CN106905422B

CN106905422B - Pathogenic gene and protein of verticillium dahliae and application thereof

Info

Publication number: CN106905422B
Application number: CN201510981897.5A
Authority: CN
Inventors: 郭惠珊; 张涛
Original assignee: Institute of Microbiology of CAS
Current assignee: Beijing Zhongke Keshi Silk Biotechnology Co.,Ltd.; Xinjiang Runtu Agricultural Technology Co ltd
Priority date: 2015-12-23
Filing date: 2015-12-23
Publication date: 2021-03-30
Anticipated expiration: 2035-12-23
Also published as: CN106905422A

Abstract

The invention relates to pathogenic protein of verticillium dahliae, which has the effect of causing cotton verticillium wilt and is protein of 1) or 2) as follows: 1) the amino acid sequence is shown as SEQ ID NO: 2; 2) converting SEQ ID NO: 2 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to the pathogenicity of the verticillium dahliae, and is derived from the protein 1). Also relates to a gene for coding the protein and application of the gene and the protein. The pathogenic gene and protein are closely related to the mechanism of verticillium dahliae causing cotton verticillium wilt, and the pathogenicity of verticillium dahliae can be reduced by knocking out the pathogenic gene.

Description

Pathogenic gene and protein of verticillium dahliae and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a pathogenic gene and protein of verticillium dahliae and application thereof.

Background

The Verticillium wilt of cotton caused by soil filamentous fungus Verticillium dahliae Kleb is a vascular bundle disease transmitted by soil, has the characteristics of rapid variation of pathogenic bacteria, wide distribution, heavy harm, long survival time, difficulty in preventing and controlling chemical pesticides and the like, is one of the most destructive diseases in the growth process of cotton, and seriously threatens the production and development of cotton. Cotton verticillium wilt was originally observed in the state of Phizinia in the United states in 1914, and was subsequently discovered in other states and various cotton-planting countries in the world (Shenyi, 1992), and was introduced into China in 1935 with the introduction of American cotton varieties, but the harm was not serious. By the 50 s of the twentieth century, verticillium wilt disease successively occurs in local cotton areas in the north and south of China, and the diffusion and spread speed is accelerated. At the end of the 80 s, verticillium wilt had spread across 478 vegetable cotton counties (cities) throughout the country. Since the 90 s, the Chinese cotton verticillium wilt is rapidly expanded and spread, particularly, the verticillium wilt continuously and greatly happens nationwide in 1993,1995,1996,2002 years, and the loss is serious. According to the report of 11 th international verticillium major conference in 2012, the average annual yield of cotton 2005-2010 is 2352 million tons, the loss caused by verticillium diseases is up to 30% of the annual yield, and the loss of each 1% yield is equivalent to $ 3.54 million lost. China is a big cotton producing country, the cotton planting area in China is nearly hundred million mu, and the cotton yield accounts for one fourth of the total cotton yield in the world. The quality of cotton production not only directly affects the income and life of cotton farmers, but also has great influence on light textile, foreign trade and national defense construction. However, the worldwide prevalence of cotton verticillium wilt seriously threatens the production and development of cotton. In Xinjiang, the main cotton production area in China, the problem of cotton yield reduction caused by verticillium wilt every year is very serious, and great economic losses are caused to cotton farmers and countries. The cotton verticillium wilt has become one of the main obstacles for the sustainable development of cotton production, is called cancer of cotton (Jianguiliang et al 2003), and has become a prominent problem which restricts the cotton production in China at present.

