Application of GhIQD1 gene in verticillium wilt resistance of plants

Abstract

The invention provides an application of a GhIQD1 gene in plant verticillium wilt resistance, and particularly provides an application of the GhIQD1 gene in regulation and control of plant verticillium wilt resistance. When the expression quantity of the GhIQD1 gene in the plant is relatively high, the verticillium wilt resistance of the plant is enhanced, and when the expression quantity of the GhIQD1 gene in the plant is relatively low or the GhIQD1 gene is not expressed, the verticillium wilt resistance of the plant is weakened. The new research finds that not only is the understanding of the plant immune system enriched, but also important guidance is provided for developing more effective disease-resistant measures and strategies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the application of the GhIQD1 gene in plant resistance to Verticillium wilt. Background Art

[0002] Cotton is one of the most important economic crops in the world, and its wide range of uses makes it a major source of income for farmers around the world. However, cotton production has always faced a serious problem, namely cotton Verticillium wilt. This disease is caused by a microorganism called Verticillium dahliae and is mainly spread through the soil. The incidence of cotton Verticillium wilt in cotton production is extremely high, and the degree of the disease is also very serious. This has brought huge losses to the yield and quality of cotton. Therefore, researchers have been working hard to explore and study cotton Verticillium wilt in order to find effective control methods to reduce the impact of the disease on cotton yield and quality.

[0003] Plant hormones also play a crucial role in plant immunity. They are internal signal molecules in plants that can regulate various growth and development processes and are also involved in the disease resistance defense mechanism of plants. Salicylic acid (SA) is a key hormone related to plant defense and plays an important role in systemic acquired resistance and broad-spectrum and durable disease resistance. Jasmonic acid (JA) plays a crucial role in the defense response of plants to necrotrophic and biotrophic fungal infections. These two pathways are mostly antagonistic, and the crosstalk balance between them affects the pathological outcome. Some studies have shown that the IQD family is involved in plant hormone regulation. SUN24 in the IQD gene family of tomatoes controls seed germination through the abscisic acid signaling pathway. The IQD gene family of moso bamboo shows a tissue-specific pattern and is also found to be involved in methyl jasmonate and drought stress. The IQD gene family of soybeans is also specifically expressed and also regulates methyl jasmonate stress.

[0004] IQD1 is a calmodulin-binding protein and is a member of the IQD gene family. It has been confirmed in Arabidopsis thaliana as a positive regulator of glucosinolate (GS) accumulation and plant defense responses to insects, and it has been demonstrated that the IQD1 overexpression line also has strong resistance to the necrotrophic fungus Botrytis cinerea. Further studies have shown that IQD1 and related proteins provide a scaffold for promoting the cellular transport of RNA along microtubule tracks, as a mechanism for controlling and fine-tuning gene expression and protein sorting, thus explaining the pleiotropic effects of IQD1 in many cellular pathways. There has been little research on the IQD1 gene in cotton, and currently there is no research on the IQD gene in cotton resistance to Verticillium wilt. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] To explore the relationship between IQD1 and plant resistance to Verticillium wilt, the inventors obtained the GhIQD1 gene sequence highly homologous to the Arabidopsis gene ATIQD1 through the CottonFGD website and verified its amplification. Then, through a series of experiments, the role of the GhIQD1 gene in cotton against V. dahliae was characterized. The research results showed that GhIQD1 was expressed in various tissues, with the highest expression level in the stem segments of Zhongzhimian 2 (ZM2) and the highest expression in the leaves of Xinluzao 36 (XL36), and the expression level increased significantly after infection with V. dahliae. Overexpression of the GhIQD1 gene in tobacco showed that these transgenic plants had improved resistance to V. dahliae. On the contrary, silencing the GhIQD1 gene in cotton by VIGS reduced the resistance to V. dahliae. Generally speaking, the present invention reveals the relationship between the GhIQD1 gene and cotton resistance to V. dahliae, providing important clues and information for further studying the adaptation mechanism of plants when resisting pathogen invasion.

[0007] Therefore, in one aspect of the present invention, the present invention proposes the use of the GhIQD1 gene in regulating plant resistance to Verticillium wilt. Through a large number of experiments, the inventors found that there is a special relationship between the GhIQD1 gene and plant resistance to Verticillium wilt, that is, when the expression level of the GhIQD1 gene in plants is relatively high, the effect of plant resistance to Verticillium wilt is enhanced, and when the expression level of the GhIQD1 gene in plants is relatively low or not expressed, the effect of plant resistance to Verticillium wilt is weakened. This new research finding not only enriches the understanding of the plant immune system but also provides important guidance for developing more effective disease resistance measures and strategies.

