阅读说明：本技术 大丽轮枝菌乙酰乳酸合成酶催化亚基基因VdILV2A的应用 (Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A ) 是由 都业娟 黄家风 邵胜楠 于 2021-09-09 设计创作，主要内容包括：本发明提供了大丽轮枝菌乙酰乳酸合成酶(AHAS)催化亚基基因VdILV2A的应用,属于功能基因技术领域。本发明以敲除突变体ΔVdILV2A和互补突变体为实验对象,分别从致病性以及支链氨基酸生物合成等方面分析VdILV2A的作用。结果表明ΔVdILV2A致病力显著下降；维管束变褐明显减弱；在玻璃纸上穿透能力延迟；并且在根和茎中的定殖都受到阻碍。VdILV2A也是支链氨基酸合成的必需基因,VdILV2A参与亮基酸合成。又鉴于AHAS是除草剂的作用靶点,VdILV2A可在防治棉花黄萎病中进行应用。因此本发明对大丽轮枝菌的防控和开发新型杀菌剂具有重要的理论价值,对促进棉花安全生产具有重要的现实意义。(The invention provides an application of verticillium dahliae acetolactate synthase (AHAS) catalytic subunit gene VdILV2A, belonging to the technical field of functional genes. According to the invention, a knockout mutant delta VdIV 2A and a complementary mutant are taken as experimental objects, and the effect of VdIV 2A is analyzed from the aspects of pathogenicity, branched chain amino acid biosynthesis and the like. The results show that the pathogenicity of delta VdIV 2A is remarkably reduced; the browning of the vascular bundles is obviously weakened; delayed penetration on cellophane; and colonization in both the root and stem is hindered. VdILV2A is also an essential gene for branched chain amino acid synthesis, and VdILV2A is involved in leucine synthesis. In addition, because AHAS is the action target of herbicide, VdIV 2A can be applied to the prevention and treatment of cotton verticillium wilt. Therefore, the invention has important theoretical value for the prevention and control of verticillium dahliae and the development of novel bactericides and has important practical significance for promoting the safe production of cotton.)

1. Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in pathogenicity of verticillium dahliae.

2. The use according to claim 1, wherein the pathogenicity of Verticillium dahliae is manifested in one or more of the following indicators: disease index, vascular bundle browning level, hyphal penetration and colonization ability on the host.

3. Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in biosynthesis of branched chain amino acid of verticillium dahliae.

4. The use of claim 3, wherein the branched chain amino acid comprises leucine.

5. The use according to any one of claims 1 to 4, wherein the nucleotide sequence of the verticillium dahliae acetolactate synthase catalytic subunit gene VdILV2A is shown as SEQ ID NO. 1.

6. A medicine for preventing and treating cotton diseases is characterized in that the main active component comprises a reagent for reducing the expression of Verticillium dahliae acetolactate synthase catalytic subunit gene VdILV 2A.

7. The medicament as claimed in claim 7, wherein the said agent comprises sulfonylurea herbicides and/or pyrimidine salicylic acid herbicides.

8. The drug according to claim 8, wherein the concentration of the sulfonylurea herbicide is not less than 819 mg/L;

the concentration of the pyrimidine salicylic acid herbicide is not lower than 409 mg/L.

9. Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A as a drug target in preventing and treating cotton verticillium wilt.

Technical Field

The invention belongs to the technical field of functional genes, and particularly relates to an application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A.

Background

Verticillium dahliae (Verticillium dahliae), belonging to the fungi of the genus Verticillium of the subdivision Deuteromycotina. The verticillium dahliae has a wide host range, and can be used for killing 660 plants of 38 families, wherein 184 crops and 153 weeds are planted in foreign reports. According to the identification of China, at least 20 host plants are 80, field crops comprise sunflower, eggplant, peppers, tomato, tobacco, potato, melon, watermelon, cucumber, peanut, kidney bean, mung bean, soybean, sesame, beet and the like, and common gramineous crops such as rice, wheat, corn, millet, sorghum and the like are not damaged. Verticillium dahliae is the main pathogen causing cotton verticillium wilt, and the generation of pathogenic toxins and duct blockage are the main pathogenic mechanisms of pathogenesis. Verticillin (VD-toxin) produced by verticillium dahliae is the main cause of withering. The reason for the blockage of the catheter is that hyphae and conidia are propagated in large quantities. Secondly, after the germs invade the host, the adjacent parenchyma cells are stimulated to generate gel, gum and invade. Thirdly, after the invasion of pathogenic bacteria, pectinase is generated, and colloidal substances and pectic substances in cells and cell walls are decomposed, so that tissues are disintegrated, and a catheter is blocked.

