CN106905423A - The Disease-causing gene of verticillium dahliae, albumen and its application - Google Patents

Abstract

The present invention relates to a pathogenic protein of Verticillium dahliae, the protein has the effect of causing Verticillium dahliae, and is the following 1) or 2) protein: 1) The amino acid sequence is as shown in SEQ ID NO:2 Protein; 2) The amino acid sequence of SEQ ID NO: 2 is substituted and/or deleted and/or added by one or several amino acid residues, and the protein derived from 1) is related to the pathogenicity of Verticillium dahliae. It also relates to the gene encoding the protein and the application of the gene and 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 Genes, Proteins and Applications of Verticillium dahliae

technical field

The invention relates to the field of biotechnology, in particular to the pathogenic gene and protein of Verticillium dahliae and application thereof.

Background technique

Verticillium dahliae Kleb. caused by the soil filamentous fungus Verticillium dahliae Kleb. is a soil-borne vascular disease. Pesticides are difficult to control and are one of the most devastating diseases in the growth process of cotton, which seriously threatens the production and development of cotton. Cotton verticillium wilt was first seen in Virginia in the United States in 1914, and was subsequently discovered in other states and cotton-growing countries around the world (Shen Qiyi, 1992). It was introduced into China with the introduction of American cotton varieties in 1935, but the damage was not serious. After the 1950s, verticillium wilt occurred successively in some cotton areas in the north and south of my country, and the speed of spread accelerated. In the late 1980s, verticillium wilt had spread to 478 cotton-growing counties (cities) across the country. Since the 1990s, the cotton verticillium wilt has spread rapidly in China, especially in 1993, 1995, 1996, and 2002, it occurred continuously nationwide and caused serious losses. According to the report of the 11th International Verticillium Conference in 2012, the average annual output of cotton in the world from 2005 to 2010 was 23.52 million tons, and the loss caused by Verticillium disease was as high as 30% of the annual output, and the loss of every 1% of the output was equivalent to Loss of $354 million. China is a big cotton producing country, with nearly 100 million mu of cotton planted in the country, accounting for a quarter of the world's total cotton production. The quality of cotton production not only directly affects the income and life of cotton farmers, but also has a major impact on textile, foreign trade and national defense construction. However, the prevalence of cotton verticillium wilt in the world seriously threatens the production and development of cotton. In Xinjiang, the main cotton production area in China, the problem of cotton production reduction caused by Verticillium wilt is very serious every year, causing great economic losses to cotton farmers and the country. Cotton verticillium wilt has become one of the main obstacles to the sustainable development of cotton production, known as "the cancer of cotton" (Jian Guiliang et al. 2003), and has become a prominent problem restricting my country's cotton production.

Verticillium dahliae Kleb. (Verticillium dahliae Kleb.) belongs to the subphylum Deuteromycetes, the order Amygium, Leuchromosporaceae, and the genus Verticillium. The host range of Verticillium dahliae is very wide, involving Brassicaceae, Rosaceae, Fabaceae, Solanaceae, Lamiaceae, Asteraceae, etc. Currently, it has reached more than 660 kinds of plants, and it is still expanding year by year. There are many explanations for the pathogenic mechanism of Verticillium wilt in cotton, among which two views of duct blockage and poisoning are the main ones. In the 1960s, people’s understanding of the pathogenic mechanism of this bacteria was that the bacteria colonized in the duct and multiplied in large numbers, and at the same time stimulated the adjacent parenchyma cells to produce gelatinous substances and invading bodies to block the duct and hinder the movement of water, resulting in Cotton plants wilt (Garber, 1966). Ma Yuanli et al. (1990) studied the distribution of Verticillium dahliae in the vessels of various parts of cotton and concluded that the potential water delivery capacity of normal secondary xylem vessels far exceeded the total water demand of plants, and the number of blocked vessels accounted for the entire The proportion of vascular bundles was not large (the largest was 17.7%), so duct blockage was not the main cause of cotton wilting. Keen et al. (1972) believed that the toxin produced by Verticillium dahliae in the metabolic process is a toxic protein, which is a complex of acidic protein-lipopolysaccharide. The complex has a damaging effect on the cell membranes of the leaves and roots of the susceptible cotton varieties, causing a large amount of intracellular K ⁺ and Na ⁺ leakage. The cell membranes of disease-resistant varieties do not have receptor sites for toxin action and are not damaged by toxins. Wang et al. (2004) isolated a new protein VdNEP with a wilting effect on cotton leaves from the hyphae of Verticillium dahliae. This protein can induce the formation of necrotic spots in tobacco leaves, and can also cause Arabidopsis to produce a disease-resistant response, so this protein may be involved in the interactive response of Verticillium dahliae to cotton infection. But it is unclear whether this protein is the same substance as the toxin protein that has been isolated before. More and more studies now show that the toxin secreted by Verticillium dahliae is the key biochemical factor leading to Verticillium wilt, and the obstruction of tissue ducts affecting water transport may aggravate the disease.