[0008] According to the embodiments of the present invention, the use may further include at least one of the following additional technical features:

[0009] According to the embodiments of the present invention, the protein expressed by the GhIQD1 gene has the amino acid sequence shown in SEQ ID NO:1.

[0010] According to the embodiments of the present invention, the plant is selected from cotton, Arabidopsis, rice, corn, wheat, sorghum, barley, oats, rye, soybeans, tobacco, rapeseed, tomatoes.

[0011] In the second aspect of the present invention, the present invention proposes the use of a reagent in improving plant resistance to Verticillium wilt, and the reagent is used to improve the activity or expression level of the GhIQD1 gene. As mentioned above, overexpression of the GhIQD1 gene can improve the effect of plant resistance to Verticillium wilt. Therefore, a reagent that can improve the activity or expression level of the GhIQD1 gene also has the effect of improving plant resistance to Verticillium wilt.

[0012] According to an embodiment of the present invention, the use may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the reagent includes jasmonic acid or a plasmid for overexpressing the GhIQD1 gene.

[0014] According to an embodiment of the present invention, the jasmonic acid is selected from methyl jasmonate.

[0015] According to an embodiment of the present invention, the plasmid has the nucleotide sequence shown in SEQ ID NO:2.

[0016] In the third aspect of the present invention, the present invention provides the use of jasmonic acid in increasing the expression level of the GhIQD1 gene in plants. The inventors found in experiments that by exogenously applying jasmonic acid, the expression level of the GhIQD1 gene in plants can be significantly increased, thereby effectively enhancing the resistance of plants to Verticillium wilt. This discovery provides a more effective and sustainable solution for developing new ways to control plant Verticillium wilt.

[0017] In the fourth aspect of the present invention, the present invention provides a method for preparing transgenic plants. According to an embodiment of the present invention, the method includes: transforming the plant to be constructed with Agrobacterium carrying the GhIQD1 gene. By using the method of the present invention, transgenic plants with high disease resistance can be successfully obtained, providing a new way to improve the resistance of plants to Verticillium wilt. By introducing the GhIQD1 gene into transgenic plants, the defense ability of plants against Verticillium wilt pathogens is effectively enhanced, providing new prospects and hopes for solving important disease problems such as Verticillium wilt.

[0018] According to an embodiment of the present invention, the method for preparing transgenic plants may further include at least one of the following additional technical features:

[0019] According to an embodiment of the present invention, the method further includes: culturing the transformed plant to be constructed to obtain the transgenic plant.

[0020] According to an embodiment of the present invention, the Agrobacterium carrying the GhIQD1 gene is obtained by introducing a plasmid carrying the GhIQD1 gene into the Agrobacterium to be transfected.

[0021] According to an embodiment of the present invention, the plasmid has the nucleotide sequence shown in SEQ ID NO:2.

[0022] According to an embodiment of the present invention, the plant is selected from cotton, Arabidopsis thaliana, rice, maize, wheat, sorghum, barley, oats, rye, soybean, tobacco, rapeseed, tomato.

[0023] In the fifth aspect of the present invention, a method for improving the Verticillium wilt resistance of plants is proposed. According to an embodiment of the present invention, the method includes: increasing the expression level of the GhIQD1 gene in the plant. As described above, overexpression of the GhIQD1 gene helps to improve the Verticillium wilt resistance of plants. Therefore, the method described in the present invention can effectively enhance the plant's defense ability against Verticillium wilt pathogens, providing new prospects and hopes for solving important disease problems such as Verticillium wilt.

[0024] According to an embodiment of the present invention, the method for preparing a plant with improved Verticillium wilt resistance may further include at least one of the following additional technical features:

[0025] According to an embodiment of the present invention, the expression level of the GhIQD1 gene in the plant is increased by the following methods: transforming the plant to be treated with Agrobacterium carrying the GhIQD1 gene; or spraying methyl jasmonate on the plant.

[0026] According to an embodiment of the present invention, the method further includes: further including: culturing the transformed plant to be treated to obtain the transgenic plant, thereby increasing the expression level of the GhIQD1 gene in the plant.