Verticillium dahliae Kleb belongs to Deuteromycotina, Aphyllophorales, Achromosporaceae, Verticillium. The verticillium dahliae has a wide host range, relates to cruciferae, rosaceae, leguminosae, solanaceae, labiatae, compositae and the like, reaches 660 plants at present, and is also expanded year by year. There are many explanations about the pathogenic mechanism of cotton verticillium wilt, which mainly includes two viewpoints of catheter blockage and poisoning. The pathogenic mechanism of the pathogen was recognized in 60 s because the bacterial cells colonized in the duct and proliferated in a large amount, and simultaneously stimulated the adjacent parenchyma cells to generate colloidal substances and invade and fill the duct to block the water, so that the cotton plants withered (Garber, 1966). After the distribution of verticillium wilt bacteria in various parts of cotton is studied by Mayueli et al (1990), it is considered that the potential water delivery capacity of normal secondary xylem conduits far exceeds the total water demand of plants, and the proportion of the number of blocked conduits to the whole vascular bundle is not large (17.7% at the maximum), so that the blockage of the conduits is not the main reason for the wilt of cotton. Keen et al (1972) have suggested that the toxin produced by verticillium wilt bacteria during metabolism is a toxic protein, an acidic protein-lipopolysaccharide complex. The compound has effect in destroying cell membrane of leaf and root tissue of infected cotton, and can make K in cell⁺And Na⁺A large amount of leakage. The cell membrane of the disease-resistant variety does not have receptor sites for toxin action and is not damaged by the toxin. Wang et al (2004) isolated a novel protein VdNEP from the hyphae of verticillium wilt pathogens which has a stunting effect on cotton leaves. The protein can induce tobacco leaf to form necrotic spot, and can also make Arabidopsis thaliana produceThe protein can participate in the interaction reaction when verticillium wilt bacteria infect cotton. However, it is not clear whether the protein is the same substance as the previously isolated toxin protein. More and more studies now show that toxins secreted by verticillium wilt bacteria are key biochemical factors causing verticillium wilt, and the generation of the disease can be aggravated by the influence of water transportation caused by the blockage of tissue ducts.

The verticillium dahliae is a soil-borne fungus and can generate a microsclerotia of a dormant structure in a dry and severe environment, so that the verticillium dahliae can survive in soil for many years. The formation of microsclerotia is therefore closely related to its pathogenicity. In 2004, Dobinson KF et al (Dobinson, K.F., et al, 2004) successfully targeted the knockout of trypsin VTP1 derived from Verticillium dahliae, tomato, using a modified EZ:: TN transposition system. The gene can promote the formation of microsclerotia, but the pathogenicity and the growth characteristics of the microsclerotia are not influenced after the gene is knocked out. In 2005, Dobinson KF et al knocked out the cytokinin-activating protein kinase gene VMK1 derived from Verticillium dahliae of lettuce and tomato. After VMK1 is knocked out, the pathogenicity of the strain to various hosts is seriously declined, and the important role of a MAP kinase mediated signal path on the pathogenicity of fungi is shown. And the knock-out of the gene reduced spore production and microsclerotia formation, suggesting that the gene may be involved in multiple cellular processes.

Due to the severity of the verticillium wilt disease and the wide range of hosts, intensive research has been carried out by scientists in many countries throughout the world. The plant obtains a series of complex defense mechanisms to protect itself in the long-term co-evolution process of the plant and pathogens, and the resistance is expressed as constitutive resistance and inducible resistance, and the inducible resistance comprises tissue structure resistance and physiological and biochemical resistance. The cotton varieties with different verticillium wilt resistances have certain differences in tissue structure, and are proved by many studies at home and abroad. The disease-resistant variety has small xylem cell gap and thick cell wall, and the xylem contains more marrow rays. In addition, the diameter of the vessel lumen and xylem fiber lumen of the disease resistant variety is smaller than that of the susceptible variety, indicating that the cotton variety's resistance to verticillium wilt is related to its solid xylem. The physiological and biochemical disease resistance of cotton has been studied, and many disease resistance related factors including phytochemicals, tannins, soluble sugars, amino acids and various enzymes have been studied. After the cotton plants are attacked by pathogenic bacteria, a plurality of antibacterial substances are generated inside the cotton plants, mainly comprising gossypol, phytochemicals, tannin and the like, and in addition, some enzymes, proteins and small molecular substances such as H202 are also generated. Their action is non-specific and is associated with the basal resistance of plants. The mechanism of the cotton for resisting the verticillium wilt is a very complex problem, and a plurality of factors are involved, so that the continuous and deep research on the rule of the disease-resistant reaction products on the gene expression level has important significance for further and deep understanding of the disease-resistant mechanism and modification of the resistance of cotton varieties by using a genetic engineering means.