Verticillium dahliae is a soil-borne fungus that can survive in the soil for many years by producing its dormant structure microsclerotia in a dry and harsh environment. Therefore, the formation of microsclerotia is closely related to its pathogenicity. In 2004, Dobinson KF et al. (Dobinson, K.F., et al., 2004) used the modified EZ::TN transposition system to successfully knock out the trypsin VTP1 of Verticillium dahliae from tomato . This gene can promote the formation of microsclerotia, but its pathogenicity and growth characteristics were not affected after knockout. In 2005, Dobinson KF et al knocked out the cytokinin-activated protein kinase gene VMK1 of Verticillium dahliae from lettuce and tomato. After knocking out VMK1, the pathogenicity of the strain to various hosts was severely reduced, indicating that the signaling pathway mediated by MAP kinase plays an important role in fungal pathogenicity. And knockout of the gene reduced the production of spores and the formation of microsclerotia, indicating that the gene may be involved in multiple cellular processes.

Due to the seriousness of cotton verticillium wilt damage and the wide range of hosts, scientific and technological workers in many countries in the world have carried out in-depth research on it. Plants have obtained a series of complex defense mechanisms to protect themselves during the long-term co-evolution with pathogens. Resistance is manifested as constitutive resistance and induced resistance, and induced resistance includes tissue structure resistance and physiological and biochemical resistance. sex. Cotton varieties with different resistance to Verticillium wilt have certain differences in tissue structure, which has been confirmed by many studies at home and abroad. The xylem of resistant cultivars had smaller intercellular spaces, thicker cell walls, and more pith rays in the xylem. In addition, the vessel lumen and fiber lumen diameters of the resistant varieties were smaller than those of the susceptible varieties, indicating that the resistance of cotton varieties to Verticillium wilt is related to their firm xylem. There have been many studies on the physiological and biochemical disease resistance of cotton, and the disease resistance-related factors that have been studied more include: phytoalexin, tannin, soluble sugar, amino acid and various enzymes. After the cotton plant is attacked by pathogens, it produces a variety of antibacterial substances, mainly including gossypol, phytoalexin, tannin, etc., and also with some enzymes, proteins, and small molecular substances such as H202. Their action is non-specific and related to the basal resistance of the plant. The mechanism of cotton resistance to Verticillium wilt is a very complicated issue involving many factors. Therefore, continuous in-depth research on the regularity of these disease resistance reaction products at the gene expression level is crucial for further understanding the disease resistance mechanism and transforming cotton by genetic engineering. Variety resistance is of great significance.

The acquisition of disease-resistant genes is an important basis for breeding disease-resistant varieties. At present, more than 39 plant disease-resistant genes have been cloned by means of molecular biology, of which there are about 20 resistant to pathogenic fungi, such as Arabidopsis thaliana Powdery mildew resistance gene RPW8, tomato fusarium wilt resistance gene Secret Ve, sorghum resistance to common rust (Puccinia Sorghi) gene Rpl-D, and NBS-LRR resistance genes have been cloned in sea island cotton. In conventional breeding, domestic and foreign cotton breeders have always paid attention to the screening and creation of resistance sources. From 1983 to 1986, Li Chengbao and others identified 161 pairs of 911 upland cotton resources against Verticillium wilt, and screened a batch of resistant (tolerant) varieties with better resistance. K.V.Srinvasin identified the resistance of 126 sea island cotton varieties, and the results showed that 85% of them showed disease tolerance and disease resistance. Some progress has been made in either finding the source of resistance through conventional methods or cloning disease-resistant genes through molecular biology methods, but no effective way to truly control cotton Verticillium wilt has been found. The fundamental reason is that the genetic background of crops such as cotton is complex, and it is difficult to conduct in-depth research at the molecular level. In addition, the strong variability in the differentiation of Verticillium dahliae species also brings many difficulties to disease-resistant genetic breeding. Therefore, it is very important to study the molecular mechanism of the interaction between Verticillium dahliae, the pathogen of Verticillium dahliae in cotton, and the host plant.

Contents of the invention

The invention provides a pathogenic gene and protein of Verticillium dahliae and its application. The pathogenic gene and protein are closely related to the mechanism of Verticillium wilt in cotton caused by Verticillium dahliae. Genes reduce pathogenicity of Verticillium dahliae.

One aspect of the present invention provides a pathogenic protein of Verticillium dahliae, named after CLP-1 (calpain clp-1), from Verticillium dahliae Kleb., said protein has the ability to cause cotton The function of verticillium wilt is the following 1) or 2) protein:

1) A protein whose amino acid sequence is shown in SEQ ID NO: 2;

2) A protein derived from 1) in which the amino acid sequence of SEQ ID NO: 2 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is associated with pathogenicity of Verticillium dahliae.