[0027] According to an embodiment of the present invention, the Agrobacterium carrying the GhIQD1 gene is obtained by introducing a plasmid carrying the GhIQD1 gene into the Agrobacterium to be transfected.

[0028] According to an embodiment of the present invention, the plasmid has the nucleotide sequence shown in SEQ ID NO:2.

[0029] According to an embodiment of the present invention, the methyl jasmonate is methyl jasmonate.

[0030] According to an embodiment of the present invention, the plant is selected from cotton, Arabidopsis thaliana, rice, corn, wheat, sorghum, barley, oats, rye, soybeans, tobacco, rapeseed, tomatoes.

[0031] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0033] Figure 1 is a multi-species protein alignment map of GhIQD1 according to an embodiment of the present invention;

[0034] Figure 2Expression levels of GhIQD1 in cotton according to embodiments of the present invention, where:

[0035] Figure 2 A is a graph showing the expression levels of GhIQD1 in the roots, stems, and leaves of cotton;

[0036] Figure 2 B is a graph showing the expression levels of GhIQD1 at 0 h, 3 h, 6 h, 12 h, 24 h, 72 h, and 120 h after infection with V. dahliae;

[0037] Figure 3 Results graph showing that overexpression of GhIQD1 according to embodiments of the present invention increases the resistance of tobacco to Verticillium dahliae, where:

[0038] Figure 3 A is a graph showing the results of PCR testing of transgenic lines;

[0039] Figure 3 B is a graph showing the expression levels of GhIQD1 in transgenic lines and wild type detected by RT-qPCR;

[0040] Figure 3 C is a phenotypic graph of transgenic tobacco 15 days after inoculation;

[0041] Figure 3 D is the disease index of transgenic tobacco 15 days after inoculation;

[0042] Figure 4 Results graph showing that silencing GhIQD1 in cotton according to embodiments of the present invention reduces the resistance of plants to Verticillium dahliae, where:

[0043] Figure 4 A is a phenotypic graph of albinism of TRV:GhCLA1 15 days after VIGS;

[0044] Figure 4 B is a graph showing the silencing efficiency of GhIQD1 detected by RT-qPCR;

[0045] Figure 4 C is a phenotypic graph of Verticillium wilt of TRV:00 plants and TRV:GhIQD1 plants 25 days after inoculation;

[0046] Figure 4 D is a graph showing the disease index of different varieties 25 days after inoculation;

[0047] Figure 4 E is a graph showing the isolation of Verticillium dahliae from TRV:00 plants and TRV:GhIQD1 plants;

[0048] Figure 4 F is a longitudinal section graph of the stems of TRV:00 plants and TRV:GhIQD1 plants;

[0049] Figure 4 G is the trypan blue staining map of the first true leaves of TRV:00 plants and TRV:GhIQD1 plants;

[0050] Figure 5 It is the result map of the influence of GhIQD1 on SA and JA signaling pathways according to the embodiments of the present invention.

[0051] Wherein:

[0052] Figure 5 A is the expression level of the GhIQD1 gene at 0, 6, 12, and 48 h after treatment with MeSA and MeJA;

[0053] Figure 5 B is the content of JA and SA in the leaves of TRV:00 plants and TRV:GhIQD1 plants 48 h after infection with V. dahliae; the values are the mean and standard deviation (SD), the error bars represent the SD of three biological replicates, and the asterisks indicate significant correlation at the 0.01 level (two-sided). All experiments were repeated at least three times. Detailed implementation manners

[0054] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0055] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0056] In this article, the terms "containing", "comprising" or "including" are open expressions, that is, including the content specified by the present invention, but not excluding other aspects of the content.

[0057] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent described event or condition may or may not occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0058] The present invention provides the use of the GhIQD1 gene in regulating plant resistance to Verticillium wilt, the use of a reagent in improving plant resistance to Verticillium wilt, the use of jasmonic acid in increasing the expression level of the GhIQD1 gene in plants, a method for preparing transgenic plants, and a method for improving plant resistance to Verticillium wilt, which will be described in detail below respectively.