The obtaining of disease-resistant genes is an important basis for breeding disease-resistant varieties, and the number of the plant disease-resistant genes cloned by a molecular biological means at present is more than 39, wherein approximately more than 20 of pathogenic fungi are resistant, such as an arabidopsis powdery mildew resistant gene RPW8, a tomato blight resistant gene secreted Ve, a sorghum common rust resistant (Puccinia Sorghi) gene Rpl-D, and NBS-LRR disease-resistant genes are cloned on island cotton. In conventional breeding, cotton breeders at home and abroad always pay attention to the screening and creation of resistance sources. 1983 and 1986, 161 Lichengbao et al, carried out the identification of verticillium wilt resistance on 911 upland cotton resources, and screened a batch of disease-resistant varieties with better resistance. K.V.Srinvasin identifies the resistance of 126 varieties of sea island cotton, and the result shows that the disease resistance and disease resistance account for 85 percent. No matter the conventional method is used for searching an antigen, or the molecular biology method is used for cloning a disease-resistant gene, certain progress is made, but an effective method for really preventing and treating cotton verticillium wilt is not found. The fundamental reasons are that the genetic background of crops such as cotton is complex, and the intensive research on molecular level is difficult, and the serious difficulty is brought to the disease-resistant genetic breeding due to the strong differentiation variability of the verticillium dahliae. Therefore, the research on the molecular mechanism of the interaction of verticillium dahliae, a verticillium wilt pathogen of cotton, and host plants is very important.

Disclosure of Invention

The invention provides a pathogenic gene and protein of verticillium dahliae and application thereof, wherein the pathogenic gene and protein are closely related to a mechanism of verticillium dahliae causing cotton verticillium wilt, and pathogenicity of verticillium dahliae can be reduced by knocking out the pathogenic gene.

In one aspect of the present invention, there is provided a pathogenic protein of Verticillium dahliae, named HiC-15(isotrichodermin C-15 hydroxyylase), derived from Verticillium dahliae Kleb, which has an effect of causing cotton Verticillium wilt and is the following protein 1) or 2):

1) the amino acid sequence is shown as SEQ ID NO: 2;

2) converting SEQ ID NO: 2 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to the pathogenicity of the verticillium dahliae, and is derived from the protein 1).

In another aspect of the present invention, there is provided a pathogenic gene (named HiC-15) of verticillium dahliae, wherein the protein encoded by the pathogenic gene has an effect of causing verticillium wilt of cotton, and is any one of the genes described in the following 1) to 4):

1) the nucleotide sequence is shown as SEQ ID NO:1 from the 5' end, 1-62, 120-722, 780-1012 and 1063-1741;

2) the nucleotide sequence is shown as SEQ ID NO: 1;

3) a gene which hybridizes under stringent conditions with a gene defined in 1) or 2) and which encodes a protein according to claim 1;

4) a gene having a homology of 90% or more with the gene defined in 1) or 2) and encoding the protein of claim 1.

SEQ ID NO:1 consists of 1742 deoxynucleotides, SEQ ID NO:1 from position 1-62 of the 5' end; 722 th bit 120; 780-1012 bits; position 1063-1741 is the ORF region encoding the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue sequence set forth in SEQ ID NO: the protein shown in 2 is named as HiC-15, and the coding gene of HiC-15 protein is named as HiC-15.

The "stringent conditions" are conditions sufficient to bring the nucleotide sequence into alignment with SEQ ID NO:1, which are well known to those skilled in the art, for example: in 0.1 XSSPE containing 0.1% SDS or 0.1 XSSC containing 0.1% SDS, hybridization was performed at 65 ℃ and the membrane was washed with the solution.

The invention also provides a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium of the gene.

By "expression cassette" is meant a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence suitable for use in a host cell, comprising regulatory elements operably linked to the nucleotide sequence of interest. The regulatory element may be a promoter, enhancer, silencer, terminator and/or other elements controlling the expression of the nucleotide sequence, such as polyadenylation sequences and the like.

In a further aspect of the present invention, there is provided a use of the gene as described above for reducing pathogenicity of Verticillium dahliae by knocking out the gene as described above in Verticillium dahliae.