Acetolactate synthase (AHAS or ALS for short, EC 2.2.1.6) is the first key enzyme in the biosynthetic pathway of 3 branched-chain amino acids, such as valine, leucine and isoleucine, catalyzed in plants and microorganisms. Because of the lack of this enzyme in mammals, inhibitors targeted for development with this enzyme, such as sulfonylurea, imidazolinone, pyrimidyloxybenzoic acid, and triazolopyrimidine sulfonamide herbicides, are biologically safe for mammals, and thus, research on AHAS has been focused more on its biochemical structure.

AHAS is composed of Catalytic Subunit (CSU) and Regulatory Subunit (RSU), under the condition of lacking RSU, the activity of AHAS enzyme is reduced to below 15% of the activity of holoenzyme, and RSU has activation effect on the catalytic activity of CSU, thus 2 subunits are necessary for realizing AHAS holoenzyme activity. The CSU sequence is conserved in most AHAS, and the RSU sequence is low in conservation among different AHAS, but the RSU has a conserved ACT structural domain containing a branched chain amino acid binding site, and the structural domain is related to functions such as metabolism, signal transduction and solute transport and is an important element for activating the catalytic activity of the CSU by the RSU. The ACT domain of RSU is capable of activating not only homologous catalytic subunits, but also heterologous catalytic subunits from different species (plants, fungi and bacteria). An important feature of AHAS is feedback inhibition, i.e. the ability of branched chain amino acid products to negatively regulate holoenzyme activity.

Since the abbreviation for the 3 branched-chain amino acids valine, leucine, isoleucine, respectively, I, L, V, the AHAS-expressing gene in bacteria and fungi is commonly denoted as ILVx, e.g., the fungal AHAS catalytic subunit gene is designated ILV 2; the genes for E.coli expressing AHAS I, AHAS II and AHAS III are denoted as ilvBN, ilvGM and ilvIH, respectively. Researches show that AHAS of pathogenic fungi not only participates in the synthesis of branched chain amino acid, but also is closely related to pathogenicity of pathogenic bacteria. The ILV2 knock-out of the CSU gene of Candida albicans (Candida albicans) resulted in a reduced survival rate and a marked reduction in pathogenicity of pathogenic bacteria (Kingsbury, 2010); knockout of the CSU gene FgILV2 of Fusarium graminearum (Fusarium graminearum) resulted in reduction of aerial hyphae and haematochrome, complete loss of pathogenicity of the Δ FgILV2 mutant (Liu et ah, 2015); the rice blast fungus MoIlV2 subcellular localization on mitochondria, MoIlV2 gene knockout caused the rice blast fungus not to produce conidiophores and conidia, the penetration of anchorage cell is reduced and pathogenicity is reduced significantly (Du et al, 2013). However, the function of the verticillium dahliae acetolactate synthase regulatory subunit gene (VDAG _09113, named VdILV2A) is not yet clear.

Disclosure of Invention

In view of the above, the invention aims to provide a new application of the verticillium dahliae acetolactate synthase catalytic subunit VdIV 2A gene, to clarify the action of the VdIV 2A gene in the pathogenic mechanism of the verticillium dahliae, and to provide a basis for preventing and treating the verticillium dahliae and developing a novel bactericide.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in pathogenicity of verticillium dahliae.

Preferably, the pathogenicity of the verticillium dahliae is expressed in one or more of the following indexes: disease index, vascular bundle browning level, hyphal penetration and colonization ability on the host.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in biosynthesis of branched chain amino acid of verticillium dahliae.

Preferably, the branched-chain amino acid biosynthesis comprises a branched-chain amino acid synthesis.

Preferably, the branched chain amino acid comprises leucine.

Preferably, the nucleotide sequence of the verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A is shown in SEQ ID NO. 1.

The catalytic subunit gene VdILV2A of AHAS is obtained by cloning and sequencing by taking the genome DNA and cDNA of the Verticillium dahliae wild strain V592 as a template. The VdIV 2A gene predicts that the encoded protein contains 641 amino acids. Sequence analysis showed that VdILV2A contains 1N-terminal Thiamine Pyrophosphatase (TPE) binding domain, 1 central TPE domain and 1C-terminal TPE binding domain (see fig. 1).

The invention provides a medicament for preventing and treating cotton diseases, which mainly comprises a reagent for reducing the expression of verticillium dahliae acetolactate synthase catalytic subunit gene VdILV 2A.