[0059] Use

[0060] The present invention provides the use of the GhIQD1 gene in regulating plant resistance to Verticillium wilt. Through a large number of experiments, the inventors found a special relationship between the GhIQD1 gene and plant resistance to Verticillium wilt, that is, when the expression level of the GhIQD1 gene in plants is relatively high, the plant's resistance to Verticillium wilt is enhanced; when the expression level of the GhIQD1 gene in plants is relatively low or not expressed, the plant's resistance to Verticillium wilt is weakened. This new research finding not only enriches the understanding of the plant immune system but also provides important guidance for the development of more effective disease resistance measures and strategies.

[0061] It should be noted that the "regulation" mentioned in the present invention can refer to either increasing or decreasing. When the GhIQD1 gene is overexpressed in plants, the plant's resistance to Verticillium wilt can be improved; when the GhIQD1 gene is silenced in plants, the plant's resistance to Verticillium wilt can be reduced.

[0062] According to the embodiments of the present invention, the use may further include at least one of the following additional technical features:

[0063] According to the embodiments of the present invention, the protein expressed by the GhIQD1 gene has the amino acid sequence shown in SEQ ID NO:1.

[0064] MGASAKWVKSLIGLKKTVKDDKEKMSGKSKKWKLWRSSSGDGIGSSWKGFKGKFKADYEGSDSSPRSEAFSAAMAAVVRAPPKDFRVVRQEWAAIRIQTAFRGFLARRALRALKGIVRIQAFVRGRQVRKQAAVTLRCMQALVRVQARVRARRVRMSIEGQAVQKILDEHRSKAELLKQAEEGWCDSKGTLDDVKIKLQLRQEGAFKRERALAYSLAQKQWRLNMDSNTRTNSSVSVPYLKNQVFDKNSWGWSWLERWMAARPWETRLMEQSQADPSEPTPPSKTCSESRKTTRPTEPCSVKVRKNNVTTRISAKPPHIGQGTRSSSSPSSEFRFEESSASSSICTSTTPVSWNTIPTSERMEKTGNSRPNYMNLTESTKAKQRAANHALRRIQMQSMDEFQLKKTAGLYDGDSKSSVGSDPTVHMSRPLYTPTRLG(SEQ ID NO:1)

[0065] The present invention provides the use of a reagent in enhancing the Verticillium wilt resistance of plants, and the reagent is used to enhance the activity or expression level of the GhIQD1 gene. As described above, overexpression of the GhIQD1 gene can enhance the Verticillium wilt resistance of plants. Therefore, a reagent that can enhance the activity or expression level of the GhIQD1 gene can also enhance the Verticillium wilt resistance of plants.

[0066] According to an embodiment of the present invention, the use may further include at least one of the following additional technical features:

[0067] According to an embodiment of the present invention, the reagent includes jasmonic acid or a plasmid for overexpressing the GhIQD1 gene.

[0068] According to an embodiment of the present invention, the jasmonic acid is selected from, but not limited to, methyl jasmonate.

[0069] According to an embodiment of the present invention, the plasmid has the nucleotide sequence shown in SEQ ID NO:2.

[0070]

[0071] The present invention provides the use of jasmonic acid in increasing the expression level of the GhIQD1 gene in plants. The inventors found in experiments that by exogenously applying jasmonic acid, the expression level of the GhIQD1 gene in plants can be significantly increased, thereby effectively enhancing the resistance of plants to Verticillium wilt. This discovery provides a more effective and sustainable solution for developing new approaches to control plant Verticillium wilt.

[0072] According to an embodiment of the present invention, the jasmonic acid is selected from methyl jasmonate.

[0073] Method

[0074] The present invention provides a method for preparing transgenic plants. According to an embodiment of the present invention, the method includes: introducing a plasmid carrying the GhIQD1 gene into Agrobacterium to be transfected, transforming the Agrobacterium into the plant to be constructed, and culturing the transformed plant to be constructed to obtain the transgenic plant. Among them, the plasmid has the nucleotide sequence shown in SEQ ID NO:2.

[0075] By using the method of the present invention, transgenic plants with high disease resistance can be successfully obtained, providing a new way to improve the resistance of plants to Verticillium wilt. By introducing the GhIQD1 gene into transgenic plants, the defense ability of plants against Verticillium wilt pathogens is effectively enhanced, providing new prospects and hopes for solving important disease problems such as Verticillium wilt.