In another aspect of the present invention, a method for reducing pathogenicity of verticillium dahliae is provided, wherein the gene is knocked out, and the method specifically comprises the following steps:

(1) two pairs of primers were used to amplify SEQ ID NO: 3 and SEQ ID NO: 4, introducing the amplified fragment into an expression vector;

(2) a polypeptide comprising SEQ ID NO: 3 and SEQ ID NO: 4, transforming agrobacterium with the expression vector of the fragment of the sequence shown in the specification;

(3) selecting agrobacterium which is successfully transformed;

(4) and (4) transfecting the verticillium dahliae by the successfully transformed agrobacterium in the step (3), and selecting a strain with resistance, namely the strain with reduced pathogenicity.

The method as described above, wherein the two pairs of primer sequences in step (1) are as follows:

an upstream primer 1:5 '→ 3' direction: GGGTTTAAUGCACCATTACGGATACAGAG (SEQ ID NO: 5);

an upstream primer 2: 5 '→ 3' direction: GGACTTAAUGAGAGAAGCAACAAGGGAGA (SEQ ID NO: 6);

a downstream primer 1:5 '→ 3' direction: GGCATTAAUTACCTATTGACCTCCATCGC (SEQ ID NO: 7);

a downstream primer 2: 5 '→ 3' direction: GGTCTTAAUAAGATTGAGCCCTATTTCCC (SEQ ID NO: 8).

The method described above, wherein the step (1) further specifically includes the following steps:

PCR amplification is carried out on the nucleotide sequence of SEQ ID NO: 3, and the downstream primers 1 and 2 amplify the gene with the sequence shown in SEQ ID NO: 4, and connecting the two amplified PCR products to an expression vector.

The method as described above, wherein the nucleic acid sequence comprising SEQ ID NO: 3 and SEQ ID NO: 4 into agrobacterium-competent cells.

The method as described above, wherein in step (3), the strains successfully transformed are selected by plating the Agrobacterium on a plate containing an antibiotic and culturing.

The method as described above, wherein the strain successfully transformed in step (4) is selected by plating the fungus strain transformed with Agrobacterium on a plate containing an antibiotic.

Experiments prove that the pathogenic gene and the protein provided by the invention have close connection with the mechanism of cotton verticillium wilt caused by verticillium dahliae, the pathogenicity of verticillium dahliae can be reduced by knocking out the pathogenic gene in verticillium dahliae, a verticillium dahliae mutant with the pathogenic gene knocked out and a wild verticillium dahliae infect plants simultaneously, the incidence rate of the cotton verticillium wilt infected by the wild verticillium dahliae mutant strain is reduced by more than 40%, and the disease index and the disease level are also greatly reduced compared with cotton infected by the wild verticillium dahliae. Therefore, the invention provides a new revelation for researching the pathogenic mechanism of verticillium dahliae and provides a new way for treating and preventing cotton verticillium wilt.

Drawings

FIG. 1 is a comparative electropherogram of HiC-15 knockout mutants at DNA level detected by PCR in example 2; lane 1 is marker, lanes 2,3,4 are wild type Verticillium dahliae V592; lane 56,7 are HiC-15 knockout mutants Vda^Δhic-15-1; lanes 8,9,10 are HiC-15 knock-out mutants Vda^Δhic-15-2. The wild type verticillium dahliae V592 can not generate a band by using two pairs of primers of F1-HptR and HptF-R1, and Fg-Rg can generate a band by amplification; in contrast, HiC-15 knock-out mutant Vda^Δhic-15-1 and Vda^Δhic-152, the two pairs of primers F1-HptR and HptF-R1 can amplify a band, and Fg-Rg cannot amplify the band, which indicates that HiC-15 is knocked out;

FIG. 2 is a bar graph of the HiC-15 knockout mutant tested for HiC-15 expression at RNA levels by qRT-PCR;

FIG. 3 is a control cotton plant not infected as in example 4;

FIG. 4 is a cotton plant infected with Verticillium dahliae V592 in example 4;

FIG. 5 shows Vda, a knockout mutant of Verticillium dahliae V592 in example 4^Δhic-15Infected cotton plants;

FIG. 6 shows the cotton plants of example 4 modified by wild type Verticillium dahliae V592 and knock-out mutant Vda^Δhic-15A plot of incidence rate 20 days after infection;

FIG. 7 shows the cotton plants obtained in example 4 by wild type Verticillium dahliae V592 and knock-out mutant Vda^Δhic-15Disease index comparison chart after 20 days of infection;

FIG. 8 shows the cotton plants obtained in example 4 by wild type Verticillium dahliae V592 and knock-out mutant Vda^Δhic-15Disease grade number comparison chart after 20 days of infection.