Preferably, the agent comprises a sulfonylurea herbicide and/or a pyrimidine salicylic acid herbicide.

Preferably, the concentration of the sulfonylurea herbicide is not lower than 819 mg/L;

the concentration of the pyrimidine salicylic acid herbicide is not lower than 409 mg/L.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A as a drug target in preventing and treating cotton verticillium wilt.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in pathogenicity of verticillium dahliae. According to the invention, a knockout mutant delta VdIV 2A and a complementary mutant are taken as experimental objects, VdIV 2A gene deletion is analyzed from the aspects of disease index of caused diseases, vascular bundle browning degree, capability of hyphae penetrating through a host, capability of colonization in the host and the like, so that the disease index of verticillium dahliae is reduced, the vascular bundle browning degree is weakened, the penetration of verticillium dahliae is delayed, and the colonization of verticillium dahliae in cotton roots and stems is hindered by knockout of VdIV 2A gene, while the complementary strain and a wild strain V592 have consistent pathogenicity. It can be seen that VdIV 2A gene is related to pathogenicity of Verticillium dahliae.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in biosynthesis of branched chain amino acid of verticillium dahliae. According to the invention, the knockout mutant delta VdIV 2A and the complementation mutant are taken as experimental objects, the influence of leucine, isoleucine and valine auxotrophic culture medium on hypha growth is clarified by adding amino acid on the auxotrophic culture medium through exogenous sources, the result shows that 5mM Leu has feedback inhibition effect on V592 and delta VdILV2A knockout mutants and the complement ECVdIV 2A, and the feedback inhibition effect of V592 is strongest compared with that of knockout and complement; 5mM Leu inhibited the expression of VdIV 2A gene. In order to clarify the influence of the catalytic subunit Δ VdILV2A gene knockout on the expression of downstream genes of AHAS in the branched-chain amino acid synthesis pathway of verticillium dahliae, the relative expression levels of the downstream genes were significantly reduced by measuring the transcriptional expression of the downstream genes by RT-qPCR, respectively, wherein 4 genes of dihydroxy-acid dehydratase essential for isoleucine, valine and leucine synthesis (VDAG-03858, VDAG-04620, VDAG-05384 and VDAG-09671), 1 gene of branched-chain amino acid transaminase (VDAG-02852) and 1 gene of 2-isopropylmalate synthase essential for leucine synthesis (VDAG — 03299), 1 gene of 3-isopropylmalate dehydratase (VDAG — 00564), and 2 genes of 3-isopropylmalate dehydrogenase (ag-05325, VDAG-032 06467). The above results indicate that the knockout of a single subunit gene of AHAS has an effect on the expression of most downstream genes in the branched chain amino acid synthesis pathway. VdIV 2A is involved in the branched-chain amino acid biosynthetic pathway.

Meanwhile, the sulfonylurea herbicide and the pyrimidine salicylic acid herbicide are respectively adopted to treat the wild type V592, and the results show that the two herbicides both obviously inhibit the growth of Verticillium dahliae, the V592 does not generate microsclerotia, the relative expression quantity of the VdIV 2A gene is adjusted, and the change of the expression quantity is concentration-dependent. Thus, it was confirmed again that VdIV 2A is an essential gene for branched chain amino acid synthesis, and VdIV 2A is involved in branched chain amino acid synthesis.

Drawings

FIG. 1 is a schematic diagram of the domain of the AHAS catalytic subunit VdILV2A gene;

FIG. 2 shows the results of determining the pathogenicity of Verticillium dahliae VdIV 2A knockout mutants to cotton, wherein FIG. 2A shows the cotton morphology and vascular bundle browning morphology of diseases caused by knockout mutants Δ VdIV 2A-1 and Δ VdIV 2A-2 and their control strains, and FIG. 2B shows the disease index broken line graphs after different strains are treated;

FIG. 3 shows the result of the determination of the penetration ability of Verticillium dahliae VdIV 2A knockout mutant to cotton;

FIG. 4 shows the result of determination of colonization of cotton by Verticillium dahliae VdIV 2A knockout mutant;

FIG. 5A is a schematic diagram showing the inoculation positions of Verticillium dahliae VdIV 2A knockout mutant, complementation strain and wild strain on FGA medium;

FIG. 5B is a colony morphology diagram of Verticillium dahliae VdIV 2A knockout mutant on FGA medium supplemented with different amino acids;

FIG. 6 shows the results of relative expression levels of VdIV 2A gene after branched-chain amino acid treatment;

FIG. 7 shows the results of relative expression levels of genes downstream of the AHAS synthetic pathway in the Verticillium dahliae Δ VdILV2A knock-out mutant;

FIG. 8A shows the results of colony morphology change after AHAS inhibitor treatment V592;

FIG. 8B shows the relative expression level of VdIV 2A gene after AHAS inhibitor treatment of V592.