[0076] The present invention provides a method for improving the resistance of plants to Verticillium wilt. According to an embodiment of the present invention, the method includes the following two ways: 1) introducing a plasmid carrying the GhIQD1 gene (the plasmid has the nucleotide sequence shown in SEQ ID NO:2) into Agrobacterium to be transfected, using the Agrobacterium to transform the plant to be treated, and culturing the transformed plant to be treated to obtain the transgenic plant, thereby increasing the expression level of the GhIQD1 gene in the plant; or 2) spraying jasmonic acid (which can be selected from but not limited to methyl jasmonate) on the plant, thereby increasing the expression level of the GhIQD1 gene in the plant.

[0077] As described above, overexpressing the GhIQD1 gene helps to improve the resistance of plants to Verticillium wilt. Therefore, using the method of the present invention can effectively enhance the defense ability of plants against Verticillium wilt pathogens, providing new prospects and hopes for solving important disease problems such as Verticillium wilt.

[0078] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0079] Example 1: Cultivation of plant growth and test strains

[0080] Select seeds of the disease-resistant control material Zhongzhimian 2 (ZM2) and the disease-susceptible control material Xinluzao 36 (XL36) with plump grains, soak them until the radicles emerge, and plant them in a culture medium of vermiculite and nutrient soil (1:2). Place 20 10×10 seedling boxes on each tray, and plant 3-4 seeds in each box. Cultivate at a temperature of 25°C, with a light intensity of 16 h of light / 8 h of darkness, and a light intensity of 120 μmol m -2s-1 Under the conditions, the relative humidity is maintained at about 60%. When the cotton seedlings grow to the stage where the cotyledons are fully unfolded, VIGS injection is carried out. When the true leaves of the cotton seedlings unfold, select the root, stem, and leaf tissues with consistent growth vigor as test materials, and take 3 biological replicates of each sample and store them frozen at -80°C in a refrigerator for tissue expression characteristic analysis. Select cotton seedlings with consistent growth conditions for inoculation treatment.

[0081] The test strain is Verticillium dahliae Vd592 (preservation number: CGMCC: 3.3758). The Vd592 strain is the dominant strain of cotton Verticillium wilt in Xinjiang and belongs to the defoliating type strain with strong pathogenicity. Streak the Verticillium dahliae 'V592' stored in a -80°C refrigerator on a PDA medium and culture at 25°C for 4-5 days. Then pick single colonies into a Czapek liquid medium, culture at 25°C, 220 rpm, and in the dark for 3-4 days, and filter with four layers of gauze. Count the spore number of the cultured bacterial liquid with a hemocytometer. Finally, suspend the spores of the Verticillium dahliae bacterial liquid with sterilized ddH2O to a final concentration of 1.0×10 7 CFU / mL for inoculation treatment. Place the washed roots of the cotton seedlings in the 'V592' spore suspension, and use sterilized ddH2O as a control treatment. Take the root tissues of 3 biological replicates of cotton plants at different time points, collect the materials and quickly freeze them in liquid nitrogen for expression analysis after inoculation.

[0082] Example 2: Cloning and sequence analysis of the GhIQD1 gene

[0083] Through transcriptome analysis of cotton at the budding stage of both susceptible and resistant varieties 72 h after inoculation, some disease resistance-related DEGs were obtained. During this process, it was found that the expression level of the gene GhIQD1 increased significantly in the resistant variety after V. dahliae infection. The full-length sequence of GH_D07G0234 was obtained through the CottonFGD website. It was highly homologous to the Arabidopsis gene ATIQD1 and was named GhIQD1. Primers were designed using Primer Premier 5, and the GhIQD1 gene sequence was amplified from the cDNA of ZM2. It was ligated to the 5×TA / Blunt-Zero Cloning Mix vector and transformed into Escherichia coli DH5α competent cells by heat shock. Single colonies were picked for PCR positive identification, and the correct ones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0084] According to the GhIQD1 verification sequence, the protein sequence was identified through the BLAST program (http: / / www.example.com). The IQD1 sequences of other species were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Multiple sequence alignment analysis was performed using DNAMAN software, and a phylogenetic tree of the gene IQD1 was constructed using the Neighbour-joining model in MEGA11 software. Protein parameters, including molecular weight, theoretical isoelectric point (pI), and amino acid composition, were calculated using the ExPASy program (http: / / www.expasy.org / ).

