CN113481216B - Multi-main corynespora CcTLS1 protein and coding gene and application thereof - Google Patents

Abstract

The application discloses a corynespora polymorpha CcTLS1 protein, a coding gene and application thereof. Experiments prove that after the CcTLS1 gene of the corynespora multocida is knocked out, the obtained knocked-out mutant of the corynespora multocida has a greatly reduced disease spot area on cucumber leaves compared with a wild strain; the hypha growth speed of the corynespora polymorpha knockout mutant is obviously lower than that of the wild corynespora polymorpha; the activity of the mutant cellobiohydrolase of the knock-out mutant of the corynebacterium polymorpha is obviously lower than that of the wild corynebacterium polymorpha. Indicating that the deletion of the CcTLS1 gene of the corynespora polymorpha knockout mutant can lead to the reduction of the infection capacity of the corynespora polymorpha on the cucumber. The CcTLS1 gene and the application thereof provided by the application have important significance in preventing and controlling the cucumber corynespora leaf spot.

Description

Multi-main corynespora CcTLS1 protein and coding gene and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a corynespora polymorpha CcTLS1 protein, a coding gene and application thereof.

Background

As a broad host range of plant pathogens, corynespora polymorpha [ Corynespora cassicola (Berk. & m.a. Curtis) c.t.wei ] infects not only crops of important economic value, such as cucumbers, tomatoes, peppers, eggplants, papaya, soybeans, rubber trees and the like, but also horticultural flower plants. Corynespora polymorpha has a variety of lifestyles including saprophytic, endophytic and necrotic. In recent years, leaf spot disease of coryneform bacteria is an important disease in vegetable production.

The corynespora polymorpha attaches the disease residues and seeds in the form of mycelium and conidium, and survives in soil, and also survives on non-host plants to become a new infection source. The corynespora polymorpha can survive for 2 years on the disease residue. After 4 months of overspray, 70% of the conidia still germinate, which can become the infection source of the next crop. Hyphae of the corynespora polymorpha can be attached to the surface layer of the seeds and can also submerge into the seeds. The corynespora polymorpha can be separated from cucumber seeds stored for 6 months and pumpkin seeds used for stocks by separation culture.

In recent years, research on pathogenic mechanisms of corynespora polymorpha has been mainly focused on biological characteristics, pathogenic differentiation, cloning of virulence-related genes, and the like. It has been reported that the coryneform polymorpha harvested from cucumber can grow at 10-35℃with an optimum growth temperature of about 30 ℃. The corynespora polymorpha spores germinate from one or both ends at a temperature range of 25-30 ℃ with a relative humidity >90%, wherein the germination rate is highest in water droplets. The corynespora polymorpha invades the cucumber leaf mainly through direct contact or stomata. Pathogenic identification and genetic diversity analysis are carried out on the corynespora multocida separated from different host plants, so that the corynespora multocida has higher host specialization. 64 strains of corynespora polymorpha isolated from Japanese perilla, cucumber, tomato, eggplant and sweet pepper were divided into 7 pathogenic groups (PG 1-PG 7). Cassiicolin is a small secreted glycoprotein, an important pathogenic agent of corynebacterium polymorpha, and in a multi-main corynebacterium isolate of different hosts and geographical origin, cassiicolin toxin comprises 6 different subtypes Cas1, cas2, cas3, cas4, cas5 and Cas 6. The invasive capacity of each strain is related to its subtype, with the strain carrying the Cas1 gene being the most aggressive to rubber trees. In addition, some strains without Cas gene also produced moderate symptoms on the rubber tree leaves, indicating the presence of insignificant effectors of corynespora polymorpha.

Like other filamentous fungal pathogens, corynebacterium polymorpha has species pathogenic agents such as cutinases, cell wall degrading enzymes, cell membranes and cell inclusion body degrading enzymes, and furthermore, can invade host plants and cause diseases by toxins (Cassiicolin) which pass through mitosisProactivatory protein kinase (MAPK), ca ²⁺ And cAMP signaling pathway. To date, other pathogenic related genes besides the two MAPK genes CCk1 and CMP1 and Cassiicolin encoded gene Cas are rarely cloned and functionally identified, and far from fully represent the pathogenic mechanism of Corynebacterium polymorphum. Thus, a large number of virulence related genes remain to be identified, cloned and functionally characterized. Therefore, it is important to study the molecular mechanism of interaction of corynespora polymorpha with host plants.

Disclosure of Invention

The application aims to provide a gene related to pathogenicity of corynespora polymorpha, and the gene is used as an action target point to realize control of the corynespora leaf spot.

In order to solve the above technical problems, a first object of the present application is to provide an application, which is any one of the following:

application of P1, protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or the content of the protein in regulating the pathogenicity of corynespora polymorpha;

application of P2, protein or a substance for regulating expression of a gene encoding the protein or a substance for regulating activity or content of the protein in regulating the size of a multi-main corynespora plaque;

application of P3, protein or a substance for regulating the expression of a protein coding gene or a substance for regulating the activity or the content of the protein in regulating the growth rate of the hyphae of the corynespora polymorpha;

p4, protein or a substance regulating the expression of a gene encoding the protein or the application of a substance regulating the activity or the content of the protein in regulating the growth and development of corynespora polymorpha;

use of P5, a protein or a substance regulating expression of a gene encoding said protein or a substance regulating activity or content of said protein in regulating the activity of a multimeric cellulase of corynespora polymorpha;

application of P6, protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in regulating the secretion of a polysaccharide hydrolase of corynebacterium polymyxa;

application of P7, protein or a substance for regulating expression of a gene encoding the protein or a substance for regulating activity or content of the protein in controlling plant diseases caused by corynespora polymorpha;

application of P8, protein or a substance for regulating expression of a gene encoding the protein or a substance for regulating activity or content of the protein in preventing and treating cucumber corynespora leaf spot caused by corynespora polymorpha;

application of P9, the protein or the protein coding gene as a target of a drug for controlling plant diseases; the plant disease is cucumber corynespora leaf spot caused by corynespora polymorpha;

the protein is any one of the following proteins:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 A protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in the A1), has more than 90 percent of identity with the protein shown in the A1) and has the function of regulating and controlling the pathogenicity of the coryneform bacteria;

a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).

Wherein, the sequence 1 in the sequence table consists of 389 amino acid residues.

In the above application, the protein is derived from corynespora polymorpha (Corynespora cassicola).

Herein, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

Herein, the identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

Herein, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 99% identity.

In the above application, the gene encoding the protein may be a DNA molecule as shown in a 1) or a 2) or a 3) as follows:

a1 A coding sequence is a DNA molecule shown as a sequence 2 in a sequence table;

a2 A DNA molecule which has 90% or more identity to the nucleotide sequence defined in a 1) and which encodes the protein A described above;

a3 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined under a 1) or a 3) and which codes for a protein as described above.

In the above application, the substance that regulates the activity or content of the protein may be a substance that regulates the expression of the gene encoding the protein.

In the above application, the substance that regulates the expression of the protein-encoding gene may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

In the above application, the modulating the gene expression may be inhibiting or reducing the gene expression, which may be achieved by gene knockout or by gene silencing.

The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.

The gene silencing refers to the phenomenon that the gene is not expressed or expressed under the condition of not damaging the original DNA. Gene silencing is premised on the fact that the DNA sequence is not altered, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of a target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression (co-suppression), gene suppression (sequencing), RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.

In the above application, the substance regulating the expression of the gene encoding the protein or the substance regulating the activity or content of the protein may be any one of the following c 1) to c 4):

c1 A nucleic acid molecule that inhibits or reduces expression of a gene encoding a protein as described above;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

c3 A recombinant vector comprising c 1) said nucleic acid molecule, or a recombinant vector comprising c 2) said expression cassette;

c4 A recombinant microorganism comprising c 1) said nucleic acid molecule, or a recombinant microorganism comprising c 2) said expression cassette, or a recombinant microorganism comprising c 3) said recombinant vector.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The present application also provides the above-mentioned protein or the above-mentioned substance regulating the expression of the gene encoding the protein or the substance regulating the activity or content of the protein.

