal ofAdvancedResearch,2023.

[0280] Zhang T,Jin Y,Zhao J H,et al.Host-induced gene silencing of thetarget gene in fungal cells confers effective resistance to the cotton wiltdisease pathogen Verticillium dahliae[J].Molecular Plant,2016,9(6):939-942.

[0281] Wei C,Qin T,Li Y,et al.Host-induced gene silencing ofthe acetolactatesynthases VdILV2 and VdILV6 confers resistance to Verticillium wilt in cotton(Gossypium hirsutum L.)[J].Biochemical and Biophysical ResearchCommunications,2020,524(2):392-397.

[0282] Chen B,Tian J,Feng Z,et al.Chitin synthases containing myosin motor-like domain are required for cell wall integrity andvirulence ofvascularwiltpathogenVerticillium dahliae[J].Phytopathology Research,2023,5(1):21.

[0283] Zhang Y,Zhang Y,Ge X,et al.Genome-wide association analysis reveals anovel pathway mediated by a dual-TIR domain protein forpathogen resistance incotton[J].Genome Biology,2023b,24(1):111.

[0284] Li C,He Q,Zhang F,et al.Melatonin enhances cotton immunity toVerticillium wilt via manipulating lignin and gossypolbiosynthesis[J].ThePlant Journal,2019a,100(4):784-800.

[0285] WickhamH.Data analysis[M].Springer International Publishing,2016.

[0286] Zhang T,Hu Y,Jiang W,et al.Sequencing ofallotetraploid cotton(Gossypium hirsutum L.acc.TM-1)provides aresource for fiber improvement[J].Nature Biotechnology,2015,33(5):531-537.

[0287] Turkan I.(2017).Emerging roles for ROS andRNS-versatile moleculesinplants.Journal ofExperimental Botany,68,4413-4416.

[0288] Qiu D, Hu W, ZhouY, et al. TaASR1-D confers abiotic stress resistance by affecting ROS accumulation and ABA signaling in transgenic wheat [J]. PlantBiotechnology Journal, 2021, 19(8): 1588-1601.

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[0290] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Positive regulation GhDWARF27 The application of the gene in improving cotton resistance to Verticillium wilt and / or breeding Verticillium wilt-resistant cotton, GhDWARF27 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 64; The positive regulation refers to increasing the content of GhDWARF27 protein in the receptor cotton.

2. The application according to claim 1, characterized in that, The positive regulation refers to promoting the receptor cotton in cotton. GhDWARF27 Gene expression.

3. Positive regulation GhDWARF27 The application of genetically modified biological materials in improving cotton resistance to Verticillium wilt and / or breeding Verticillium wilt-resistant cotton, the aforementioned GhDWARF27 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 64; The biomaterials include any one or more of the following: 1) Contains GhDWARF27 Expression cassette of gene CDS sequence; 2) Contains GhDWARF27 Recombinant expression vectors of gene CDS sequences; 3) A recombinant expression vector containing the expression cassette described in 1); 4) Contains GhDWARF27 Engineered bacteria with CDS gene sequences; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3); The positive regulation refers to increasing the content of GhDWARF27 protein in the receptor cotton.

4. The application according to claim 3, characterized in that, The GhDWARF27 The nucleotide sequence of the gene CDS sequence is shown in SEQ ID NO.

63.

5. The application according to claim 3, characterized in that, The positive regulation refers to promoting the receptor cotton in cotton. GhDWARF27 Gene expression.

6. A method for cultivating transgenic cotton, characterized in that, The transgenic cotton includes Verticillium wilt-resistant cotton or Verticillium wilt-susceptible cotton models; the cultivation steps of the Verticillium wilt-resistant cotton include: increasing the content of GhDWARF27 protein in the recipient cotton to obtain the Verticillium wilt-resistant cotton; The cultivation steps of the Verticillium wilt-susceptible cotton model include: reducing the content of GhDWARF27 protein in the recipient cotton to obtain the Verticillium wilt-susceptible cotton model; The GhDWARF27 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 64.

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