Detailed Description

The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Agrobacterium tumefaciens EHA105(Elizabeth E.hood. New Agrobacterium tumefaciens plasmids for gene transfer to plants. transgenic Research, July 1993, Volume 2, Issue 4, pp 208-.

Verticillium dahliae V592(Feng-Gao, Bang-JunZHou, A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an institutional microorganisms Formation and pathogenesis. PLoS ONE 5(12): e15319.) in the examples described below was publicly available from the institute of microorganisms from the national academy of sciences twenty years from the filing date, and was used only for the repetition of experiments related to the present invention and not for other uses.

In the following examples "wild-type" means an organism that does not contain a heterologous nucleic acid molecule, being a non-transformed or non-transgenic organism.

EXAMPLE 1, HiC-15 construction of knockout vectors

The genomic DNA of Verticillium dahliae V592 was extracted by the CTAB method, and the following primers were used to extract the genomic DNA as a template

C_xPCR amplification with Hotstart DNA polymerase (Agilent): an upstream primer 1:5 '→ 3' direction: GGGTTTAAUGCACCATTACGGATACAGAG (SEQ ID NO: 5); an upstream primer 2: 5 '→ 3' direction: GGACTTAAUGAGAGAAGCAACAAGGGAGA (SEQ ID NO: 6); a downstream primer 1:5 '→ 3' direction: GGCATTAAUTACCTATTGACCTCCATCGC (SEQ ID NO: 7); a downstream primer 2: 5 '→ 3' direction: GGTCTTAAUAAGATTGAGCCCTATTTCCC (SEQ ID NO: 8). Two PCR products were ligated simultaneously with the USER enzyme mix (New England Biolabs) to the pGKO-HPT vector (Tian, L., Chen, J., Wang, J., and Dai, X.2011, which was publicly available from the institute of microbiology, national academy of sciences, twenty years since the filing date, and which was used only for the repetition of the experiments related to the present invention and not for other usesIn application), the sequencing result shows that the recombinant vector containing the DNA molecules shown in SEQ ID No.3 and SEQ ID No.4 is determined as the final knockout vector. The HiC-15 knockout mutant is obtained by transferring agrobacterium-mediated transformation into wild type verticillium dahliae V592.

EXAMPLE 2, HiC-15 obtaining of knockout mutants

1) Genetic transformation of fungi by Agrobacterium-mediated method

The related culture medium:

(a) chazuke culture medium (30g/L sucrose, 3g/L NaNO)₃,0.5g/L MgSO₄-7H₂O,0.5g/L KCl,100mg/L FeSO₄-7H₂O,1g/L K₂HPO₄pH 7.2). LB liquid medium: 10g of peptone, 5g of yeast extract and 1000ml of water, adjusting the pH value to 7,121 ℃ by using NaOH, and sterilizing for 20min by using high-pressure steam.

(b) LB liquid medium: 10g of peptone, 5g of yeast extract and NaCl₅g, adding water to 1L, sterilizing with steam at 121 deg.C for 20 min. Solid medium plus 1.5% agar.

(c) MM basal medium: 10mL of K-bufer (200g/L K)₂HPO₄,145g/L KH₂PO₄，H₃PO₃pH adjusted to 7.0), 20mL M-N buffer (30g/L MgSO)₄·7H₂O,15g/L NaCl), 1mL of 1% CaCl₂·2H₂O (w/v), 10mL of 20% sucrose (w/v), 1mL of 0.1% FeSO₄(w/v)，0.5g NH₄NO₃Distilled water was added to 1L. Steam sterilizing at 113 deg.C for 20 min.