The present application also provides a method for reducing the pathogenicity of corynespora polyvidans, comprising reducing the pathogenicity of corynespora polyvidans by inhibiting or reducing the expression level of the gene encoding the protein or the activity of the protein in the corynespora polyvidans of interest; the protein is any one of the following:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 A protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in the A1), has more than 80 percent of identity with the protein shown in the A1) and has the function of regulating and controlling the pathogenicity of the coryneform bacteria;

a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).

In the above method, the gene encoding the protein may be a DNA molecule as shown in a 1) or a 2) or a 3) as follows:

a1 A coding sequence is a DNA molecule shown as a sequence 2 in a sequence table;

a2 A DNA molecule which has 80% or more identity to the nucleotide sequence defined in a 1) and which encodes the protein A described above;

a3 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined under a 1) or a 3) and which codes for a protein as described above.

In the above, the protein tag (protein-tag) refers to a polypeptide or protein which is fusion expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

Above, the 80% identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The application also provides a method for preparing the recombinant corynespora polyrhiza, which comprises inhibiting or reducing the expression quantity of the coding gene of the protein in the target corynespora polyrhiza or the activity of the protein to obtain the recombinant corynespora polyrhiza.

The use of substances which inhibit the expression of genes encoding said proteins or which reduce the activity of said proteins for the preparation of inhibitors of coryneform bacteria also falls within the scope of the present application.

The application of the substance or the method for reducing the pathogenicity of the corynespora polymorpha in preventing and controlling plant diseases caused by the corynespora polymorpha also belongs to the protection scope of the application. The plant disease caused by the corynespora polymorpha may be cucumber corynespora leaf spot.

Experiments prove that after the CcTLS1 gene of the corynespora multocida is knocked out, the obtained knocked-out mutant of the corynespora multocida has a greatly reduced disease spot area on cucumber leaves compared with a wild strain; the hypha growth speed of the corynespora polymorpha knockout mutant is obviously lower than that of the wild corynespora polymorpha; the activity of the mutant cellobiohydrolase of the knock-out mutant of the corynebacterium polymorpha is obviously lower than that of the wild corynebacterium polymorpha. Indicating that the deletion of the CcTLS1 gene of the corynespora polymorpha knockout mutant can lead to the reduction of the infection capacity of the corynespora polymorpha on the cucumber. The CcTLS1 gene and the application thereof provided by the application have important significance in preventing and controlling the cucumber corynespora leaf spot.

Drawings

FIG. 1 is a graph showing the results of PCR identification of CcTLS1 knockout mutants in example 1. Wherein, WT is wild type corynebacterium polymyxa HG14102524, P is pCAMBIA1300- ΔCcTLS1, and ΔCcTLS1 is CcTLS1 knockout mutant.

FIG. 2 is a graph showing the results of PCR identification of the CcTLS1 complementation mutant in example 2. Wherein, WT is wild type coryneform bacterium HG14102524, P is pCAMBIA1300-cΔCcTLS1, and cΔCTLS1 is CcTLS1 complementation mutant.

FIG. 3 shows the spot diameters of ΔCcTLS1 (knockout strain), cΔCcTLS1 (complementation strain) and HG14102524 (wild type CcHB) measured by the crisscross method in example 3.

FIG. 4 is a graph showing the results of measuring the growth rates of ΔCcTLS1, cΔCTLS1 and HG14102524 every 24 hours by the crisscross method in example 4.

FIG. 5 is a graph showing the results of measuring the cellulase activities of ΔCcTLS1, cΔCTLS1 and HG14102524 in example 5.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

1. Strain and vector

The strain HG14102524 of Corynebacterium polymorphum (Corynespora cassicoa) in the following examples is described in non-patent document "Zhu Fa," resistance of Corynebacterium polymorphum (Corynespora cassicola) to boscalid and its mechanism study, "the biological material is available to the public from the national academy of agricultural sciences, 2018," and is used only for repeated experiments related to the present application, but is not used for other purposes.

Agrobacterium strain AGL-1, product of Beijing Bomaide Gene technology Co., ltd., product No. BC302-01 was used in the following examples.

The pPIC9K-His plasmid in the examples below was obtained as VECT2430 from Beijing Wash Vietnam Biotechnology Co., ltd.

The pCAMBIA1300 plasmid in the examples below was obtained as VECT0070 from Beijing Wash Vietnam Biotechnology Co., ltd.

NEO-GFP plasmids in the following examples are described in the non-patent literature: "Zhang Yu, a novel fungicide, cyanomycoester, inhibits the mechanism of Fusarium asiaticum (Fusarium asiaticum) trichothecene toxin synthesis.

2. Culture medium

The PDA culture medium used in the following examples was prepared by the following method: 200g of potato, 20g of glucose and 15g of agar, dissolving in water, fixing the volume to 1L, and sterilizing by steam at 121 ℃ for 20min.

The PDA solid medium containing antibiotics (hygromycin 150. Mu.g/ml, cephalosporin 600. Mu.g/ml) used in the following examples was a medium based on PDA medium, to which hygromycin and cephalosporin were added, the concentration of hygromycin in the medium being 150. Mu.g/ml, and the concentration of cephalosporin being 600. Mu.g/ml.

The PDA solid medium containing antibiotics (G418150. Mu.g/ml, cephalosporin 600. Mu.g/ml) used in the following examples was a basal medium based on PDA medium to which G418 and cephalosporin were added, wherein the concentration of G418 was 150. Mu.g/ml and the concentration of cephalosporin was 600. Mu.g/ml.

The preparation method of the LB liquid medium used in the following examples comprises the following steps: 10g of peptone, 5g of yeast extract and 5g of NaCl are dissolved in water, the volume is fixed to 1L, and steam sterilization is carried out for 20min at 121 ℃.

The LB liquid medium (50. Mu.g/ml kanamycin, 50. Mu.g/ml rifampicin) containing antibiotics used in the following examples was a medium based on LB liquid medium, to which kanamycin and rifampicin were added, the concentration of kanamycin in the medium being 50. Mu.g/ml, and the concentration of rifampicin in the medium being 50. Mu.g/ml.

The preparation method of the MM minimal medium used in the following examples comprises: 10mL of K-Bufer (pH 7.0), 20mL of M-Nbuffer,1mL of 1% (w/v) CaCl ₂ ·2H ₂ O,10mL of 20% (w/v) sucrose, 1mL of 0.1% (w/v) FeSO ₄ ，0.5g NH ₄ NO ₃ Dissolving in distilled water, and fixing volume to 1L. Steam sterilizing at 113 deg.C for 20min. Wherein, K-bufer comprises solute and solvent, the solvent is water, the solute and its concentration are: k (K) ₂ HPO ₄ Is 200g/L, KH ₂ PO ₄ At a concentration of 145g/L with H ₃ PO ₃ The pH was adjusted to 7.0. The M-Nbbuffer consists of a solute and a solvent, wherein the solvent is water, and the solute is: mgSO (MgSO) ₄ ·7H ₂ The concentration of O is 30g/L, and the concentration of NaCl is 15g/L.

The MM liquid medium (50. Mu.g/ml kanamycin, 50. Mu.g/ml rifampicin) containing antibiotics used in the following examples was a basic medium based on MM minimal medium, and kanamycin and rifampicin were added to the basic medium at a concentration of 50. Mu.g/ml and rifampicin at a concentration of 50. Mu.g/ml.

The preparation method of the IM culture medium used in the following examples comprises the following steps: 10mL of K-Bufer (pH 7.0), 20mL of M-Nbuffer,1mL of 1% (w/v) CaCl ₂ ·2H ₂ O,2.5mL of 20% (w/v) NH ₄ NO ₃ 1mL of 0.1% (w/v) FeSO ₄ 5mL of glycerol, 5mL of 2mol/L of sucrose, 2mL of 100mmol/L of acetosyringone, 40mL of 1mol/L MES (pH 5.3) were dissolved in distilled water and the volume was fixed to 1L. Steam sterilizing at 113 deg.C for 20min.

The CM co-culture medium used in the following examples was a medium obtained by adding agar powder to a basal medium based on an IM medium. The agar powder content in the culture medium was 1.5% (w/v). Steam sterilizing at 113 deg.C for 20min.