Another aspect of the present invention provides a pathogenic gene (named as CLP-1) of Verticillium dahliae, the protein encoded by this gene has the effect of causing cotton verticillium wilt, which is any of the following 1) to 4) One of the genes:

1) The nucleotide sequence is as shown in the 1-347th, 438-746th, 808-1311th and 1368-2901th positions from the 5' end in SEQ ID NO: 1;

2) A gene whose nucleotide sequence is shown in SEQ ID NO: 1;

3) a gene that hybridizes with the gene defined in 1) or 2) and encodes the protein of claim 1 under stringent conditions;

4) A gene that has more than 90% homology with the gene defined in 1) or 2) and encodes the protein of claim 1.

SEQ ID NO: 1 is composed of 2901 deoxynucleotides. In the sequence table, the 1-347th position from the 5' end of SEQ ID NO: 1; the 438-746th position; the 808-1311th position; the 1368-2901st position The ORF region encodes the protein of the amino acid residue sequence described in SEQ ID NO: 2, the protein shown in SEQ ID NO: 2 is named CLP-1, and the gene encoding the CLP-1 protein is named CLP-1.

The "stringent conditions" are conditions sufficient to allow the nucleotide sequence to hybridize to the gene sequence shown in SEQ ID NO: 1, and these conditions are well known to those skilled in the art, for example: in 0.1×SSPE containing 0.1% SDS or Hybridize at 65°C in 0.1×SSC solution containing 0.1% SDS, and wash the membrane with this solution.

The present invention also provides recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria of the above-mentioned genes.

"Expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising regulatory elements operably linked to the desired nucleotide sequence. The regulatory elements may be promoters, enhancers, silencers, terminators and/or other elements controlling the expression of the nucleotide sequences, such as polyadenylation sequences and the like.

Another aspect of the present invention provides the use of the above-mentioned genes, and knocking out the above-mentioned genes in Verticillium dahliae reduces the pathogenicity of Verticillium dahliae.

Another aspect of the present invention provides a method for reducing the pathogenicity of Verticillium dahliae, knocking out the gene described in claim 2, specifically comprising the following steps:

(1) using two pairs of primers to amplify the sequences shown in SEQ ID NO:3 and SEQ ID NO:4 respectively, and import the fragments obtained after the amplification into the expression vector;

(2) Transforming Agrobacterium with an expression vector containing a fragment of the sequence shown in SEQ ID NO:3 and SEQ ID NO:4 obtained in step (1);

(3) select the successfully transformed Agrobacterium;

(4) Transfect Verticillium dahliae with the successfully transformed Agrobacterium described in step (3), and select a strain with resistance, that is, a strain with reduced pathogenicity.

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

Upstream primer 1: 5'→3' direction: GGGTTTAAUGATGAATACTTCGCACCACG (SEQ ID NO:5);

Upstream primer 2: 5'→3' direction: GGACTTAAUGTCAGTGGTGCTGCCATCAA (SEQ ID NO: 6);

Downstream primer 1: 5'→3' direction: GGCATTAAUACGCAAACCCAGGGCAAAAC (SEQ ID NO: 7);

Downstream primer 2: 5'→3' direction: GGTCTTAAUAACTCACGCGGCGGGATACT (SEQ ID NO: 8).

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

Through the upstream primer 1 and the upstream primer 2, the gene of the sequence shown in SEQ ID NO: 3 is amplified by PCR using the Verticillium dahliae genomic DNA as a template, and the downstream primer 1 and the downstream primer 2 use the dahliae Verticillium genomic DNA is used as a template to PCR amplify the gene of the sequence shown in SEQ ID NO:4, and the two amplified PCR products are connected to the expression vector.

The method described above, wherein, in step (2), the expression vector containing the fragments of the sequences shown in SEQ ID NO:3 and SEQ ID NO:4 is introduced into Agrobacterium competent cells by means of electric shock transformation.

The above-mentioned method, wherein, in step (3), by coating the Agrobacterium on a plate containing antibiotics and culturing, the strains that have successfully transformed are screened.

The method described above, wherein, in step (4), the fungal strain transformed by Agrobacterium is spread on a flat plate containing antibiotics to cultivate and screen for successfully transformed bacterial strains.