Detailed Description

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in pathogenicity of verticillium dahliae.

In the invention, the nucleotide sequence of VdIV 2A is shown as SEQ ID NO. 1. In order to verify whether the deletion of VdIV 2A can influence the pathogenicity of Verticillium dahliae, the invention constructs a VdIV 2 mutant strain delta VdIV 2A of the Verticillium dahliae with VdIV 2A gene knocked out by taking a wild strain V592 as a basic strain, and performs gene complementation on the delta VdIV 2A to obtain a complemented strain ECVdIV 2A. The construction method of the Verticillium dahliae mutant strain delta VdIV 2A with the VdIV 2A gene knocked out is completed by utilizing the principle of homologous recombination, and concretely, the method can be seen in the prior art to obtain a knock-out and complementary mutant (WANG S, XING H, HUA C, GUO H S, ZHANG J. animal mutant-step cloning mutants (ATMT) -based gene-deletion method for Verticillium dahliae. phytopathology,2016,106(6): 645) 652.). The invention respectively takes delta VdIV 2A, a complementary strain ECVdIV 2A and a wild strain V592 as experimental objects, and respectively measures the following indexes: the disease index, the vascular bundle browning degree, the hyphal penetrability and the colonization ability on the host, and the results show that the disease indexes of diseases caused by the knockout mutant delta VdIV 2A are respectively 55.0-57.0; the pathogenicity of the complementary strain is obviously restored to the level of the wild strain, and the disease index is higher than 90; when the strain is inoculated for 30 days, the infected plant is cut and observed, and the browning degree of the knockout mutant delta VdIV 2A vascular bundle is obviously reduced; the VdIV 2A knockout mutant does not penetrate when inoculated at the 3 rd day and can penetrate through the cellophane at the 5 th day, which shows that the VILV 2A knockout causes the delayed penetrating capability of the verticillium dahliae on the cellophane; the VdIV 2A gene knockout can prevent Verticillium dahliae from colonizing in cotton roots and stems. The experimental result shows that the pathogenicity of verticillium dahliae can be reduced by knocking out the VdIV 2A gene, namely the VdIV 2A gene is related to the pathogenicity of verticillium dahliae.

The invention provides application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A in biosynthesis of branched chain amino acid of verticillium dahliae. The branched chain amino acid preferably comprises leucine.

In the invention, in order to clarify the effect of VdIV 2A in the branched chain amino acid synthesis pathway, a knockout mutant delta VdIV 2A, a complementation strain and a wild strain V592 are cultured on an auxotrophic culture medium, and exogenous amino acids are added, so that the result shows that 5mM Leu has feedback inhibition effect on both the V592 knockout mutant and the delta VdIV 2A knockout mutant as well as the complement ECVdILV2A, and the feedback inhibition effect of V592 is strongest compared with that of the knockout and the complement; and 5mM Leu inhibits the expression of VdIV 2A gene, and VdIV 2A participates in the branched chain amino acid biosynthesis pathway of Leu, which shows that the VdIV 2A gene is an important component in the branched chain amino acid synthesis pathway. In order to clarify the influence of the catalytic subunit Δ VdILV2A gene knockout on the expression of downstream genes of AHAS in the branched-chain amino acid synthesis pathway of verticillium dahliae, the relative expression levels of the downstream genes were significantly reduced by measuring the transcriptional expression of the downstream genes by RT-qPCR, respectively, wherein 4 genes of dihydroxy-acid dehydratase essential for isoleucine, valine and leucine synthesis (VDAG-03858, VDAG-04620, VDAG-05384 and VDAG-09671), 1 gene of branched-chain amino acid transaminase (VDAG-02852) and 1 gene of 2-isopropylmalate synthase essential for leucine synthesis (VDAG — 03299), 1 gene of 3-isopropylmalate dehydratase (VDAG — 00564), and 2 genes of 3-isopropylmalate dehydrogenase (ag-05325, VDAG-032 06467). The above results indicate that the knockout of a single subunit gene of AHAS has an effect on the expression of most downstream genes in the branched chain amino acid synthesis pathway.

