阅读说明：本技术 一种燕麦叶枯病菌基因CvCAO1及其应用 (Oat leaf blight bacterium gene CvCAO1 and application thereof ) 是由 张祥辉 于汇琳 王璐 孙颖 焦文莉 刘金亮 潘洪玉 于 2019-10-08 设计创作，主要内容包括：一种燕麦叶枯病菌CvCAO1基因及其应用属微生物基因工程技术领域,本发明提供的来自燕麦叶枯病菌的控制毒素形成的CvCAO1基因,其DNA序列如SEQ ID No:1所示；提供的CvCAO1基因所编码的蛋白质,其氨基酸序列如SEQ ID No:2所示；CvCAO1基因可在植物抗燕麦叶枯病基因工程领域中应用；通过对燕麦叶枯病菌的控制毒素形成的蛋白质CvCAO1进行缺失、突变或修饰,而使其毒素形成受限,可作为靶标在设计和筛选抗燕麦叶枯病药剂中应用。(The invention provides a CvCAO1 gene of oat leaf blight bacteria and application thereof, belonging to the technical field of microbial genetic engineering, the DNA sequence of the CvCAO1 gene formed by control toxin of oat leaf blight bacteria is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the CvCAO1 gene is shown as SEQ ID No. 2; the CvCAO1 gene can be applied to the field of plant gene engineering for resisting bacterial leaf blight of oat; the protein CvCAO1 formed by controlling the toxin of the bacterial leaf blight of oat is deleted, mutated or modified, so that the toxin formation is limited, and the protein CvCAO1 can be used as a target to be applied to designing and screening of anti-bacterial leaf blight medicaments of oat.)

1. A fusarium oxysporum f.avenaceum (Cochliobolus victoriae) CvCAO1 gene, comprising: the DNA sequence is shown in SEQ ID No. 1.

2. A protein coded by a CvCAO1 gene of Hitacea avenae (Cochliobolus victoriae), which is characterized in that: the amino acid sequence is shown in SEQ ID No. 2.

3. The application of the gene CvCAO1 of the bacterial leaf blight of oat (Cochliobolus victoriae) of claim 1 in regulating the production of bacterial toxin of oat leaf blight.

4. An application of the gene CvCAO1 of the bacterial leaf blight of oat (Cochliobolus victoriae) as a target in designing and screening an anti-bacterial leaf blight medicament of oat of claim 1.

5. Use of a protein encoded by the CvCAO1 gene of claim 2 to treat a deficiency in the formation of a toxin of bacterial leaf blight.

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to discovery of a gene for controlling mycotoxin formation in the field of plant protection and application of a protein coded by the gene.

Background

Oat leaf blight is caused by a helminthosporium cucumerinum, which mainly damages overground parts such as leaves, leaf sheaths and glumes. The leaf infection forms yellow to yellow brown spots with inconspicuous edges at the early stage, and then gradually enlarges, the middle part is brown, and the periphery is slightly yellow. When the disease is more spots, the disease gradually dies from the tip of the leaf to the bottom. The disease mostly starts from the lower lobe and spreads upward. Moreover, the bacterial blight of oat can generate a toxin-victorin, which has a toxic effect on oat varieties with specific genotypes after being diluted by one million times, so that the concept of host-specific toxins is also provided and widely researched. The toxin victorin is one of essential elements for successfully infecting oats with fusarium oxysporum f.sp.avenae. Strains that do not produce victorin are not pathogenic to oats, and isolated victorin can also infect oats. The susceptibility of oats to victorin is mainly determined by Vb genes of the oats, the oats containing the Vb genes show susceptibility, and the oats without the Vb genes show disease resistance. The Vb gene is the same gene with the Pc2 gene of oat rust resistance, so the susceptibility of the oat leaf blight is related to a resistance gene. And victorin is capable of causing defense responses in oats, including callose accumulation, oxygen burst, and programmed cell death. The victorin mainly comprises 5 structures, namely B, C, D, E and victorine, the structure C is the main structure, and the activity ratio reaches 85% -90%. The bacterial leaf blight of oat causes great loss to the production of oat every year, and particularly, the generation of the toxin victorin has great influence on the safety of human and animals. The generation mechanism of the toxin victorin is not clear at present, which causes great difficulty in controlling the bacterial leaf blight of oat.

The CAO1 (hopper amine oxidase 1) gene is an unknown functional gene in the bacterial leaf blight of oat, and the function of the CAO1 gene is analyzed to evaluate the effect of the CAO1 gene in the production of the bacterial leaf blight toxin of oat, so that the potential prevention and treatment target can be identified, and the CAO1 (hopper amine oxidase 1) gene is used for screening a novel medicament for preventing and controlling the bacterial leaf blight of oat.

Disclosure of Invention

The invention aims to provide a gene for controlling mycotoxin formation and a protein coded by the gene.