The pathogenic gene and protein provided by the present invention have been proved by experiments to be closely related to the mechanism that Verticillium dahliae causes cotton verticillium wilt, and can make Verticillium dahliae by knocking out the pathogenic gene of the present invention in Verticillium dahliae The pathogenicity of the mycobacteria is reduced, and the Verticillium dahliae mutant and the wild type Verticillium dahliae that have knocked out the pathogenic gene of the present invention infect the plant simultaneously, and the cotton that is infected by the Verticillium dahliae mutant strain is similar Compared with the wild type Verticillium dahliae infection, the incidence of Verticillium dahliae in cotton was reduced by more than 50%, and the disease index and disease grade were also greatly reduced. Therefore, the invention provides new enlightenment for studying the pathogenic mechanism of Verticillium dahliae, and provides a new approach for treating and preventing cotton verticillium wilt.

Description of drawings

Fig. 1 is the comparative electrophoresis of the CLP-1 knockout mutant detected by PCR at the DNA level in Example 2; Swimming lane 1 is a marker, and swimming lanes 2, 3, and 4 are wild-type Verticillium dahliae V592; Swimming lanes 5, 6, 7 is the CLP-1 knockout mutant Vda ^Δclp-1 -1; lanes 8, 9, and 10 are the CLP-1 knockout mutant Vda ^Δclp-1 -2. The two pairs of primers F1-HptR and HptF-R1 could not amplify bands in wild-type Verticillium dahliae V592, but Fg-Rg could amplify bands; on the contrary, CLP-1 knockout mutant Vda ^{Δclp -1} -1 and Vda ^Δclp-1 -2 can amplify bands with the two pairs of primers F1-HptR and HptF-R1, but Fg-Rg cannot amplify bands, indicating that CLP-1 is knocked out Lost;

Figure 2 is a blot of the expression of CLP-1 in the RNA level CLP-1 knockout mutant detected by Northern hybridization in Example 2; the figure shows that CLP-1 is knocked out in the Northern detection knockout mutant, not express. Lanes 1 and 2 are wild-type Verticillium dahliae V592 that can express CLP-1; lanes 3 and 4 are knockout mutants Vda ^Δclp-1 -1 and Vda ^Δclp-1 -2 that cannot express CLP-1.

Fig. 3 is the uninfected control cotton plant in embodiment 3;

Fig. 4 is the cotton plant after being infected by Verticillium dahliae V592 in embodiment 3;

Fig. 5 is the cotton plant after being infected by the knockout mutant Vda ^Δclp-1 of Verticillium dahliae V592 in embodiment 3;

Fig. 6 is a comparison chart of the incidence of cotton plants infected by wild-type Verticillium dahliae V592 and the knockout mutant Vda ^Δclp-1 for 20 days in Example 3;

Fig. 7 is a comparison chart of the disease index of cotton plants in Example 3 after being infected by wild-type Verticillium dahliae V592 and the knockout mutant Vda ^Δclp-1 for 20 days;

Fig. 8 is a graph comparing the disease grades of cotton plants infected by wild-type Verticillium dahliae V592 and the knockout mutant Vda ^Δclp-1 for 20 days in Example 3.

detailed description

The specific implementation manners of the present invention will be described in more detail below in conjunction with the accompanying drawings and examples, so as to better understand the solution of the present invention and its advantages in various aspects. However, the specific embodiments and examples described below are for the purpose of illustration only, rather than limiting the present invention.

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Agrobacterium tumefaciens EHA105 (Elizabeth E. Hood. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Research, July 1993, Volume 2, Issue 4, pp 208-218) in the following examples is available to the public for twenty years from the filing date Obtained from the Institute of Microbiology, Chinese Academy of Sciences, this biological material is only used for repeating related experiments of the present invention, and cannot be used for other purposes.

Verticillium dahliae V592 (Feng-Gao, Bang-JunZhou, A GlutamicAcid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity.PLoS ONE5(12):e15319.) in the following examples from the application The public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences 20 years after the date of the date. The biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

"Wild type" in the following examples means an organism that does not contain heterologous nucleic acid molecules, which is a non-transformed or non-transgenic organism.

Embodiment 1, the construction of CLP-1 knockout vector

Utilize the CTAB method to extract Verticillium dahliae V592 genomic DNA, use the following primers to use the genomic DNA as a template, use C _x Hotstart DNA polymerase (Agilent) for PCR amplification: upstream primer 1: 5'→3' direction: GGGTTTAAUGATGAATACTTCGCACCACG (SEQ ID NO:5); upstream primer 2: 5'→3' direction: GGACTTAAUGTCAGTGGTGCTGCCATCAA (SEQ ID NO :6); downstream primer 1: 5'→3' direction: GGCATTAAUACGCAAACCCAGGGCAAAAC (SEQ ID NO:7); downstream primer 2: 5'→3' direction: GGTCTTAAUAACTCACGCGGCGGGATACT (SEQ ID NO:8). USER enzyme mix (New England Biolabs) was used to simultaneously connect the two PCR products to the pGKO-HPT vector (Tian, L., Chen, J., Wang, J., Wang, J., and Dai, X.2011, since Twenty years from the date of application, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes). The sequencing results show that it contains SEQ ID No: 3 and SEQ ID No. : The recombinant vector of the DNA molecule shown in 4 was determined as the final knockout vector. Agrobacterium-mediated transformation was used to transform wild-type Verticillium dahliae V592 into a CLP-1 knockout mutant.