EXAMPLE 1 acquisition of the C.polymorpha CcTLS1 knockout mutant

1. Construction of pCAMBIA 1300-DeltaCcTLS 1 knockout vector

The amino acid sequence of CcTLS1 in the coryneform bacteria is shown in the sequence 1 of the sequence table, the CDS sequence for encoding the CcTLS1 protein is shown in the sequence 2 of the sequence table, and the gene sequence of CcTLS1 in the coryneform bacteria is shown in 2049-3270 of the sequence 4 of the sequence table. The CcTLS1 protein contains no domain, as analyzed by the uniprotKB database (https:// www.uniprot.org/uniprot /), and the specific biological function of the C.polymorpha CcTLS1 gene is not known.

The binary vector pCAMBIA1300 was digested with restriction enzyme XhoI to remove hygromycin resistance gene and self-ligated with T4 ligase, and the resulting plasmid was designated pCAMBIA1300-XhoI.

Extracting genome DNA of corynespora polymorpha HG14102524 by using CTAB method, using the genome DNA as template, and usingMaxdnapolyrase, PCR amplified with primer pair 1 (primer pair consisting of F1 and R1) to obtain the 5' fragment of the CcTLS1 gene (containing positions 6 to 1088 of sequence 3):

F1：5’-CCATGATTACGAATTCATCCGGTCTGTTGGGTCG-3' (underlined sequence EcoR I enzyme recognition site sequences);

R1：5’-AACCGCCTCTCCCCGGTACCGTTGTCAGTTGTGAGGAGTATT-3' (underlined sequence is Kpn I enzyme recognition site sequence).

Using the genomic DNA as a templateMaxdnapolyrase, amplified by PCR with primer pair 2 (primer pair consisting of F2 and R2) to obtain the 3' fragment of the CcTLS1 gene (containing positions 2868 to 4160 of sequence 3):

F2：(the sequence indicated by double underlines is the Xba I enzyme recognition site sequence);

R2：(the sequence indicated by double underlining is the PstI enzyme recognition site sequence).

By means ofThe plasmid miniextract kit extracts pPIC9K-His plasmid as template by using +.>Maxdnapolyrase, amplified by PCR with primer pair 3 (primer pair consisting of F3 and R3) to obtain hygromycin resistance gene expression cassette fragment (containing positions 1109-2861 of sequence 3):

F3：5’-GGGGTACCGGGGAGAGGCGGTTTGC-3' (underlined sequence is Kpn I enzyme recognition site sequence);

R3：(the sequence indicated by double underlines is the Xba I enzyme recognition site sequence).

Three PCR products (the 5 'fragment of the CcTLS1 gene, the 3' fragment of the CcTLS1 gene and the hygromycin resistance gene expression cassette fragment) were ligated into the pCAMBIA1300-XhoI vector using In-FusionHDCloningkits (Takara), specifically: 1) The hygromycin resistance gene expression cassette fragments obtained by PCR amplification and pCAMBIA1300-XhoI are respectively subjected to double digestion by restriction enzymes Kpn I and Xba I, and an In-fusion seamless cloning kit (product of Bao Ri doctor materials technology (Beijing) Co., ltd., product number 639648) is connected to obtain a plasmid 1; 2) The plasmid 1 was subjected to linear digestion with EcoR I and Kpn I at a multiple cloning site In front of the promoter region of the hygromycin resistance gene, and the 5' -end fragment of the CcTLS1 gene obtained by PCR amplification was ligated with an In-fusion seamless cloning kit (Takiday doctor materials technology (Beijing) Co., ltd., cat# 639648) to obtain a plasmid 2; 3) The multicloning site of the plasmid 2 behind the terminator region of the hygromycin resistance gene is subjected to double-enzyme tangential linearization by using Xba I and Pst I, and the 3' -end fragment of the CcTLS1 gene obtained by PCR amplification is connected by using an In-fusion seamless cloning kit, so that the obtained expression vector is: the restriction endonuclease KpnI and Xba I recognition site fragments (small fragments including KpnI recognition site and Xba I recognition site) of the pCAMBIA1300-XhoI vector were replaced with hygromycin resistance gene expression cassette fragments, the restriction endonuclease EcoRI and KpnI recognition site fragments (small fragments including EcoRI recognition site and KpnI recognition site) of the pCAMBIA1300-XhoI vector were replaced with the CcTLS1 gene 5 '-end fragments, and the restriction endonuclease Xba I and Pst I recognition site fragments (small fragments including Xba I recognition site and Pst I recognition site) of the pCAMBIA1300-XhoI vector were replaced with the CcTLS1 gene 3' -end fragments, to give a recombinant expression vector named pCBIA 1300- ΔcTL1 containing the CcTLS1 gene 5 '-end, the CcTLS1 gene 3' -end and the hygromycin resistance gene expression cassette. The amino acid sequence of pCAMBIA 1300-delta CcTLS1 is shown as a sequence 3 in a sequence table, wherein 1109-2861 is a hygromycin resistance gene expression cassette fragment, 6-1088 is a CcTLS1 gene 5 'end fragment, and 2868-4160 is a CcTLS1 gene 3' end fragment.

2. Acquisition of CcTLS1 knockout mutants

1. Construction of CcTLS1 knockout mutants

The pCAMBIA 1300-delta CcTLS1 constructed in the above way is transformed into wild type corynespora polymorpha HG14102524 by using agrobacterium-mediated transformation to obtain a CcTLS1 knockout mutant. The method comprises the following specific steps:

(1) The single colony of positive Agrobacterium (recombinant Agrobacterium containing pCAMBIA 1300-. DELTA.CcTLS 1) was picked up by transferring pCAMBIA 1300-. DELTA.CcTLS 1 into Agrobacterium AGL-1 (manufactured by Beijing Bomaide Gene technology Co., ltd.) and placed in 5ml of LB liquid medium (50. Mu.g/ml kanamycin, 50. Mu.g/ml rifampicin) containing antibiotics, and placed on a shaking table at 28℃for cultivation at 200rpm for 24 hours. Adding 1ml of bacterial liquid cultured for 24 hours into 20ml of MM liquid culture medium (50 mug/ml kanamycin, 50 mug/ml rifampicin) containing antibiotics, placing on a shaking table at 28 ℃ for continuous culture for 24 hours at 200rpm, centrifuging at 4000rpm on a centrifuge for 10min, collecting bacterial cells, washing the bacterial cells twice with IM culture medium, re-suspending the bacterial cells with the IM culture medium, and adjusting OD _600nm To 0.25, an agrobacterium solution a was obtained.

(2) At the same time use ddH ₂ Brushing wild type multi-main corynespora strain HG14102524 spores on PDA culture medium to obtain spore suspension, resuspending spores with IM liquid culture medium +200μM AS (product of Acetosyringone, beijing Soy Bao technology Co., ltd., product No. A8110), counting by blood cell counting plate, and regulating spore concentration to 1.0X10 ^6-7 The spores/ml, a fungal spore suspension A was obtained.

(3) Mixing the agrobacterium liquid A obtained in the step (1) and the fungus spore suspension A obtained in the step (2) uniformly according to a volume ratio of 1:1 (1 ml is taken respectively), coating 200 mu 1 of each culture dish on cellophane on a CM co-culture medium plate, and culturing at 26 ℃ for 36 hours to obtain a co-culture A.

(4) Co-culture A was rinsed with 3ml of sterile water and plated at 500. Mu.l/plate on PDA solid medium (hygromycin 150. Mu.g/ml, cephalosporin 600. Mu.g/ml) containing antibiotics for 5-7 days to obtain CcTLS1 knock-out mutants.

2. PCR identification of CcTLS1 knockout mutant

The CTAB method is used for respectively extracting the genomic DNA of the CcTLS1 knockout mutant obtained in the step 1 and the genomic DNA of the wild type corynespora polymorpha HG 14102524.

PCR amplification was performed on the genomic DNA of wild type coryneform bacterium HG14102524, pCAMBIA 1300-. DELTA.CcTLS 1 (see step one of this example) and the genomic DNA of the CcTLS1 knockout mutant, respectively, using four primer pairs, and the results are shown in FIG. 1:

the first primer pair amplifies hygromycin resistance gene (Hph gene), consisting of FhphU and FhphL:

FhphU：5’-TGTCCTGCGGGTAAATAGC-3’；

FhphL：5’-TTGTTGGAGCCGAAATCC-3’。

the CcTLS1 knockout mutant genomic DNA was successfully amplified with the first primer pair to a sequence equal to the expected size (468 bp), indicating successful insertion of the hygromycin resistance gene cassette.