As AHAS is the target of the action of the herbicide, in order to verify the action relationship between the VdIV 2A gene and the herbicide, the wild type strain V592 is inoculated with the sulfonylurea herbicide and the bispyribac-sodium, and the colony morphology is observed to find that the tribenuron-methyl and the bispyribac-sodium obviously inhibit the growth of Verticillium dahliae under the action of 1638.4mg/L, and the V592 does not generate microsclerotia; and the results of respectively treating V592 with 3 tribenuron-methyl and bispyribac-sodium with different concentrations show that the relative expression quantity of the VdILV2A gene is in a down-regulation trend, and the down-regulation is more obvious when the concentration is higher. This indicates that VdIV 2A gene is the target of action of pesticide. Therefore, the invention provides the application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A as a drug target in preventing and treating cotton verticillium wilt.

The invention provides a bactericide for preventing and treating cotton diseases, which mainly comprises a reagent for reducing the expression of verticillium dahliae acetolactate synthase catalytic subunit gene VdILV 2A.

In the present invention, the agent preferably includes a sulfonylurea herbicide and/or a pyrimidine salicylic acid herbicide. The concentration of the sulfonylurea herbicide is preferably not less than 819mg/L, more preferably 900-1638.4 mg/L, and most preferably 1638.4 mg/L. The concentration of the pyrimidine salicylic acid herbicide is preferably not less than 409mg/L, more preferably 500-1638.4 mg/L, and most preferably 1638.4 mg/L.

The method for using the bactericide is not particularly limited, and the conventional spraying method of the bactericide known in the field can be adopted. The bactericide achieves the purpose of preventing and controlling the occurrence and development of diseases by reducing the pathogenicity of the VdIV 2A gene.

The following examples are provided to describe in detail the application of the verticillium dahliae acetolactate synthase catalytic subunit gene VdILV2A provided by the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction method of Verticillium dahliae mutant strain delta VdIV 2A with VdIV 2A gene knocked out

A primer is designed according to the upstream-downstream homologous ratio of VdIV 2A gene (nucleotide sequence is shown as SEQ ID NO: 1), a knockout vector is constructed by using homologous recombination technology, and a wild strain V592 is used as an initial strain to construct a mutant strain, specifically referring to the prior art (WANG S, XING H, HUA C, GUO H S, ZHANG J. animal mutant-step cloning strategy strains (ATMT) -base gene-deletion method for Verticillium dahliae. phytopathology,2016,106(6): 652.) to obtain VdIV 2A gene knockout Verticillium (delta VdILV2A-1, delta. 2A-2).

Example 2

Construction method of verticillium dahliae VdILV2A gene complementary mutant ECVdILV2A

Using the Verticillium dahliae mutant strain Δ VdIV 2A constructed in example 1 as an initial strain, a complementary mutant of the Verticillium dahliae VdIV 2A gene (VdILV 2A-1, ECILVdIV 2A-2) was obtained by referring to the prior art (WANG S, XING H, HUA C, GUO H S, ZHANG J. An improved single-step cloning site transformation (ATMT) -based gene-deletion method for Verticillium dahliae. Phytopathology,2016,106(6): 652).

Example 3

In order to clarify the influence of the VdIV 2A gene knock-out on the virulence of Verticillium dahliae, the virulence of the VdIV 2A gene knock-out mutant on cotton was determined, using wild strain V592 as a control. The pathogenicity assay was as follows:

the knockout mutant, the complementation strain and V592 constructed as above are inoculated into Czapek-Dox liquid medium and cultured for 5d at 26 ℃ and 200 r/min. After the 5 th main leaf of the cotton seedling grows out, 200mL of cotton with the concentration of 1.0X 10 is inoculated in each pot by using a root soaking inoculation method⁷CFU/mL of bacterial solution, 3 hydroponic boxes (36 cotton seedlings in total) were repeated for each strain. The disease classification standard is that the disease is observed every day after inoculation, the disease is counted from the beginning of the disease, the disease is generally recorded every 3d until one month after the disease, and the disease classification standard is as follows: level 0: the disease is not developed; level 1: 1-2 leaves of the plant are attacked; and 2, stage: 1 true leaf onset; and 3, level: 2 true leaves are attacked; 4, level: 3 or more than 3 true leaves. Disease index was calculated according to formula I.

Disease index [ [ sigma ] disease plant number of each stage x stage/(total plant number x highest disease stage) ] × 100 formula I

Disease index is the average of 3 biological experiments.