Example 2, Obtaining of CLP-1 Knockout Mutants

1) Genetic transformation of fungi by Agrobacterium-mediated method

Related media:

(a) Chase medium (30g/L sucrose, 3g/L NaNO ₃ , 0.5g/L MgSO ₄ -7H ₂ O, 0.5g/L KCl, 100mg/L FeSO ₄ -7H ₂ O, 1g/L K ₂ HPO ₄ , pH 7.2). LB liquid medium: 10g of peptone, 5g of yeast extract, 1000ml of water, adjust the pH to 7 with NaOH, and sterilize by high-pressure steam for 20min at 121°C.

(b) LB liquid medium: _peptone 10g, yeast extract 5g, NaCl 5g, add water to 1L, steam sterilize at 121°C for 20min. Solid medium plus 1.5% agar.

(c) MM basic medium: 10mL K-bufer (200g/L K ₂ HPO ₄ , 145g/L KH ₂ PO ₄ , H ₃ PO ₃ to adjust the pH to 7.0), 20mL MN buffer (30g/L MgSO ₄ ·7H ₂ O, 15g/L NaCl), 1 mL 1% CaCl ₂ 2H ₂ O (w/v), 10 mL 20% sucrose (w/v), 1 mL 0.1% FeSO ₄ (w/v), 0.5 g NH ₄ NO ₃ , add distilled water to 1L. 113°C, steam sterilization for 20min.

(d) IM induction medium: 10 mL K-bufer (pH 7.0), 20 mL MN buffer, 1 mL 1% CaCl ₂ ·2H ₂ O (w/v), 2.5 mL 20% NH ₄ NO ₃ (w/v), 1mL 0.1% FeSO ₄ (w/v), 5mL glycerol, 5mL 2mol/L sucrose, 2mL 100mmol/L acetosyringone, 40mL 1 mol/L MES (pH 5.3), add distilled water to 1L. 113°C, steam sterilization for 20min.

(e) CM co-cultivation medium: add 1.5% agar powder to IM medium, steam sterilize at 113° C. for 20 minutes.

specific steps

(1) Pick a single colony of Agrobacterium and put it into 5ml of LB liquid medium containing antibiotics (50ug/ml kanamycin, 50ug/ml rifampicin), place it on a shaker at 28°C, and cultivate it at 200rpm for 24 hours, and At the same time, use a toothpick to pick 3 V592 bacterium cakes on the PDA solid medium and put them into a conical flask containing 80ml Chase medium, place on a shaker at 26°C, and cultivate at 200rpm.

(2) Take 1ml of the Agrobacterium cultured for 24 hours and add it to 20ml of MM liquid medium containing antibiotics (50ug/ml kanamycin, 50ug/ml rifampicin), place on a shaker at 28°C, 200rpm After continuing to cultivate for 24 hours, centrifuge at 4000rpm for 10 minutes to collect the bacteria, wash the bacteria twice with IM medium, and then resuspend with IM medium to adjust OD ₆₀₀ ≈0.25. After 48 hours of cultivation, the wild-type large Verticillium verticillium V592 was filtered with four layers of sterilized gauze, and the filtered bacterial solution was placed in a centrifuge at 4000rpm, centrifuged for 10min, and the spores were resuspended with IM+AS, counted on a hemocytometer, and the spore concentration was adjusted to 1.0×10 ^6-7 spores/ml.

(3) Mix the Agrobacterium bacteria liquid and the fungal spore suspension at a volume ratio of 1:1 (1ml each), spread 200μl of cellophane on a CM medium plate per petri dish, and incubate at 26°C for 36 hours.

(4) Rinse the co-culture with 3ml sterile water, and apply 500ul/plate on the plate containing antibiotics (hygromycin 50ug/ml, carbenicillin 200ug/ml, cephalosporin 200ug/ml, 20ug/ml) Fluorouracil) on PDA solid medium for 5-7 days.