The second primer pair amplifies the CcTLS1 gene, consisting of CcTLS1F and CcTLS 1R:

CcTLS1F：5’-ATGCGTTCCTCAATCATCATCGC-3’；

CcTLS1R：5’-TTACAATGGCTGTCGTACTGGG-3’。

the absence of amplification of the genomic DNA of the CcTLS1 knockout mutant to the target band with the second primer pair indicates successful CcTLS1 gene knockout;

the third primer pair amplifies the right border of the knockout vector to the hygromycin resistance gene cassette T-DNA sequence consisting of FrbU and FhphL:

FrbU：5’-CCTCTTCGCTATTACGCC-3’；

FhphC：5’-CGATTTCGGCTCCAACAA-3’。

pCAMBIA1300- ΔCcTLS1 was successfully amplified to the right border T-DNA sequence (2000 bp) of the knockout vector with the third primer pair.

The fourth primer pair amplified the 5' -homology arm upstream of the CcTLS1 gene to the hygromycin resistance gene cassette T-DNA sequence (2153 bp) consisting of F5F and Fhphl:

F5F：5’-CAAGGATGCGAGCCGAAAGG-3’；

FhphL：5’-TTGTTGGAGCCGAAATCC-3’。

the fourth primer pair was amplified only in the genomic DNA of the CcTLS1 knockout mutant, and the band size (2153 bp) was expected to be consistent, indicating that the hygromycin gene was inserted in the correct position.

The above results demonstrate that the CcTLS1 knockout mutant based on Corynebacterium polymyxa HG14102524 was successfully constructed and was designated as ΔCcTLS1.

EXAMPLE 2 acquisition of the anaplerotic mutant of Isaria polymorpha CcTLS1

1. Construction of pCAMBIA 1300-cDeltaCcTLS 1 complementation vector

Extracting genome DNA of corynespora polymorpha HG14102524 by using CTAB method, using the genome DNA as template, and usingMaxdnapolyrase, amplified by PCR with primer pair 4 (primer pair consisting of F4 and R4) to obtain the CcTLS1 gene and its 5' 3265kb fragment (containing positions 6-3270 of sequence 4):

F4：5’-CCATGATTACGAATTCGCTCGGGATTTGGCACACG-3' (underlined sequence is EcoRI enzyme recognition site sequence);

r4:5'-CTAGAGGATCCCCGGGTACCTTACAATGGCTGTCGTACTGGG-3' (Kpn I enzyme recognition site sequence is underlined).

Using the genomic DNA as a templateMaxdnapolyrase, amplified by PCR with primer pair 5 (primer pair consisting of F5 and R5) to obtain the 3' -end 985kb fragment of the CcTLS1 gene (containing the 4467-5451 th bits of sequence 5):

F5：5’-GTTCTTCTGAAGACCAAAAGAGCATTAGTTCATCA-3' (underlined indicates that the sequence is reverse complement to R6);

R5：(the sequence indicated by double underlining is the Sbf I enzyme recognition site sequence).

By means ofThe NEO-GFP plasmid is extracted from the plasmid miniprep kit as template by using +.>Maxdnapolyrase, amplified by PCR with primer pair 6 (primer pair consisting of F6 and R6), amplified NEO resistance gene expression cassette fragment (containing positions 3292-4456 of sequence 5):

F6：(the underlined sequence is Kpn I enzyme recognition site sequence, the double underlined sequence is Xba I enzyme recognition site sequence);

R6：5’-CTTTTGGTCTTCAGAAGAACTCGTCAAGAAGGC-3' (underlined indicates that the sequence is reverse complement to F5).

Three PCR products (CcTLS 1 gene and its 5 '-end 3265kb fragment, ccTLS1 gene 3' -end 985kb fragment and NEO resistance gene expression cassette fragment) were ligated to pCAMBIA1300-XhoI vector using In-FusionHDCloningkits (Takara), specifically: (1) Carrying out double enzyme digestion on pCAMBIA1300-XhoI by using restriction enzymes Kpn I and EcoRI, and connecting the pCTLS 1 gene and a 3265kb fragment at the 5' end thereof obtained by PCR amplification with an In-fusion seamless cloning kit to obtain a plasmid (1); (2) The plasmid (1) was digested with XbaI and SbfI, and the NEO-resistant gene expression cassette fragment obtained by PCR amplification and the CcTLS1 gene 3' -end 985kb fragment were simultaneously ligated by an In-fusion seamless cloning kit, and the resulting expression vector was: the restriction endonuclease Kpn I and EcoR I recognition site fragments (including Kpn I recognition site and EcoR I recognition site fragments) of the pCAMBIA1300-XhoI vector are replaced with the CcTLS1 gene and a 3265kb fragment at the 5 'end thereof, and the NEO resistance gene expression cassette fragment and a 985kb fragment at the 3' end of the CcTLS1 gene are homologous joined to replace the restriction endonuclease Xba I and Sbf I recognition site fragments of the pCAMBIA1300-XhoI vector (including Xba I recognition site and Sbf I recognition site fragments) to thereby obtain a recombinant expression vector comprising the CcTLS1 gene and a 3265kb fragment at the 5 'end thereof, a 985kb fragment at the 3' end of the CcTLS1 gene and a NEO resistance gene expression cassette fragment, which is named pCAMBIA1300-cΔCTLS1. The sequence verification shows that the amino acid sequence of pCAMBIA 1300-cDeltaCcTLS 1 is shown as sequence 4 in a sequence table, wherein, the 6 th to 3270 th sites are the CcTLS1 gene and a 3265kb fragment at the 5 'end thereof, the 3292 th to 4456 th sites are NEO resistance gene expression cassette fragments, and the 4467 th to 5451 th sites are 985kb fragments at the 3' end of the CcTLS1 gene.

2. Acquisition of CcTLS1 anaplerotic mutants

1. Construction of CcTLS1 anaplerotic mutants

The complementation vector pCAMBIA1300-cΔCcTLS1 constructed as described above was transformed into the corynebacterium polymyxa CcTLS1 knockout mutant ΔCcTLS1 (see example 1) using Agrobacterium-mediated transformation to obtain the CcTLS1 complementation mutant.

(1) The positive Agrobacterium single colony was picked up and placed in 5ml of LB liquid medium (50. Mu.g/ml kanamycin, 50. Mu.g/ml rifampicin) containing antibiotics, and placed on a shaking table at 28℃for culturing at 200rpm for 24 hours, and transferred into Agrobacterium tumefaciens competent cell AGL-1 (product of Beijing Bomaide Gene technology Co., ltd., product No. BC 302-01) with pCAMBIA1300-cΔCcTLS1. Adding 1ml of bacterial liquid cultured for 24 hours into 20ml of MM liquid culture medium (50 mug/ml kanamycin, 50 mug/ml rifampicin) containing antibiotics, placing on a shaking table at 28 ℃ for continuous culture for 24 hours at 200rpm, centrifuging at 4000rpm on a centrifuge for 10min, collecting bacterial cells, washing the bacterial cells twice with IM culture medium, re-suspending the bacterial cells with the IM culture medium, and adjusting OD _600nm To 0.6, the agrobacterium solution B was obtained.

(2) At the same time use ddH ₂ O and sterilizing brush to obtain spore suspension, re-suspending spore with IM liquid culture medium +200μM AS (product of Beijing Soy Bao technology Co., ltd., product No. A8110), counting with blood cell counting plate, and regulating spore concentration to 1.0X10 ^6-7 And (3) spores/ml, to obtain fungal spore suspension B.

(3) Uniformly mixing the agrobacterium liquid B obtained in the step (1) and the fungus spore suspension B obtained in the step (2) according to a volume ratio of 1:1 (1 ml is taken respectively), and culturing for 36 hours at 26 ℃ according to cellophane coated on a CM co-culture medium plate with 200 mu 1 per culture dish to obtain a co-culture B.

(4) Co-culture B was rinsed with 3ml of sterile water and plated at 500 ul/plate on PDA solid medium with antibiotics (G418150. Mu.g/ml, cephalosporin 600. Mu.g/ml) for 5-7 days to obtain CcTLS1 complementation mutant.