On day 30, the straw was cut and vascular bundles were photographed using a stereomicroscope (Olympus SZX7, Japan).

The results show that disease indexes of diseases caused by the knockout mutants delta VdIV 2A-1 and delta VdIV 2A-2 are 57.0 and 55.0 respectively; the pathogenicity of the complementary strain is obviously restored to the level of the wild strain, and the disease index is higher than 90. When inoculated for 30 days, infected plants are subjected to stalk cutting observation, and the browning degree of the delta VdIV 2A-1 and delta VdIV 2A-2 vascular bundles of knockout mutants is obviously reduced (figure 2).

Example 4

In order to analyze whether the reduced pathogenicity of verticillium dahliae caused by the VdIV 2A gene knockout is related to the host penetrating ability of verticillium dahliae, a cellophane penetrating experiment is carried out on a VdIV 2A knockout mutant strain by taking V592 as a control, and the specific method is as follows:

and (4) paving the glassine paper which is subjected to high-temperature sterilization treatment and has the size basically consistent with that of the culture dish on the poured MM minimal medium. Picking hyphae with toothpick, inoculating to the center of cellophane, culturing for 3d, 4d and 5d respectively, throwing away cellophane, allowing the strain to grow for 7d, and repeating for 3 strains if the bacterial colony can grow on the culture dish.

The results showed that the VdIV 2A knock-out mutant was able to penetrate the glassine paper only when inoculated at 5d (FIG. 3). This indicates that the knockout of VdILV2A gene delays the penetration of verticillium dahliae and reduces the infectivity of verticillium dahliae.

Example 5

In order to determine the influence of the VdIV 2A gene on the infection and colonization ability of Verticillium dahliae, the fungal biomass was determined by qPCR on cotton plants inoculated with wild type strains V592, VdIV 2A knock-out mutants and complementary strains. And (3) inoculating different strains, and taking roots (5 cm below red stem lines), stems (5 cm above half of cotton seedlings) and leaves of cotton at 26d, and respectively extracting total DNA of the roots, the stems and the leaves. Primers for qPCR [ Internaltrescripted spacer (ITS) -F (5'-TGTTGCTTCGGCGGCTCGTT-3', SEQ ID NO:6) and ITS-R

(5'-GCGTTTCGCTGCGTTCTTCA-3') (SEQ ID NO:7) ] was designed based on the ITS1 and ITS2 regions of Verticillium dahliae ribosomal RNA (Z29511). The total DNA of each sample was quantitated by normalization using a constitutively expressed beta-tubulin gene (DQ266153) as an internal reference gene (internal reference gene: beta-tubulin-F: TCACCAGCCGTG GCAAGGTTG, SEQ ID NO: 8; beta-tubulin-R: AGCAAAGGGCGGTCTGGAC GTTG, SEQ ID NO: 9). Each reaction was set to 3 replicates.

RT-qPCR is adopted to detect the expression condition of VdIV 2A in different tissues of the colonized cotton, a kit method is adopted to extract RNA of different tissues of the cotton, cDNA obtained by reverse transcription is taken as a template to carry out RT-qPCR detection, and the designed primer sequences are as follows:

VdILV2A-qPCR-F：ATGTCGCCTGTCACCTCGC(SEQ ID NO:2)；

VdILV2A-qPCR-R：ATACAACCTCTGGCGCTCTCC(SEQ ID NO:3)；

β-tubulin-F：TCACCAGCCGTGGCAAGGTTG(SEQ ID NO:4)；

β-tubulin-R：AGCAAAGGGCGGTCTGGACGTTG(SEQ ID NO:5)；

verticillium dahliae beta-tubulin (DQ266153) is an internal reference gene.

Reaction system (10 μ L):

the reaction procedure for RT-qPCR detection was as follows: 2min at 50 ℃; 2min at 95 ℃; 953s, 30s at 60 ℃ for 40 cycles. The resulting data were analyzed using 7500Fast Real-Time PCR System at 2^-ΔΔCtIs calculated by the method of (1).

The results show that the Δ VdILV2A mutant had significantly lower biomass in both root and stem tissues than the V592 strain and the 2 complement strains (fig. 4). The results show that the VdIV 2A gene knockout can lead the verticillium dahliae to be inhibited from colonizing in cotton roots and stems; the results are consistent with the disease index and the degree of vascular bundle browning caused by the Δ VdILV2A mutant on cotton seedlings.