(d) IM induction medium: 10mL of K-buff (pH 7.0), 20mL of M-N buffer, 1mL of 1% CaC1₂·2H₂O (w/v), 2.5mL of 20% NH₄NO₃(w/v), 1mL of 0.1% FeSO₄(w/v), 5mL of glycerol, 5mL of 2mol/L sucrose, 2mL of 100mmol/L acetosyringone, 40mL of 1mol/L MES (pH 5.3), and distilled water to 1L. Steam sterilizing at 113 deg.C for 20 min.

(e) CM co-culture medium: adding 1.5% agar powder into IM culture medium, sterilizing with steam at 113 deg.C for 20 min.

The concrete operation steps

(1) A single colony of Agrobacterium was picked and placed in 5ml LB broth containing antibiotics (50ug/ml kanamycin, 50ug/ml rifampicin) and cultured on a shaker at 28 ℃ for 24 hours at 200rpm, while at the same time the V592 cake on 3 PDA solid media was picked with a toothpick and placed in a flask containing 80ml of agar and cultured on a shaker at 26 ℃ at 200 rpm.

(2) Adding 1ml of 24 hr cultured Agrobacterium into 20ml MM liquid medium containing antibiotics (50ug/ml kanamycin, 50ug/ml rifampicin), placing on a shaking table at 28 deg.C, culturing at 200rpm for 24 hr, centrifuging at 4000rpm for 10min, collecting thallus, washing with IM medium twice, re-suspending with IM medium, adjusting OD₆₀₀About.0.25, filtering the wild Verticillium dahliae V592 cultured for 48 hours by four layers of sterilized gauze, placing the filtered bacterial liquid on a centrifuge at 4000rpm, centrifuging for 10min, re-suspending spores by IM + AS, counting by a blood counting plate, adjusting the concentration of the spores to be 1.0 × 10^6-7Spores per ml.

(3) And (3) mixing the agrobacterium liquid and the fungal spore suspension according to the proportion of 1: 1 volume ratio (1 ml each) of the mixture was applied to a petri dish of 200. mu.l cellophane coated on a CM medium plate and incubated at 26 ℃ for 36 hours.

(4) The co-culture was washed with 3ml of sterile water and plated at 500 ul/plate on PDA solid medium containing antibiotics (hygromycin 50ug/ml, carbenicillin 200ug/ml, cephamycin 200ug/ml, 20ug/ml pentafluorouracil) for 5-7 days.

2) Identification of HiC-15 knockout mutants by PCR (FIG. 1)

A. HiC-15 knockout mutant genome DNA is extracted by a CTAB method.

B. Three pairs of primers were used for PCR amplification. A first pair: a primer F1: 5'-ACATCCTGTTCAAACGGCTC-3' (SEQ ID NO:9) and a primer HptR on the HPT BOX are designed at the upstream of an upstream fragment SEQ ID NO.3 of the target gene HiC-15: 5'-AAATTTTGTGCTCACCGCCTGGAC-3' (SEQ ID NO:10) were subjected to upstream fragment PCR amplification. The second pair: one primer designed downstream of the downstream fragment of the target gene SEQ ID NO.4 is named as R1: 5'-GGAGTGTTGCCACCGAATGC-3' (SEQ ID NO:11), and the other primer HptF: 5'-TCTCCTTGCATGCACCATTCCTTG-3' (SEQ ID NO:12) on the HPT BOX is used for amplifying the downstream fragment. If primer pair 1 and primer pair 2 are both capable of amplifying a fragment of the same size as expected, it is indicated that recombination is occurring at the location of the gene of interest. A third pair of primers: fg: 5'-CGAAATCGATGGATCCTGGCTAACATCAGCCATCTG-3' (SEQ ID NO:13), Rg: 5'-AGGCTACGTAGGATCCCGTCATCAAACCCCAACTCT-3' (SEQ ID NO:14) was used to amplify the knocked-out target gene, and the failure to amplify the fragment surface target gene was indeed successful.