2. PCR identification of CcTLS1 anaplerotic mutant

The CTAB method is used for respectively extracting the genomic DNA of the CcTLS1 anaplerotic mutant and the genomic DNA of the wild corynespora polymorpha HG 14102524.

PCR amplification was performed on wild-type C.polymorpha HG14102524 genomic DNA, pCAMBIA1300-cΔCcTLS1 (see step one of this example), and CcTLS1 back-complemented mutant genomic DNA using four pairs of primers, and the results are shown in FIG. 2:

the first primer pair amplifies Neo gene, consisting of Neo-F and Neo-R:

NEO-F：5’-GTCGACAGAAGATGATATTG-3’；

NEO-R：5’-TCAGAAGAACTCGTCAAGAAGGCG-3’。

the successful amplification of the genomic DNA of the CcTLS1 back-complemented mutant with the first primer pair was identical to the expected size (1165 bp), indicating successful insertion of the Neo-resistance gene cassette.

The second primer pair amplifies the CcTLS1 gene, consisting of CcTLS1F and CcTLS 1R:

CcTLS1F：5’-ATGCGTTCCTCAATCATCATCGC-3’；

CcTLS1R：5’-TTACAATGGCTGTCGTACTGGG-3’。

the genomic DNA of the CcTLS1 reintegration mutant was amplified to the target band (1222 bp) with the second primer pair, indicating that the reintegration was successful.

The third primer pair amplifies the right border of the complementing vector back to the Neo-resistance gene cassette T-DNA sequence consisting of frduc and Neo-R:

M13R：5’-CACACAGGAAACAGCTATGAC-3’；

NEO-R：5’-TCAGAAGAACTCGTCAAGAAGGCG-3’。

pCAMBIA1300-cΔCcTLS1 amplified the plasmid vector left border T-DNA sequence (4487 bp) with the third primer pair.

The fourth primer pair amplified the 5' -homology arm upstream of the CcTLS1 gene to the NEO resistance gene cassette sequence (2153 bp), consisting of F4F2 and (NNEO) NEO-R:

F4F2：5’-GAATTAGCTGCGCTCATGCAC-3’；

NEO-R：5’-TCAGAAGAACTCGTCAAGAAGGCG-3’。

the fourth primer pair was only amplified in the CcTLS1 back-filled mutant genomic DNA, and the band size (4571 bp) was expected to be consistent, indicating that the Neo gene was inserted in the correct position.

The above results indicate that the make-up mutant was successfully constructed and was designated cΔCcTLS1.

Example 3 detection of pathogenicity of mutants

The mutants to be tested were the CcTLS1 knockout mutant ΔCcTLS1 constructed in example 1 (abbreviated knockout strain ΔCcTLS 1) and the complementation mutant cΔCcTLS1 constructed in example 2 (abbreviated complementation strain cΔCTLS 1), and the control was wild-type coryneform multi-master strain HG14102524 (abbreviated wild-type HG 14102524).

The pathogenicity is identified by an in vitro patch culture method, and the specific steps are as follows: a clean moisturizing box is prepared, and a proper amount of clean water is sprayed to moisturize the box. And the leaf backs of the collected cucumber leaves are upwards and respectively arranged in a moisturizing box (three pieces and one box). The activated coryneform bacteria (knockout strain Δcctls1, anaplerotic strain cΔcctls1 and wild type HG 14102524) were used to form a cake at the edge of the colony with a 0.5cm punch, the cake was inoculated onto cucumber leaves (hyphae facing down) with a sterilized forceps holder, and each of the coryneform bacteria was attached to one cake. After the bacterial cake is cut, a proper amount of clear water is sprayed, moisture is preserved, and when the bacterial cake is ill, the diameter of the bacterial plaque is measured by a crisscross method, and the result is shown in figure 3.

Through the method, the fact that the pathogenicity of the CcTLS1 knockout mutant delta CcTLS1 to the cucumber is obviously weakened is verified, and the pathogenicity of the complementation mutant c delta CcTLS1 is recovered. The CcTLS1 gene is described as being associated with the pathogenicity of corynespora polymorpha.

EXAMPLE 4 determination of the growth Rate of mutant hyphae

And (3) respectively picking bacterial cakes from the colony edges of the cultured multiple main coryneform bacteria CcTLS1 knockout mutant delta CcTLS1 and the reintegration mutant c delta CcTLS1 by adopting a 5mm puncher, picking the bacterial cakes by adopting a sterilized toothpick, sticking the hyphae face down to the center of a PDA flat plate, setting 3 flat plate repetitions for each bacterial strain, and measuring the colony diameter by adopting a cross method every 24 hours. Wild type corynespora polymorpha HG14102524 was used as a control.

The results are shown in FIG. 4, which demonstrates that CcTLS1 knockout mutant ΔCcTLS1 grows slowly in early growth (0-24 h) of C.polymorpha, and that rapid growth occurs in wild-type HG14102524 and the complementing mutant cΔCcTLS1. The CcTLS1 gene is involved in the growth of early hyphae of Isaria polymorpha.

EXAMPLE 5 mutant cellulase Activity assay

The multimeric cellulase activity of wild-type coryneform bacteria HG14102524, the CcTLS1 knockout mutant Δcctls1 and the complementation mutant cΔcctls1 was determined by plate assay:

a7 mm diameter multi-master coryneform bacteria cake (. DELTA.CcTLS 1, cDELTA.CcTLS 1, HG 14102524) was prepared by a punch, and the three bacteria cakes were inoculated on a plate with sterilized toothpicks, respectively, and cultured at 28℃for 3d. Carboxymethyl cellulose plates were stained with 0.1% Congo red dye for 30min, followed by decolorizing with 1mol/L sodium chloride solution for 30min, observation and photographing. Each treatment was repeated 3 times.

The results are shown in FIG. 5, which demonstrates that the CcTLS1 knockout mutant ΔCTLS1 grows slowly on carboxymethyl cellulose plates, although secreting cellulases, demonstrating that the CcTLS1 gene affects secretion of the polysaccharide hydrolase of Leuconostoc mesenteroides.

Therefore, the CcTLS1 gene provided by the application can be used for preventing and controlling plant diseases, in particular to cucumber corynespora leaf spot caused by corynespora polymorpha. In addition, the gene provided by the application can be used as a target of a drug for controlling plant diseases. Those skilled in the art can follow the teachings and teachings of the present specification to develop pharmaceuticals for controlling plant diseases, particularly corynespora polymorpha.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of vegetable and flower at national academy of agricultural sciences

<120> Isaria polymorpha CcTLS1 protein and coding gene and application thereof

<130> GNCSY212011

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 389

<212> PRT

<213> Isaria mulina (Corynespora cassiicola)