2) PCR identification of CLP-1 knockout mutants (Figure 1)

A. Genomic DNA of CLP-1 knockout mutant was extracted by CTAB method.

B. PCR amplification using three pairs of primers. The first pair: design a primer F1: 5'-GGCTGACCTTGTCGGTGTCT-3' (SEQ ID NO: 9) upstream of the upstream fragment of SEQ ID NO: 3 of the target gene CLP-1, and a primer HptR on the HPT BOX: 5' - AAATTTTGTGCTCACCGCCTGGAC-3' (SEQ ID NO: 10) for upstream fragment PCR amplification. The second pair: a primer redesigned downstream of the target gene downstream fragment SEQ ID NO: 4, named R1: 5'-CCGTTGGTGGGTAGGTATCA-3' (SEQ ID NO: 11), another primer HptF on the HPT BOX: 5'-TCTCCTTGCATGCACCATTCCTTG-3' (SEQ ID NO: 12) was used to amplify the downstream fragment. If both primer pair 1 and primer pair 2 can amplify fragments of the same size as expected, it indicates that recombination occurs at the position of the target gene. The third pair of primers: Fg: 5'-CGAAATCGATGGATCCCAGGATCAAAAGCCCTCTAC-3' (SEQ ID NO: 13), Rg: 5'-AGGCTACGTAGGATCCTTACCACTGCGGTGCTTACA-3' (SEQ ID NO: 14) is used to amplify the knockout target gene, but cannot amplify The surface target gene was indeed knocked out successfully.

Example 3. Detection of CLP-1 expression in CLP-1 knockout mutants by Northern hybridization

A. Trizol reagent method to extract fungal tissue RNA

(1) Grind the fungal material into powder with liquid nitrogen in a mortar, add 1ml RNAVzol per 50-100mg of tissue, homogenize in a centrifuge tube until completely lysed; place at room temperature for 5 minutes.

(2) Add 0.2 times the volume of chloroform (0.2 ml chloroform to 1 ml RNAVzol) into the centrifuge tube containing the lysate, shake and mix well with a shaker for 30 seconds, and place at room temperature for 2 to 3 minutes.

(3) 4°C, 14000rpm, centrifuge for 10min, absorb the upper aqueous phase into a new centrifuge tube, and absorb about 0.5-0.55ml per ml of RNAVzol.

(4) Add 0.5ml of isopropanol per ml of the original RNAVzol, invert several times, mix well, and precipitate at room temperature for 10 minutes.

(5) 4°C, 1,4000rpm, centrifuge for 10min, RNA precipitation can be seen at the bottom of the tube. Discard the supernatant, add 1ml of 75% ethanol to each ml of the original RNAVzol, and mix gently by inversion to wash the RNA pellet. Discard the liquid, being careful not to discard the RNA pellet. Inverted at room temperature to dry (5 ~ 10min).

(6) Add an appropriate amount of 50% deionized formamide to dissolve the RNA precipitate, and store it at -80°C for later use.

B. RNA electrophoresis

(1) Related reagents

20×SSC (175.3g NaCl, 88.2g trisodium citrate, adjust pH to 7.0 with HCl, dilute to 1L, autoclave), 10×MOPS (41.8g MOPS, 6.56g NaAc, 20ml 0.5M EDTA, use Adjust the pH to 7.0 with NaOH, dilute to 1L, put in a brown bottle, autoclave or filter sterilize. After autoclaving, it will appear light yellow (available, yellow is not available), 37% formaldehyde (Formaldehyde), deionized formamide (Formamide deionized), methylene blue staining solution (0.03% methylene blue, 0.3M NaAc, pH5.2).

(2) RNA electrophoresis

(a) Preparation of 1.2% agarose formaldehyde denaturing gel:

(b) Processing of RNA samples before loading:

Sample buffer mix Volume: 25.5μl 10×MOPS 5μl 37% Methanol 8μl Deionized formamide 12.5μl

Mix equal volumes of the Sample buffer mix and the RNA sample, denature at 65°C for 5 minutes, place on ice for 5 minutes, add loading buffer, mix well and load the sample.

(c) RNA electrophoresis. The buffer used is 1×MOPS, 120V for about 3h.

(3) Membrane transfer (upward capillary transfer method)

(a) Use a rubber plate with a length and width larger than the gel as a platform, put it in a plastic plate, pour 20×SSC into it, cut a piece of filter paper that is as wide as the platform and longer than the platform, and soak one end of the filter paper in the plastic Put it on the platform slowly until the other end is also soaked in 20×SSC, and drive out the air bubbles between the filter paper and the platform;

(b) Cut a piece of nylon membrane whose length and width are slightly larger than the gel, cut off the upper left corner, completely soak in sterile water, take out the membrane, and then immerse it in 20×SSC for at least 5 minutes;

(c) After electrophoresis, cut off the useless part of the gel, and cut off a corner in the upper left corner as an orientation mark. Rinse the gel in 20×SSC for a while;

(d) Put the gel upside down in the center of the filter paper on the platform, drive out the air bubbles between the gel and the filter paper, surround the gel with Parafilm, and do not touch the sample on the gel;

(e) Wet the gel with 20×SSC, place the wet nylon membrane on top of the gel, make the cut corners of the two overlap, and drive out the air bubbles between the nylon membrane and the gel;

(f) Wet 5 pieces of filter paper with the same size as the nylon membrane with 20×SSC, place on the nylon membrane, and drive out the air bubbles between the filter paper and the nylon membrane;