[0085] The CDS length of the calmodulin-binding protein GhIQD1 gene cloned from the disease-resistant variety ZM2 of upland cotton is 1314 bp, encoding 437 amino acids. The total number of negatively charged residues (Asp + Glu): 41, and the number of positively charged residues (Arg + Lys): 82. The isoelectric point pI is 10.48. The instability coefficient of this protein is 53.68. Two conserved functional structural sites of the IQ calmodulin-binding motif were found in GhIQD1. Multiple amino acid sequence alignment showed that GhIQD1 was highly conserved among different cotton genera and was highly consistent with the IQD1 of poplar and mulberry as Figure 1 shown.

[0086] Example 3: Analysis of GhIQD1 expression pattern

[0087] Select the two-leaf stage to analyze the expression levels of the GhIQD1 gene in the root, stem, and leaf tissues of Zhongzhimian 2 (ZM2) and Xinluzao 36 (XL36). At 0, 3, 6, 12, 24, 72, and 120 hours after inoculation, harvest the roots of at least five plants from different treatments and quickly freeze them in liquid nitrogen. Extract total RNA from cotton seedlings using the FastPure Plant Total RNA Isolation Kit (Nanjing Novizan Biotech Co., Ltd.). Synthesize the first-strand cDNA using the PrimeScript TM RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Perform RT-qPCR using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on the Roche Light Cycler 480 system (Roche, Basel, Switzerland). Using GhUBQ 7 (SEQ ID NO:3) as the internal reference gene, perform RT-qPCR using the ABI7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA), and use the 2 -ΔΔCt -method to calculate the relative expression levels of the target genes. The RT-qPCR assays were set up with three biological replicates.

[0088] ATGCAGATCTTCGTCAAAACCCTAACGGGGAAGACTATAACCCTAGAGGTCGAGTCTTCGGACACCATTGACAATGTCAAAGCTAAGATCCAAGACAAGGAAGGCATTCCACCTGACCAACAACGTCTTATCTTCGCCGGCAAGCAACTCGAAGATGGCCGCACCTTAGCCGACTACAACATCCAGAAGGAATCCAC TCTACACCTT GTCCTCCGTCTCAGGGGAGGTGCGAAGAAGAGAAAGAAGAAGACCTACACCAAGCCCAAGAAGATCAAGCACAAGAAGAAGAAGGTCAAG CTCGCTGTCC TCCAGTTTTA CAAGGTGGAT GAGTCCGGGA AGGTTCAGAGACTGAGAAAAGAGTGCCCTAATGCGGAATGTGGCGCCGGGACCTTCA TGGCTAACCATTTTGATAGGCACTATTGCGGTAAGTGTGGGCTCACTTACGTTTACCAG AAGG CTGGTGGGGA TTGA(SEQ ID NO:3)

[0089] Analysis of the expression levels of GhIQD1 in the roots, stems, and leaf tissues of ZM2 and XL36 revealed that GhIQD1 was expressed in all tissues of cotton, and the expression level of GhIQD1 in ZM2 was higher than that in XL36 in both roots and leaves. However, the expression level of GhIQD1 in XL36 was higher than that in ZM2 in leaves, as follows Figure 2 A. The expression pattern of GhIQD1 after infection with Verticillium dahliae was analyzed. The results showed that the gene expression level began to increase compared with the control group 3 h after inoculation with Verticillium dahliae, and the expression level of GhIQD1 was significantly upregulated 72 h after inoculation with Verticillium dahliae, as follows Figure 2 B.

[0090] Example 4: Overexpression of GhIQD1 enhances the resistance of tobacco to V. dahliae

[0091] To explore the role of the GhIQD1 gene in plant resistance to Verticillium dahliae, a plant expression vector CaMV35s:GhIQD1 was constructed and transformed into tobacco. A specific primer fragment of 1314 bp was designed according to the CDS sequence of the GhIQD1 gene and ligated to the pCAMBIA2300 empty vector plasmid, and then transformed into tobacco by the Agrobacterium-mediated method (for the specific method, refer to LI-MING Q, YANW, YU W, et al. Establishment of high efficient tobacco genetic transformation system for functional genes[J]. Journal of Xinjiang University (Natural Science Edition), 2008.).