<400> 1

Met Arg Ser Ser Ile Ile Ile Ala Thr Phe Ala Leu Thr Ala Leu Ala

1 5 10 15

Val Pro Val Pro Ser Pro Glu Asn Asp Leu Leu Ala Arg Asp Val Thr

20 25 30

Pro Gln Ser Ala Pro Leu Leu Ala Leu Glu Arg Gly Leu Ser Arg Thr

35 40 45

Gly Asp Gly Thr Leu Ser Tyr Arg Asp Val Glu Glu Glu Arg Gly Leu

50 55 60

Gly Arg Phe Asn Gly Lys Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg

65 70 75 80

Glu Glu Ser Glu Asp Asp Leu Val Glu Glu Arg Gly Leu Gly Arg Phe

85 90 95

Asn Gly Lys Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg Glu Glu Glu

100 105 110

Asp Glu Glu Tyr Leu Val Glu Glu Arg Gly Leu Gly Arg Phe Asn Gly

115 120 125

Lys Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg Glu Glu Asp Glu Glu

130 135 140

Asp Glu Glu Asp Leu Glu Glu Arg Gly Leu Gly Arg Phe Asn Gly Lys

145 150 155 160

Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg Gly Leu Gly Arg Phe Asn

165 170 175

Gly Lys Arg Glu Glu Ala Glu Glu Asp Ser Val Glu Glu Arg Gly Leu

180 185 190

Gly Arg Phe Asn Gly Lys Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg

195 200 205

Glu Glu Ser Glu Asp Asp Leu Val Glu Glu Arg Gly Leu Gly Arg Phe

210 215 220

Asn Gly Lys Arg Glu Glu Ser Glu Asp Asp Leu Val Glu Glu Arg Gly

225 230 235 240

Leu Gly Arg Phe Asn Gly Lys Arg Glu Glu Ser Glu Asp Asp Leu Val

245 250 255

Glu Glu Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg Glu Glu Ser Glu

260 265 270

Asp Asp Leu Val Glu Glu Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg

275 280 285

Glu Glu Ser Glu Asp Asp Leu Val Glu Glu Arg Gly Leu Gly Arg Phe

290 295 300

Asn Gly Lys Arg Gly Leu Gly Arg Phe Asn Gly Lys Arg Gly Leu Gly

305 310 315 320

Arg Phe Asn Gly Lys Arg Asp Glu Asp Ala Asp Glu Glu Leu Glu Lys

325 330 335

Arg Gln Gly Leu Gly Arg Phe Asn Gly Lys Arg Glu Ala Ser Glu Glu

340 345 350

Glu Pro Lys Asn Val Val Arg Ser Val Tyr Asp Gly Val Val Glu Met

355 360 365

Ile Glu Ala Leu Lys Thr Arg Gly Phe Ser Ser Ala Asn Ser Ile Pro

370 375 380

Val Arg Gln Pro Leu

385

<210> 2

<211> 1170

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgcgttcct caatcatcat cgcaaccttc gctctgaccg ctttggccgt ccccgtccca 60