(g) Cut a stack of paper towels with a thickness of 10 cm and slightly smaller than the filter paper, put them on the filter paper, put a glass plate on the paper towels, press a 500g weight on it, and transfer overnight;

(h) Peel off the paper towel, filter paper and Parafilm above the gel, turn over the gel and the nylon membrane, put the gel side up on a plastic plate, and mark the position of the gel injection hole on the nylon membrane with a pencil;

(i) Peel off the gel from the nylon membrane and discard it, soak the nylon membrane in 20×SSC for 5 minutes, take out the nylon membrane, put it on wet filter paper, and fix RNA by UV crosslinking;

(j) Stain with methylene blue until a clear band of RNA is seen, rinse with distilled water for decolorization, wrap in plastic wrap, and store at 4°C until use.

(4) Probe labeling

(Amersham company random primer labeling kit, RediprimeTMII Random PrimerLabelling System).

(a) Take 25ng of DNA to be labeled and add sterile water to amplify the volume to 45μl.

(b) Incubate at 98°C for 5 minutes to denature DNA.

(c) Centrifuge to pool the DNA at the bottom of the centrifuge tube and place on ice.

(d) Add the denatured DNA to the labeling mixture and mix gently until the pellet is completely melted (do not blow it with a gun).

(e) Add 1 μl of Klenow (to prevent inactivation of Klenow in the labeling mixture), and centrifuge.

(f) Add 5 μl α-32P-dCTP, pipette gently with a gun to evenly, and centrifuge.

(g) React at 37°C for 30 minutes; at 98°C for 5 minutes, denature the labeled DNA probe, centrifuge, and place on ice.

(h) Take an appropriate amount of probes for hybridization, and store the remaining probes in a refrigerator at 4°C.

C. hybridization

Preparation of hybridization buffer

Hybridization buffer: 20.4ml required volume Final concentration 8.6ml 43mM 20% SDS 7ml 7% 5%BSA 4ml 1% 0.5M EDTA 0.8ml 20mM

(a) Pre-hybridization: Pour the hybridization buffer into the hybridization tube and preheat at 65°C for 15 minutes, then put it into the cross-linked and fixed membrane and pre-mix at 65°C for 1-2 hours;

(b) Hybridization: put the labeled probe into a hybridization tube, and hybridize overnight at 65°C;

(c) Membrane washing: Wash the membrane 2 to 3 times with membrane washing buffer 2×SSC/0.2% SDS at 65°C/15 min;

(d) Pressing: Take the washed membrane out of the hybridization tube, transfer it to two layers of plastic film, detect the strength of the hybridization signal, put the membrane into a phosphor screen, and press it for several hours or overnight according to the strength of the signal;

(e) Detect hybridization signal: scan the phosphor screen.

The result is shown in Figure 2.

Pathogenicity detection of embodiment 3 knockout mutants

The pathogenicity of the strain was identified by infecting hydroponic cotton. Select plump cotton seeds, soak them in 15% sodium hypochlorite for 30 minutes, rinse them with sterile water 2-3 times, soak them in sterile water overnight, spread them in a culture box to keep them moist, wait until the buds grow to 3cm, and plant them in a germination box middle. The seedlings with cotyledons were transferred to a plastic box (8-10 cm high) filled with clear water, and cultivated at 25° C. under light for 16 hours and darkness for 8 hours. When the true leaves grow out, replace the clear water with 1/3 of the MS culture solution, replace the culture solution once a week, and inoculate when a true leaf is flattened. The Verticillium dahliae V592 strain and the CLP-1 knockout mutant Vda ^Δclp-1 stored at -80°C were activated on a PDA plate for 3-4 days, and the bacterial blocks were picked from the edge of the colony and put into Chase culture medium at 25°C. Shake culture at 220rpm for 5 days, filter, centrifuge the filtrate at 5000rpm for 5min, dilute the spores with water, count on a hemocytometer, and adjust the concentration to 1×10 ⁷ spores/ml. Add the adjusted concentration of spore suspension into an empty plastic box, and soak the roots of cotton seedlings for 40 minutes. Afterwards, 1/3 of the MS culture solution was used to light for 16 hours at 25°C, and the cotton seedlings were continued to be cultivated for 8 hours in the dark. 12 seedlings were planted in each box, and each variety had 3 replicates. The control cotton seedlings were soaked in water for 40 minutes. After 20 days, the incidence was observed.