Example 6

To determine the effect of leucine, isoleucine and valine auxotrophy on hyphal growth of Δ VdILV2A, the complementation strain and the wild-type strain V592, the effect of leucine, isoleucine and valine auxotrophy on hyphal growth was further elucidated by adding different concentrations (1mM, 5mM) of exogenous amino acids (leucine, isoleucine and valine) to FGA solid medium, and the inoculation positions of the three strains on FGA solid medium are shown in fig. 5A.

In order to clarify the role of AHAS catalytic subunit gene in branched chain amino acid synthesis pathway, we found that 5mM Leu has feedback inhibition effect on both V592 and Δ VdILV2A knockout mutants and the complement ECVdILV2A, V592 has the strongest feedback inhibition effect compared to knockout and complement, while isoleucine and valine do not produce the same result (fig. 5B).

The expression level of VdIV 2A gene in wild type strain V592 was determined by RT-qPCR as described in example 5. The results showed that 5mM Leu inhibited the expression of VdIV 2A gene, and VdIV 2A was involved in the branched-chain amino acid biosynthetic pathway of Leu (FIG. 6).

The above results indicate that VdIV 2A gene is an important component in the branched-chain amino acid synthesis pathway.

And detecting the relative expression quantity of genes at the downstream of the AHAS synthetic pathway in the delta VdILV2A gene knockout mutant by adopting an RT-qPCR method.

Results the Ct value (Threshold cycle) indicated is defined as the number of cycles required to reach a detection Threshold beyond which the fluorescence signal is detected, and the data of the results are analyzed using the 7500Fast Real-Time PCR System, 2^-ΔΔCtIs calculated by the method of (1).

The primer sequences used were as follows:

TABLE 1 primers for RT-qPCR detection of downstream genes in AHAS synthetic pathway

The reaction conditions and the reaction procedures of RT-qPCR detection are the same as above.

The results are shown in FIG. 7. In order to clarify the influence of the catalytic subunit Δ VdILV2A gene knockout on the expression of downstream genes of AHAS in the branched-chain amino acid synthesis pathway of verticillium dahliae, the relative expression levels of the downstream genes were significantly reduced by measuring the transcriptional expression of the downstream genes by RT-qPCR, respectively, wherein 4 genes of dihydroxy-acid dehydratase essential for isoleucine, valine and leucine synthesis (VDAG-03858, VDAG-04620, VDAG-05384 and VDAG-09671), 1 gene of branched-chain amino acid transaminase (VDAG-02852) and 1 gene of 2-isopropylmalate synthase essential for leucine synthesis (VDAG — 03299), 1 gene of 3-isopropylmalate dehydratase (VDAG — 00564), and 2 genes of 3-isopropylmalate dehydrogenase (ag-05325, VDAG-032 06467). The above results indicate that the knockout of a single subunit gene of AHAS has an effect on the expression of most downstream genes in the branched chain amino acid synthesis pathway.

Example 7

AHAS inhibitor treatment experiments

2 herbicides of different chemical types are selected, namely tribenuron-methyl (sulfonylurea herbicide) and bispyribac-sodium (pyrimidine salicylic acid herbicide), liquid medicines with the concentration of 12.8-1638.4 mg/L are respectively prepared and respectively inoculated to the bacterial colony of a wild type strain V592, and the influence of the liquid medicines on the growth of the strain is observed.

The colony morphology is observed, the tribenuron-methyl and the bispyribac-sodium obviously inhibit the growth of the Verticillium dahliae under the action of 1638.4mg/L, and V592 does not generate microsclerotia (figure 8A).

V592 was treated with tribenuron-methyl and bispyribac-sodium at 3 concentrations (409.6mg/L, 819.2mg/L and 1638.4mg/L), respectively, and the relative expression level of VdILV2A gene was determined by the same method as described in example 5.

The results showed that the relative expression level of VdILV2A gene was down-regulated, and the down-regulation was more pronounced at higher concentrations (fig. 8B). These results indicate that tribenuron-methyl and bispyribac-sodium can inhibit the expression of VdIV 2A gene. Again, VdILV2A was shown to be an essential gene for branched chain amino acid synthesis.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> river university