tcacctgaga acgatcttct cgcacgcgac gtcaccccgc agtccgcacc gctcttggcc 120

cttgagaggg gactatcgcg cactggcgat ggcactctat cataccgcga cgtcgaagaa 180

gagcgagggc tcggccgatt caatggaaag cgaggcctcg gtcgctttaa cggaaagcga 240

gaagagtctg aagatgacct tgttgaggaa agggggcttg gccgcttcaa cggcaagcga 300

ggtctcggcc gattcaacgg caagcgggag gaggaagacg aggagtatct tgtagaggag 360

aggggtctcg gacgcttcaa tggcaagcga ggtctcggcc gattcaacgg caagcgtgag 420

gaagatgagg aggacgagga agatcttgag gagaggggtc tcggccgctt caacggtaag 480

cggggtctcg gccgattcaa tggaaagaga ggcctcggtc gcttcaatgg caaacgcgaa 540

gaagctgagg aagactctgt agaggagagg ggtctcggac gtttcaacgg caagcgaggt 600

ctcggccgct tcaacggcaa acgcgaggag tccgaggatg accttgtgga ggagcgtggc 660

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtggc 720

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtggc 780

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtgga 840

cttggccgct tcaacggcaa gcgcgaggaa tctgaggacg atctcgtgga ggagcgtggt 900

cttggccgct tcaacggcaa gcggggcctt ggtcgcttca atggcaagag gggtctcggc 960

aggttcaacg gcaagaggga cgaggatgct gatgaggagc ttgagaagcg ccagggtctc 1020

ggccgcttca atggcaagag ggaagcgtct gaggaggaac ctaagaacgt tgttcgcagt 1080

gtgtatgacg gtgttgttga gatgatcgag gcattgaaga ccaggggatt cagcagtgcg 1140

aacagcatcc cagtacgaca gccattgtaa 1170

<210> 3

<211> 12054

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

aattcatccg gtctgttggg tcgaccacga acaacttccg ctgggagggg ccgcaaaatc 60

ctcgcttcgc ctctcgtttc tttgttttgg caggtactgg ttcggatgtt gtaaaatgca 120

gcagcccttt ctcatcatct cctatacccc tgagtccgtc tggaatccct acaggcagaa 180

aaagtctcag aagcttcttg gtgggaaact tacgtggcgg atagcatagg atttacgctt 240

caaggtcctt atcgtgcatg gtcacatcat ttggtccagg tggccatgca atccctcggt 300

tagctaatcc ttccgttgcc cagtatgcca tcttcacaaa agggcgcgta gaagtattga 360

tatcactgtg atcaagtatg tggctgcccc gttcctgcgt tagaatggtc atcgaaggca 420

tgtctccagt gtagaaagtc tgcccaatgt tcgcttcgga tcatggtgag atgaacgaac 480

cctgaacagg ctagcactct caaggtgcaa gtcagggatc caatcgcaaa gttgccacta 540

tattgaaatc tattttcatt tatctcggcg cgtaggcaca tgtcgtgtag cgtcaccgat 600

aagacagaaa cctcgaataa gagtggacga cggagattca gtctcagtgt gggtacacca 660

atagcaatgc gacgttgggt gaagcagctt gggttgagcc taaagcatcc tagtccggcg 720

cttagcagag cagccccgca cgcacagctt cctgcaatgc acccaccatc acgacaacgc 780

atgacaacgg cgcttcaggt accagggcca atcctccgct ctagtcgatg cctacaccgg 840

ggatgatggc atgacggccc aagcccttga attaaacata aaagtcacct tgccccccaa 900

gtttcacgga tcaggatctg gatccactcc aaactcgttc tttgcaacat accccttctc 960

tacaggctgt gcctactttg actattacgt gctgtgcaca ttaatctttt atgcgctttt 1020

tgtattaacc tcataaataa aaaatcaacc caacaaaccg aaatttaata ctcctcacaa 1080

ctgacaactg acaacggtac ggggatccgg ggagaggcgg tttgcgtatt ggctagagca 1140

gcttgccaac tgatattgaa ggagcatttt ttgggcttgg ctggagctag tggaggtcaa 1200

caatgaatgc ctattttggt ttagtcgtcc aggcggtgag cacaaaattt gtgtcgtttg 1260

acaagatggt tcatttaggc aactggtcag atcagcccca cttgtagcag tagcggcggc 1320

gctcgaagtg tgactcttat tagcagacag gaacgaggac attattatca tctgctgctt 1380

ggtgcacgat aacttggtgc gtttgtcaag caaggtaagt ggacgacccg gtcatacctt 1440

cttaagttcg cccttcctcc ctttatttca gattcaatct gacttaccta ttctacccaa 1500

gcatccaaat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa 1560

agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca 1620

gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct 1680

acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc 1740

ttgacattgg ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg 1800

tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctccagccg gtcgcggagg 1860

ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac 1920

cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc 1980

atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc 2040

tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcatgcgg 2100

atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga 2160

gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcctcttc tggaggccgt 2220

ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag 2280

gatcgccgcg cctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct 2340

tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc 2400

gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga 2460

ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga 2520

gggcaaagga atagagtaga tgccgaccgg gaaccagttt cgagtttctc cataataatg 2580

tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc atgtgttgag 2640

catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc 2700

taattcctaa aaccaaaatc cagtactaaa atccagatcc cccgaattaa ttcggcgtta 2760

attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt 2820

acaccacaat atatcctgcc accagccagc caacagctcc ctctagaaga ccaaaagagc 2880

attagttcat caaagctagt cgaagttgat aggttcgttg tctcgaagac tttgcacggg 2940

ttccaatagc tagatgctgg atgcgatact gtatgcactt ggagaattct ttgagcaccc 3000

tttcgaaatt ccttttgtac ctaaggcctt tcgcctagtt ggtgatgggg actgtagcgg 3060

tggactatga gcaaatacaa agcgtctggt ccttagagct atgaaagcct agtgtcctag 3120

gtttaagata ctactgacaa taatattcct cttatgctct acgaatattt cctctagtaa 3180

ccttagggtt ttttttacag caataaggct gggagcatgt gtattagacc tgacctatgg 3240

actgcctggg caatacttga gtgcggtgcg gaacagtact gctgtatacg ttggtgagac 3300

tatttccaaa attgagactt tattgtttgg acctttttag agcatctgtt tcgactctgt 3360

cttgcggttg taaaggctct gggccgagcc aattttgctc aatcaagtac tgaggctgta 3420

ctggagtatt tagtccttgt gcttaccatt ggtggaatat gtatctgaaa tgggcttagg 3480

tgctcatgaa tcttgccgat caaataccta ttttcgcaat cgcctgcctg tttccggccg 3540

agttttgggt agtcagcctg gaaaggcaag ggcttgtgta gacacgtgat gtctgccaag 3600

cactaagcgc tgtgatggac agggcccccc gtccatccag ttcctgacag cccgactcca 3660

ccagttccta ctctgtgcaa cccccgagtc tcacatcgct ggttgccttc accaaaatct 3720

ttgtcgattc gtgaattcgc aattatatcc agcgacaaat gtatcagaat cagagatctc 3780

atagcaagct gaggtttcag ccaccggtgt ctaaccgccg aaaggctgtg ccacatgggc 3840

atcacccgga ccccagtaag ctcatgcaac tataactgta cactagtgtc tgcaagcatt 3900

ttagcctgca tcgcgcccac catccgggcg cttatcacac gttctcattg ggattgacct 3960

tcctggcgct tatacgctgt tcctgtgagg actgtcagac tccagtagct tggtatgccg 4020

ccattcagat tacgctacca ttctcaatat cgtcgttgtc aattcgagcc ctcatccttg 4080

gcatgcggct ctgtggcgta gacgtgaaaa ctgcgcgtat ccttcgttat gaatggctgt 4140

ttttacgctg ccaatgagat ctgcaggcat gcaagcttgg cactggccgt cgttttacaa 4200

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 4260

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 4320

agcctgaatg gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt 4380

cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 4440

gtttattaga ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 4500

tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc 4560

gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt 4620

cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg 4680

tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca 4740

tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg 4800

acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga 4860

agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc 4920

ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac 4980

tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt 5040

gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg 5100

agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc 5160

gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg 5220

agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc 5280

gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc 5340

ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga 5400

atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt 5460

cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc 5520

gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc 5580

ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg 5640

tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa 5700

taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg 5760

tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat 5820

gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa 5880

gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc 5940

atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg 6000

tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata 6060

tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg 6120

ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt 6180

gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg 6240

agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac 6300

gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat 6360

gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac 6420

gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca 6480

actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca 6540

agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat 6600

gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa 6660

ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta 6720

agcggctggg ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg 6780

gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 6840

ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg 6900

ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc 6960

ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt 7020

cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt 7080

tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga 7140

cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct 7200

ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg 7260

agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg 7320

agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac 7380

gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga 7440

gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta 7500

catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga 7560

cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta 7620

ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta 7680

cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 7740

cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat 7800

cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac 7860

ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc 7920

tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat 7980

tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga 8040

gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg 8100

cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac 8160

ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 8220

ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc 8280

gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 8340

acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 8400

atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg 8460

cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc 8520

agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 8580

gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 8640

cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 8700

atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 8760

taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 8820

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 8880

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 8940

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 9000

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 9060

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 9120

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 9180

tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 9240

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 9300

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 9360

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 9420

aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa 9480

aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc 9540

gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata 9600

ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt 9660

cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt 9720

ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca 9780

gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg 9840

gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat 9900

gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga 9960

gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa 10020

gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg 10080

ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc 10140

attttctccc accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca 10200

gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta 10260

tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg 10320

aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca 10380

aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg 10440

tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 10500

atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 10560

gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 10620

ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg 10680

gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga 10740

cgtttttaat gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg 10800

attttggttt taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat 10860

actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct 10920

tatctgggaa ctactcacac attattatgg agaaactcga gcttgtcgat cgacagatcc 10980

ggtcggcatc tactatctca ttgccccccg ggatctgcga aagctcgaga gagatagatt 11040

tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat 11100

agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata 11160

tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 11220

ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 11280

tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 11340

atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 11400

tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 11460

acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt 11520

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 11580

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 11640

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 11700

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 11760

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttggcaagc 11820

tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 11880

ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 11940

agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 12000

gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacg 12054

<210> 4

<211> 13346

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

aattcgctcg ggatttggca cacgtgacgt ggaagccatg acagtgtcca tgacagtgta 60

gaagtcggtt gactcccttt tcatgcaatg attatttgat aggaatagat cgccgatgaa 120

tcatcccacc gcctttgttg ttgttcatat atattttgga ttactggcgt gtacgtatcg 180

tcagtgttgg gcaatctact gctctcaagc atgacttgta agcgcccgga gatagcggta 240

cagattgtca cgtcaactta tggatattat tgagtgatat cgcgccagta ttccatatca 300

aagtttctac tggctacaga ttggagttgt gtgtgctagg agtagggagc cttacgttaa 360

ttggcctcat atggtctgtt gccccacaaa gccaccttcc ctttcagtct ttacatacct 420

acctgaaagg gttttattcc gaagaagtgg atcacaagag gggtaggaat tttttccgaa 480

acgactatac ctaacgcagc agtgagacac atcgcgactc acgattgatg tagtatcagt 540

aacaaatcga cgataataga gaaaaaaatc tttcggaaga agtgaggttt ttcgatggtt 600

ctgcagcgaa tagatactct tgtgttgatt tacccgctgc caatacgttg ttttttgggg 660

ctaaattacc taaggttggt ccaagataac agcgtgcttt gtcgagctag atttttcttg 720

tgaggctctg ccaattctct tgtcattctt ggatgctgca tgaggcatct gtacgggctc 780

ggtgaagttt acgtgcgggg gctaattacc acgcatttac gaagggtccg cggtaacaca 840