Through the above method, we verified that the pathogenicity of the CLP-1 knockout mutant to cotton was significantly weakened

Compute incidence and condition indices:

Incidence rate = number of cases/total number of investigations × 100% (see Figure 6)

Disease index=∑[number of disease grades×the number of diseased leaves (ears, strains) at this level]/(total number of surveys×the highest number of disease grades)×100 (see Figure 7)

Disease grading standard:

Level 0 plant health has no symptoms;

Level 1: 0.1%-25% leaves wilt;

Level 2: 25%-50% of the leaves wilt;

Grade 3: 50%-75% of the leaves wilt;

Grade 4: 75%-100% of the leaves wilt or die;

Statistical diagram of disease classification standard of cotton infected by wild-type Verticillium dahliae V592 and cotton infected by knockout mutant Vda ^Δclp-1 (see FIG. 8 ).

From the result graph of the above method, we can see that the incidence, disease index and disease classification of the cotton infected by the knockout mutant Vda ^Δclp-1 are significantly lower than those infected by the wild-type Verticillium dahliae V592, indicating that CLP The -1 gene is associated with the pathogenicity of Verticillium dahliae.

Finally, it should be noted that obviously, the above-mentioned embodiments are only examples for clearly illustrating the present invention, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims (10)

1. a kind of pathogenic protein of verticillium dahliae, it is characterised in that the albumen has causes cotton The effect of verticillium wilt, is following protein 1) or 2)：

1) amino acid sequence such as SEQ ID NO：Protein shown in 2；

2) by SEQ ID NO：2 amino acid sequence by one or several amino acid residues substitution And/or missing and/or addition and caused a disease the related protein as derived from 1) to verticillium dahliae.

2. a kind of Disease-causing gene of verticillium dahliae, it is characterised in that the albumen of the gene code has Cause the effect of cotton verticillium wilt, be following 1) to any described gene in 4)：

1) nucleotide sequence such as SEQ ID NO：From 5 ' end 1-347,438-746 in 1 Position, 808-1311 and the gene shown in 1368-2901；

2) nucleotide sequence such as SEQ ID NO：Gene shown in 1；

3) under strict conditions with 1) or 2) described in the gene recombination and coding claim 1 for limiting The gene of albumen；

1) or 2) 4) there is more than 90% homology and coding claim 1 with the gene for limiting The gene of the albumen.

3. recombinant vector, expression cassette, transgenic cell line or weight containing gene described in claim 2 Group bacterium.

4. the purposes of gene as claimed in claim 2, it is characterised in that knocked out in verticillium dahliae Gene described in claim 2 makes the pathogenic reduction of verticillium dahliae.

5. it is a kind of to reduce the pathogenic method of verticillium dahliae, it is characterised in that to knock out claim 2 Described gene, specifically includes following steps：

(1) SEQ ID NO are expanded respectively using two pairs of primers:3 and SEQ ID NO:Sequence shown in 4, The fragment that will be obtained after amplification imports expression vector；

(2) SEQ ID NO are contained by what is obtained in step (1):3 and SEQ ID NO:Shown in 4 The expression vector conversion Agrobacterium of the fragment of sequence；

(3) the successful Agrobacterium of conversion is chosen；

(4) by the successful Agrobacterium infection verticillium dahliae of conversion described in step (3), choosing Take resistant bacterial strain, the bacterial strain of as pathogenic reduction.

6. method as claimed in claim 5, it is characterised in that two pairs are drawn described in step (1) Thing sequence is as follows：

Sense primer 1：5 ' → 3 ' directions：GGGTTTAAUGATGAATACTTCGCACCAC G；

Sense primer 2：5 ' → 3 ' directions：GGACTTAAUGTCAGTGGTGCTGCCATCA A；

Anti-sense primer 1：5 ' → 3 ' directions：GGCATTAAUACGCAAACCCAGGGCAAA AC；

Anti-sense primer 2：5 ' → 3 ' directions：GGTCTTAAUAACTCACGCGGCGGGATAC T。

7. method as claimed in claim 6, it is characterised in that step is also specifically included in (1) Following steps：

By the sense primer 1 and the sense primer 2 with verticillium dahliae genomic DNA as mould Plate PCR amplification SEQ ID NO:The gene of sequence shown in 3, the anti-sense primer 1 and the downstream are drawn Thing 2 with verticillium dahliae genomic DNA be template PCR amplifications SEQ ID NO:Sequence shown in 4 Gene, by amplification after two kinds of PCR primers be all connected to expression vector.

8. method as claimed in claim 5, it is characterised in that turned by electric shock in step (2) The method of change described will contain SEQ ID NO:3 and SEQ ID NO:The expression of the fragment of sequence shown in 4 Vector introduction Agrobacterium competent cell.

9. method as claimed in claim 5, it is characterised in that by will be described in step (3) Agrobacterium is coated and cultivated on the flat board containing antibiotic, the successful bacterial strain of screening conversion.

10. method as claimed in claim 5, it is characterised in that by by agriculture bar in step (4) Fungal bacterial strain after bacterium conversion is coated and cultivated on the flat board containing antibiotic the screening successful bacterium of conversion Strain.