<120> application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1926

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtcgcctg tcacctcgcc ccttctgctt cgtcagcagg ccacatccca cgtccgccgc 60

ttcagccgag ggcgacgacg gccgttcaga tggagagcgc cagaggttgt atcccctgat 120

aattcagtca gcggtgccag cgccattctg cacgcacttc aggccgcaga cgtcactcat 180

ctcttcgtca atctcggctc ggaccaccca gctttcctga cggcgtttgc catgaaggcg 240

tatcccaggg tcaaggtcgt cacgtcgccg aatgagatga atgcgctgtc ggcagcctcg 300

ggctatgcca tggtcactgg gaagccagca gctgtactcg ttcacgtcga atgcggcaca 360

caggctttgg cgggcgcggt gcacaatatc agtaagggcc gcttgccggt ggtgatagtt 420

gccggcaccg tgcccatcac gatggaagga gagctaccgg ggagtcgtaa cgagtgcgtg 480

gatatcccag accagcgctc tatcgtccgt caatacatga agtatgatca tgagatccga 540

tctccgcaca atgctgtaca gattgtgctc cgggctcttc agatggcgac aagtacgccg 600

cagggcccaa catatgtcat cgcatcgcga gagacactcg aagcacaaac atcgatccaa 660

agcatcaagc cttcgcaaga tccagagaag aacctctggg tggagaaaat cggcctccac 720

atgcaagacg tcgcgcgctt gggcgacatt ctcatcaggg ccaagtcccc tctgatcgtc 780

acgagttatg ccggccggaa tcctgctgcc tttcgagcgc ttcacgattt ggcccggcgc 840

ctcgctatcc ccgtgcacga gaatgcgccg gtggttaaca atttcccgac gactagtttc 900

ctccaccaag gccaaacttg gaacggcggc gggcagctcg aggccttggc cgaagcagac 960

gtggtcctcg tcgtcgactc ggatgttcca tggataccgt ccgagtctag gccgagcgat 1020

gaggcccgcg tcttccacct cgactgcgat cctctgaaag gcagcgccac gctctggagt 1080

cttccggccg agaagcgctg gatgtgtgat gcatcggtgg cgttgaaaca gctagacgag 1140

tacgtcaaga cgagcctgga actctttact gaagctgcaa agagtaggat caacgtcagg 1200

acatctctgc tgacacggcg tttcgaggca cgagcgcgga gactcgaagc agaagaagtg 1260

gcctcttcag acggaaaggt cacggtgccg tactttatga gccgcttccg caaggcgaca 1320

gaaggtctcg acgtattagc gctcaacgag agcatgacca atcttggcaa ggtcgctgac 1380

cacctacggc acgaccaaca caactcactg ttgggcagtg gtggcggctc gctcgggtgg 1440

tattctgggg ctgctgttgg ggtggccatg gcgttgggag aagaggccgc tgtgggtgat 1500

gcggccgacc gtcttgtcac ggcgtttgtg ggagatggaa cgttcctgtt tggggtgcct 1560

gccagtgcct actggatggc catgcggtat gcgacgccgt acctaaccgt cgtctggaat 1620

aacggcggct ggacgagccc gaagaatgcc tgcttgagga ttcatcctga gatgaaagat 1680

tttgtgcgac gtagcgatgg cggaggacgg catgatggca gcagggcaac agctttggct 1740

gaggcgatgg gcgtcagcat ttcgccgtca ccttcgtttg gaaagattgc tgagggtgcc 1800

ggtgatgctt ggtggacagt cgtgaagagg gcagacgagg tcgatggagc catcgcggag 1860

gccgttcgga tggtgaggga gctcaagcga tgtgcagtga ttgaggtagc cattgccagt 1920

gtgtga 1926

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgtcgcctg tcacctcgc 19

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atacaacctc tggcgctctc c 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcaccagccg tggcaaggtt g 21

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcaaagggc ggtctggacg ttg 23

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgttgcttcg gcggctcgtt 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcgtttcgct gcgttcttca 20

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaccagccg tggcaaggtt g 21

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agcaaagggc ggtctggacg ttg 23

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agagctcgag gccctcatct c 21

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctcctccgtc gtcagtccg 19

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgctttcga ggtcatttgg catg 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gacgagaatt tgttcagcgt ctcg 24

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atggcagacc aacatcaaca gg 22

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cttgcgcagg aatagcgaga ag 22

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgagagggca tgatttcccc g 21

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcagggattg ccctcccac 19

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gaccccatca agaagtacaa gccc 24

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggcagacttt ggttaccatc gc 22

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagtcccagt tcccaagacc 20

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gtcagtggtc gcaagagtgc aatc 24

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

attgactggg ccaatgtcgg 20

<210> 23

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gtggatgcgc ataaagggc 19

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgtctgaca caaagacgtt tcgc 24

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cgccgacaat gtctgtggtg 20

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgtagtgttt ggaggcgacc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccgaggaggt gttcttggag 20