The gene for controlling the formation of the toxin is derived from bacterial blight of oat (Cochlioblastusvictoriae) FI3, is named CvCAO1, and has a DNA sequence shown as SEQ ID No. 1. The DNA sequence is an open reading frame of a CvCAO1 gene and consists of 2332 nucleotides, wherein the DNA sequence comprises 4 intron sequences.

The invention provides a protein coded by a CvCAO1 gene, the amino acid sequence of which is shown as SEQ ID No. 2 and consists of 709 amino acids.

The bacterial leaf blight of oat (Cochliobolus victoriae) CvCAO1 gene can be applied to regulation and control of production of bacterial leaf blight toxin of oat.

The protein coded by the CvCAO1 gene can be applied to the defect of forming bacterial leaf blight of oat.

The protein encoded by the CvCAO1 gene which controls the formation of toxin and is derived from the bacterial leaf blight of oat (Cochliobolus victoriae) FI3 is deleted, mutated or modified, so that the formation of the toxin is blocked, and the protein can be used as a target to be applied to designing and screening of anti-bacterial leaf blight medicaments.

The invention proves that the deletion of the CvCAO1 gene can cause that the toxin of the bacterial leaf blight of oat (Cochliobolus victoriae) FI3 can not be formed, and the CvCAO1 gene is the gene necessary for forming the toxin of the bacterial leaf blight of oat (Cochliobolus victoriae) FI 3. At present, the influence of CvCAO1 gene on the formation of toxin is not reported, and the research is the first report. Therefore, screening compounds capable of preventing the gene expression and the protein expression, modification and positioning of the gene can effectively control the occurrence of the bacterial leaf blight of oat, thereby being beneficial to developing a novel bactericide, namely an important application of the CvCAO1 gene provided by the invention is as follows: the expression of the gene and the expression, modification and positioning of the protein product coded by the gene can be used as an important candidate target site for the design and screening of the oat leaf blight resistant medicament.

Drawings

FIG. 1 is a schematic diagram showing domain prediction of CvCAO1 protein

Wherein: a conserved Cu _ amine _ oxid functional domain was found.

FIG. 2 is a schematic diagram showing the knockout strategy (gene replacement by homologous recombination) of the gene FI3CvCAO1 of Hitacea avenae (Cochliobolus victoriae)

Wherein: cv FI3 is a wild type strain of the alternaria avenae, and delta Cvcao1 is a deletion mutant of a Cvcao1 gene; primers F1/R1 and F2/R2 are respectively used for amplifying the upstream and downstream sequences of the CvCAO1 gene and are used as homologous arms for knockout; primers F/R, U/NLC37, NLC38/D were used to verify the mutants.

FIG. 3 is the PCR-verified electrophoresis chart of the deletion mutant of CvCAO1 gene

Wherein: F/R, U/NLC37 and D/NLC38 are used as primers; 1 is a wild type bacterial strain FI3 of oat leaf blight bacteria (Cochliobolus victoriae), and 2 and 3 are CvCAO1 gene deletion mutants; (1) F/R is the amplification result of partial CvCAO1 gene, (2) U/NLC37 is the amplification result of the upstream sequence of CvCAO1 gene plus partial hygromycin sequence, and (3) D/NLC38 is the amplification result of the downstream sequence of CvCAO1 gene plus partial hygromycin sequence.

FIG. 4 is a photograph showing a comparison of toxin production measurement among a deletion mutant of CvCAO1 gene, a wild-type strain and a negative control strain (a strain incapable of producing a toxin)

Wherein: the liquid in the test tube is culture solution of each strain, the leaf is oat seedling leaf, and if the strain can generate toxin, the oat leaf can be bent after 24h culture; 1 is a wild type strain of bacterial blight of Avena sativa, 2 and 3 are strains of bacterial blight of Avena sativa which cannot produce toxins (negative control), and 4 and 5 are deletion mutant strains of CvCAO1 gene.

FIG. 5 is an ESI EIC spectrum of the deletion mutant of the CvCAO1 gene and the toxin produced by the wild type strain

Wherein: inoculating each strain in a liquid Fries' culture medium, culturing for 15 days, and performing toxin determination; the standard substance is a pure toxin standard substance, WT is a wild type bacterial strain of the oat fusarium oxysporum, and delta CvCAO1 is a deletion mutant bacterial strain of CvCAO1 gene.

Detailed Description

In order to better describe the invention, the following is further illustrated by specific examples, the methods of which, unless otherwise specified, are conventional.

The bacterial strain FI3 of the bacterial strain of oat leaf blight (Cochliobolus victoriae) of the present invention is Babara Gillian Turgeon of the university of Cornell, USA,

the provider contact way is as follows:

Dept.of Plant Pathology&Plant-Microbe Biology,334Plant Science Bldg.,Cornell University,Ithaca,NY 14850,United States.E-mail:[email protected]。