Example 3

Verticillium dahliae V592 and Vda extracted by CTAB method^Δhis-15The genomic DNA was subjected to reverse transcription using SuperScript III reverse transcriptase transcription kit (Promega Corporation) to obtain V592 and Vda^Δhis-15The cDNA of (1) was analyzed by qRT-PCR using One-Step RT-PCR kit (ABM, Inc.) and the apparatus was 1000series Thermal Cycling Platform (Bio-Rad Laboratories, Inc.). Two pairs of primers were used in the experiment, one pair being HiC-15-q-f: 5'-GCAAGTTTGGTGATGTGGAGGAA-3' (SEQ ID NO:15), HiC-15-q-r: 5'-GCACGGAGAATGGGCAGAA-3' (SEQ ID NO: 16); the other pair is an internal reference primer: VdELF-F: 5'-CCATTGATATCGCACTGTGG-3' (SEQ ID NO:17), VdELF-R: 5'-TGGAGATACCAGCCTCGAAC-3' (SEQ ID NO: 18). The results are shown in FIG. 2.

Example 4 pathogenicity assay of knockout mutants

The pathogenicity of the hydroponic cotton is identified by the infection of the strain. Soaking plump cotton seeds in 15% sodium hypochlorite for 30min, washing with sterile water for 2-3 times, soaking in sterile water for germination overnight, spreading in a culture box, and storing in a germination box until the bud grows to 3 cm. Transferring the seedling with cotyledon to plastic box (height 8-10cm) filled with clear water, and culturing at 25 deg.C under light for 16 hr and dark for 8 hr. When true leaves grow out, the clear water is changed into 1/3 MS culture solution, the culture solution is changed once per week, and inoculation is carried out when 1 true leaf is flattened. Verticillium dahliae V592 strain stored at-80 ℃ and HiC-15 knockout mutant Vda^Δhis-15Activating with PDA plate for 3-4 days, selecting bacteria block from colony edge, placing into Chachien culture solution, shaking at 25 deg.C and 220rpm for 5 days, filtering, centrifuging filtrate at 5000rpm for 5min, diluting spore with clear water, and measuring hemocytometerCounting plates, adjusting the concentration to 1 × 10⁷Spores per ml. Adding the spore suspension with adjusted concentration into an empty plastic box, and soaking the cotton seedling for 40 min. Then, the cotton seedlings are continuously cultured for 8 hours in the dark by using an MS culture solution of 1/3 at the temperature of 25 ℃ under the illumination for 16 hours. 12 seedlings were planted in each box, each variety was replicated 3 times, and control cotton seedlings were soaked in clean water for 40 min. After 20, the disease was observed (see FIGS. 3 to 5).

By the method, the HiC-15 knockout mutant is verified to have obviously weakened pathogenicity on cotton

Calculating the morbidity and disease index:

incidence rate is the number of attacks/survey total x 100% (see fig. 6)

Disease index ∑ disease number × number of diseased leaves (ear, strain) of the disease level ]/(total number of surveys × number of highest disease level) × 100 (see fig. 7)

Grading disease standard:

grade 0 plant health is asymptomatic;

leaf wilting at 0.1% -25% level 1;

grade 2 leaf wilting of 25% -50%;

3-grade leaf wilting of 50% -75%;

leaf wilting or death at level 4 of 75% -100%;

cotton infected by wild type verticillium dahliae V592 and knockout mutant V592^ΔisotC-15HOStandard statistical plots of disease grading for infested cotton (see FIG. 8).

From the results of the above method, we can see that the knockout mutant V592^ΔisotC-15HOCompared with the wild verticillium dahliae V592 infected cotton, the incidence rate, disease index and incidence grade of the infected cotton are obviously reduced, which shows that HiC-15 gene is related to the pathogenicity of the verticillium dahliae.

Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

1. Use of a pathogenic gene of verticillium dahliae, characterized in that the pathogenic gene is knocked out in verticillium dahliae to reduce the pathogenicity of the verticillium dahliae, the pathogenic gene being any one of the genes of 1) to 2) as follows:

1) the nucleotide sequence is shown as SEQ ID NO:1 from the 5' -end of the 1-62 th, 120-722 th, 780-1012 th and 1063-1741 th gene;

2) the nucleotide sequence is shown as SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.