[0092] The experimental results are as Figure 3 shown. T0 generation plants of transgenic GhIQD1 tobacco were obtained, a total of 10 plants ( Figure 3 as shown in Figure A). RT-qPCR was performed on the GhIQD1 gene in 10 transgenic tobacco lines, and tobacco lines OE3, OE6, and OE8 with stable and high expression levels of GhIQD1 were selected ( Figure 3 as shown in Figure B) to propagate the T2 generation. Tobacco seedlings of WT, OE3, OE6, and OE8 with a seedling age of 20 d were treated by root soaking with a Vd592 spore suspension at a concentration of 1×10 7 spores / mL. Fourteen days after the treatment, as Figure 3 shown in Figure C, the symptoms of necrosis and chlorosis in transgenic leaves were significantly alleviated compared with those of the wild type, and the disease index 18 days after inoculation was also significantly lower than that of wild-type tobacco plants ( Figure 3 as shown in Figure D). These results indicate that overexpression of GhIQD1 increases the resistance of tobacco to V. dahliae.

[0093] Example 5: Silencing the GhIQD1 gene reduces the resistance of upland cotton to V. dahliae

[0094] To study the role of the GhIQD1 gene in cotton resistance to V. dahliae, gene silencing of GhIQD1 was performed by VIGS. The specific steps are as follows:

[0095] A specific amplification fragment of 500 bp was designed according to the open reading frame sequence of the GhIQD1 gene. Restriction enzyme digestion sites of EcoR1 and BamH1 were introduced at the 5' ends of the upstream and downstream primers respectively, and using the gene-cloned Escherichia coli as a template, the target fragment for inhibiting gene expression was obtained by PCR amplification and ligated to the recombinant plasmid and the tobacco rattle virus pTRV2 empty vector plasmid. The plasmid was double-digested with EcoRI and BamH1, and the target fragment on the recombinant vector and the linearized pTRV2 plasmid were recovered. The target fragment and the linear plasmid were ligated using the seamless ligase from Vazyme Co., Ltd. (Nanjing). The expression vector pTRV2:GhIQD1 was constructed and transformed into Escherichia coli competent cells. After verification by double digestion, 10 μL of the correctly verified recombinant plasmid was taken and transformed into 'GV3101' Agrobacterium competent cells by the freeze-thaw method. Single colonies were picked for colony PCR identification. The correctly identified bacterial solution was further propagated and glycerol with a concentration of 70% was added to preserve the bacteria, which was stored at -80 °C for later use. Cotton seedlings with two fully expanded cotyledons and consistent growth were selected, and the Agrobacterium cells were activated and resuspended, and the cotton cotyledons were infected by the injection method. The plants infected with TRV:GhIQD1 (the vector for silencing GhIQD1), TRV:00 (the empty vector), and TRV:GhCLA1 (the vector for labeling GhCLA1) were used as the experimental group, negative control, and positive control respectively. About 15 days after infection, when the leaves of the positive control plants showed albino phenotypes, the second true leaves and roots of the experimental group, negative, and positive controls were sampled. Each sample had 3 replicates, and the RT-qPCR technique was used to detect the gene silencing efficiency.

[0096] The steps of the trypan blue staining experiment are as follows: The leaves were immersed in 5 ml of lactophenol trypan blue solution (250 μg / ml). Trypan blue, 25% (w / v) lactic acid, 25% water-saturated phenol, 25% glycerol, H2O, slow-release vacuum infiltration for 5 minutes, and then osmosis for 5 minutes. Then the samples were heated in boiling water for 2 minutes and cooled, and then chloral hydrate solution (25 g dissolved in 10 ml of water) was used for decolorization. After multiple exchanges of the chloral hydrate solution, the samples were equilibrated in 70% glycerol for several hours.

[0097] The experimental results are as Figure 4 shown, Figure 4 A shows the albino phenotype that appeared in the TRV:GhCLA1 plants 15 days after VIGS silencing, indicating that the VIGS system can work normally in upland cotton in this experiment; Figure 4 B shows the result graph of measuring the GhIQD1 level using RT-qPCR, proving that it was effectively silenced in the TRV:GhIQD1 cotton; When the silenced experimental group cotton and the control cotton were inoculated with Vd592, the results are as Figure 4As shown in C, it shows that the leaves are yellowing and wilting, and this phenomenon is more obvious in TRV:GhIQD1; compared with the negative control (TRV:00) plants, the disease index of TRV:GhIQD1 is higher than that of the negative control in both susceptible and resistant varieties ( Figure 4 as shown in D); similarly, the results of the fungal recovery experiment are shown in Figure 4 E, indicating that the fungal biomass colonization is more extensive in TRV:GhIQD1; compared with TRV:00, the cell death area in the cotton leaves of TRV:GhIQD1 is larger ( Figure 4 as shown in F), and the longitudinal section of the cotton stem segments of TRV:GhIQD1 plants shows a higher degree of browning ( Figure 4 as shown in G). These results indicate that knocking down TRV:GhIQD1 reduces the resistance of Gossypium hirsutum to V. dahliae, indicating that the gene silencing effect in cotton is significant.