tctcggacta tcacacgcat gaacggtgaa ctgatgctat tggctgttag taaactaccc 900

aaggccttgt cagaatccaa gaacaaggat gcgagccgaa aggaccgtcg gactcagaca 960

acgcgatccg gtctgttggg tcgaccacga acaacttccg ctgggagggg ccgcaaaatc 1020

ctcgcttcgc ctctcgtttc tttgttttgg caggtactgg ttcggatgtt gtaaaatgca 1080

gcagcccttt ctcatcatct cctatacccc tgagtccgtc tggaatccct acaggcagaa 1140

aaagtctcag aagcttcttg gtgggaaact tacgtggcgg atagcatagg atttacgctt 1200

caaggtcctt atcgtgcatg gtcacatcat ttggtccagg tggccatgca atccctcggt 1260

tagctaatcc ttccgttgcc cagtatgcca tcttcacaaa agggcgcgta gaagtattga 1320

tatcactgtg atcaagtatg tggctgcccc gttcctgcgt tagaatggtc atcgaaggca 1380

tgtctccagt gtagaaagtc tgcccaatgt tcgcttcgga tcatggtgag atgaacgaac 1440

cctgaacagg ctagcactct caaggtgcaa gtcagggatc caatcgcaaa gttgccacta 1500

tattgaaatc tattttcatt tatctcggcg cgtaggcaca tgtcgtgtag cgtcaccgat 1560

aagacagaaa cctcgaataa gagtggacga cggagattca gtctcagtgt gggtacacca 1620

atagcaatgc gacgttgggt gaagcagctt gggttgagcc taaagcatcc tagtccggcg 1680

cttagcagag cagccccgca cgcacagctt cctgcaatgc acccaccatc acgacaacgc 1740

atgacaacgg cgcttcaggt accagggcca atcctccgct ctagtcgatg cctacaccgg 1800

ggatgatggc atgacggccc aagcccttga attaaacata aaagtcacct tgccccccaa 1860

gtttcacgga tcaggatctg gatccactcc aaactcgttc tttgcaacat accccttctc 1920

tacaggctgt gcctactttg actattacgt gctgtgcaca ttaatctttt atgcgctttt 1980

tgtattaacc tcataaataa aaaatcaacc caacaaaccg aaatttaata ctcctcacaa 2040

ctgacaacat gcgttcctca atcatcatcg caaccttcgc tctgaccgct ttggccgtcc 2100

ccgtcccatc acctgaggta tggaatttta tgaactttca aattgagact atactaatta 2160

tactcctaga acgatcttct cgcacgcgac gtcaccccgc agtccgcacc gctcttggcc 2220

cttgagaggg gactatcgcg cactggcgat ggcactctat cataccgcga cgtcgaagaa 2280

gagcgagggc tcggccgatt caatggaaag cgaggcctcg gtcgctttaa cggaaagcga 2340

gaagagtctg aagatgacct tgttgaggaa agggggcttg gccgcttcaa cggcaagcga 2400

ggtctcggcc gattcaacgg caagcgggag gaggaagacg aggagtatct tgtagaggag 2460

aggggtctcg gacgcttcaa tggcaagcga ggtctcggcc gattcaacgg caagcgtgag 2520

gaagatgagg aggacgagga agatcttgag gagaggggtc tcggccgctt caacggtaag 2580

cggggtctcg gccgattcaa tggaaagaga ggcctcggtc gcttcaatgg caaacgcgaa 2640

gaagctgagg aagactctgt agaggagagg ggtctcggac gtttcaacgg caagcgaggt 2700

ctcggccgct tcaacggcaa acgcgaggag tccgaggatg accttgtgga ggagcgtggc 2760

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtggc 2820

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtggc 2880

cttggccgct tcaacggcaa gcgcgaggag tccgaggatg accttgtgga ggagcgtgga 2940

cttggccgct tcaacggcaa gcgcgaggaa tctgaggacg atctcgtgga ggagcgtggt 3000

cttggccgct tcaacggcaa gcggggcctt ggtcgcttca atggcaagag gggtctcggc 3060

aggttcaacg gcaagaggga cgaggatgct gatgaggagc ttgagaagcg ccagggtctc 3120

ggccgcttca atggcaagag ggaagcgtct gaggaggaac ctaagaacgt tgttcgcagt 3180

gtgtatgacg gtgttgttga gatgatcgag gcattgaaga ccaggggatt cagcagtgcg 3240

aacagcatcc cagtacgaca gccattgtaa ggtacccggg gatcctctag agtcgacaga 3300

agatgatatt gaaggagcac tttttgggct tggctggagc tagtggaggt caacaatgaa 3360

tgcctatttt ggtttagtcg tccaggcggt gagcacaaaa tttgtgtcgt ttgacaagat 3420

ggttcattta ggcaactggt cagatcagcc ccacttgtag cagtagcggc ggcgctcgaa 3480

gtgtgactct tattagcaga caggaacgag gacattatta tcatctgctg cttggtgcac 3540

gataacttgg tgcgtttgtc aagcaaggta agtgaacgac ccggtcatac cttcttaagt 3600

tcgcccttcc tccctttatt tcagattcaa tctgacttac ctattctacc caagcatcga 3660

tatgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt 3720

cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc 3780

agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact 3840

gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt 3900

gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 3960

ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 4020

gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 4080

catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 4140

agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga 4200

cggcgatgat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa 4260

tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga 4320

catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt 4380

cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct 4440

tgacgagttc ttctgaagac caaaagagca ttagttcatc aaagctagtc gaagttgata 4500

ggttcgttgt ctcgaagact ttgcacgggt tccaatagct agatgctgga tgcgatactg 4560

tatgcacttg gagaattctt tgagcaccct ttcgaaattc cttttgtacc taaggccttt 4620

cgcctagttg gtgatgggga ctgtagcggt ggactatgag caaatacaaa gcgtctggtc 4680

cttagagcta tgaaagccta gtgtcctagg tttaagatac tactgacaat aatattcctc 4740

ttatgctcta cgaatatttc ctctagtaac cttagggttt tttttacagc aataaggctg 4800

ggagcatgtg tattagacct gacctatgga ctgcctgggc aatacttgag tgcggtgcgg 4860

aacagtactg ctgtatacgt tggtgagact atttccaaaa ttgagacttt attgtttgga 4920

cctttttaga gcatctgttt cgactctgtc ttgcggttgt aaaggctctg ggccgagcca 4980

attttgctca atcaagtact gaggctgtac tggagtattt agtccttgtg cttaccattg 5040

gtggaatatg tatctgaaat gggcttaggt gctcatgaat cttgccgatc aaatacctat 5100

tttcgcaatc gcctgcctgt ttccggccga gttttgggta gtcagcctgg aaaggcaagg 5160

gcttgtgtag acacgtgatg tctgccaagc actaagcgct gtgatggaca gggccccccg 5220

tccatccagt tcctgacagc ccgactccac cagttcctac tctgtgcaac ccccgagtct 5280

cacatcgctg gttgccttca ccaaaatctt tgtcgattcg tgaattcgca attatatcca 5340

gcgacaaatg tatcagaatc agagatctca tagcaagctg aggtttcagc caccggtgtc 5400

taaccgccga aaggctgtgc cacatgggca tcacccggac cccagtaagc tcctgcaggc 5460

atgcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 5520

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 5580

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct 5640

tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga 5700

tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag 5760

ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc 5820

tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg 5880

ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc 5940

tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa 6000

tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct 6060

gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca 6120

cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc 6180

ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct 6240

agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc 6300

cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg 6360

catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg 6420

cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac 6480

cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt 6540

gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg 6600

cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt 6660

gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa 6720

ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg 6780

atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa 6840

atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa 6900

gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat 6960

gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga 7020

aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc 7080

tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg 7140

gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg 7200

accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg 7260

acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc 7320

tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg 7380

ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg 7440

cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc 7500

ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca 7560

caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg 7620

ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac 7680

aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag 7740

cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac 7800

caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata 7860

catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg 7920

ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca 7980

tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca 8040

atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg 8100

gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc 8160

gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct 8220

gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag 8280

ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc 8340

cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga 8400

gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg 8460

gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc 8520

gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca 8580

cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac 8640

ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag 8700

gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag 8760

atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg 8820

taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt 8880

ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag 8940

gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc 9000

aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat 9060

ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc 9120

gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta 9180

gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca 9240

aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac 9300

cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac 9360

tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag 9420

cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc 9480

gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca 9540

ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat 9600

caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 9660

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 9720

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 9780

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 9840

atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 9900

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 9960

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 10020

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 10080

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 10140

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 10200

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 10260

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 10320

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 10380

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 10440

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 10500

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 10560

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 10620

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 10680

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 10740

cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca 10800

ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc 10860

ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca 10920

agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg 10980

ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg 11040

cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg 11100

ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa 11160

tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt 11220

tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg 11280

agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt 11340

ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac 11400

cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta 11460

gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat 11520

tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa 11580

agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct 11640

aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca 11700

tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc 11760

gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt 11820

tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct 11880

cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag 11940

ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt 12000

gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga 12060

attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta 12120

ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca 12180

tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac acattattat 12240

ggagaaactc gagcttgtcg atcgacagat ccggtcggca tctactatct cattgccccc 12300

cgggatctgc gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt 12360

gtcctctcca aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg 12420

attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg 12480

tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 12540

cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 12600

aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 12660

atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 12720

ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat 12780

cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 12840

tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 12900

ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 12960

tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 13020

gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 13080

cgagagtgtc gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct 13140

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 13200

gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 13260

acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 13320

aggaaacagc tatgaccatg attacg 13346

Claims (4)

1. The use of inhibiting the expression level of a gene encoding corynebacterium polymorphum CcTLS1 or reducing the protein activity of corynebacterium polymorphum CcTLS1, characterized in that: the application is any one of the following:

p1, application in weakening pathogenicity of corynespora polymorpha to cucumber;

p2, application in preventing and controlling cucumber corynespora leaf spot caused by corynespora polymorpha;

the CcTLS1 is a protein of any one of the following:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1);

the method for inhibiting the expression level of the encoding gene of the coryneform bacterium CcTLS1 or reducing the protein activity of the coryneform bacterium CcTLS1 comprises the step of introducing any one of the following substances c 1) to c 4) into cucumber:

c1 A nucleic acid molecule for inhibiting or reducing the expression of the coding gene of the CcTLS1, wherein the coding sequence of the coding gene is a DNA molecule shown as a sequence 2 in a sequence table;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

c3 A recombinant vector comprising the expression cassette of c 2);

c4 A recombinant microorganism comprising the recombinant vector of c 3).

2. Protein CcTLS1, characterized in that: the CcTLS1 is a protein of any one of the following:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1).

3. A method for reducing the pathogenicity of coryneform bacteria, comprising reducing the pathogenicity of coryneform bacteria by inhibiting the expression level of the gene encoding the protein CcTLS1 of claim 2 or reducing the activity of the protein CcTLS1 of claim 2 in coryneform bacteria of interest.

4. A method for preparing a recombinant coryneform bacterium, comprising inhibiting the expression level of a gene encoding the protein CcTLS1 of claim 2 in a coryneform bacterium of interest or reducing the activity of the protein CcTLS1 of claim 2, to obtain a recombinant coryneform bacterium.