[0098] Example 6: GhIQD1 gene is involved in regulating SA and JA signaling pathways

[0099] Hormones play a crucial regulatory role in the process of plants responding to various stresses. It is currently known that the SA- and JA-mediated signaling pathways are involved in plant immune responses, and members of the IQD family mediate the regulation of SA and JA, suggesting that GhIQD1 may be involved in disease resistance related to hormone responses. To explore whether the GhIQD1 gene responds to the SA-mediated disease resistance signaling pathway, cotton seedlings treated with different hormones were used as materials to analyze the expression changes of the GhIQD1 gene in cotton after SA induction.

[0100] Specifically, using ZM2 as the material, the effects of hormone treatment on gene expression were studied. Cotton seedlings were cultured under the condition of a light intensity of 120 μmol m -2s-1 in a light incubator with a relative humidity maintained at about 70%. At the two-leaf stage, the seedlings were sprayed with 100 μM methyl jasmonate (MeJA) and 2 mM methyl salicylate (MeSA). Cotton leaves were collected at 0, 6, 12, and 48 h after treatment and frozen in liquid nitrogen before RNA extraction. SA and JA levels were measured in the leaves of TRV:00 and TRV:GhIQD1 at 48 h after inoculation. High-performance liquid chromatography was used, and three biological replicates were set up for the experiment.

[0101] The changes in the expression level of GhIQD1 in the leaves of ZM2 after MeSA and MeJA treatment were detected. The experimental results are shown in Figure 5 as follows, showing that the expression level of GhIQD1 was not significant at 6, 12, and 24 h after SA treatment in ZM2. The expression levels in ZM2 after MeJA treatment all showed a significant upregulation. These results indicate that MeSA and MeJA have specific regulatory effects on the expression of GhIQD1.

[0102] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0103] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of the GhIQD1 gene in regulating plant resistance to Verticillium wilt.

2. The use according to claim 1, wherein The protein expressed by the GhIQD1 gene has the amino acid sequence shown in SEQ ID NO: 1; Optionally, the plant is selected from cotton, Arabidopsis thaliana, rice, corn, wheat, sorghum, barley, oats, rye, soybean, tobacco, rape, tomato.

3. Use of a reagent in enhancing plant resistance to Verticillium wilt, where the reagent is used to enhance the activity or expression level of the GhIQD1 gene.

4. The use according to claim 3, wherein, The reagent includes jasmonic acid or a plasmid for overexpressing the GhIQD1 gene; Optionally, the jasmonic acid is selected from methyl jasmonate; Optionally, the plasmid has the nucleotide sequence shown in SEQ ID NO:

2.

5. Use of jasmonic acid in increasing the expression level of the GhIQD1 gene in plants.

6. A method for preparing a transgenic plant, characterized in that, It includes: Transforming the plant to be constructed with Agrobacterium carrying the GhIQD1 gene.

7. A method for improving the Verticillium wilt resistance of plants, characterized in that, It includes: Increasing the expression level of the GhIQD1 gene in the plant.

8. The method according to claim 7, wherein The increase in the expression level of the GhIQD1 gene in the plant is carried out in the following manner: Transforming the plant to be treated with Agrobacterium carrying the GhIQD1 gene; or Spraying jasmonic acid on the plant.

9. The method according to claim 6 or 8, characterized in that, It further includes: Culturing the transformed plant to be constructed or the plant to be treated to obtain the transgenic plant; Optionally, the Agrobacterium carrying the GhIQD1 gene is obtained by introducing a plasmid carrying the GhIQD1 gene into the Agrobacterium to be transfected; Optionally, the plasmid has the nucleotide sequence shown in SEQ ID NO:

2.

10. The method according to claim 8, wherein The jasmonic acid is selected from methyl jasmonate.

11. The method according to any one of claims 6 to 8, characterized in that, The plant is selected from cotton, Arabidopsis thaliana, rice, corn, wheat, sorghum, barley, oats, rye, soybean, tobacco, rape, tomato.

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