Application of gene FoPDCD5 in regulation and control of pathogenicity of banana fusarium oxysporum
阅读说明：本技术 基因FoPDCD5在调控香蕉枯萎病菌致病力中的应用 (Application of gene FoPDCD5 in regulation and control of pathogenicity of banana fusarium oxysporum ) 是由 李云锋 聂燕芳 蒙姑 李华平 王振中 于 2019-10-17 设计创作，主要内容包括：本发明公开一种基因FoPDCD5在调控香蕉枯萎病菌致病力中的应用,属于植物基因工程领域。本发明利用同源重组方法将该基因从香蕉枯萎病菌中敲除,获得敲除突变体ΔFopdcd5；通过构建基因回补载体,将其导入ΔFopdcd5原生质体；利用随机插入的方法将该基因回补到敲除突变体中,获得回补突变体ΔFopdcd5-com。致病性测定表明,敲除突变体ΔFopdcd5的致病性显著降低；回补突变体ΔFopdcd5-com的致病性则恢复到野生型水平。本发明证实FoPDCD5是香蕉枯萎病菌分生孢子产生、应对氧化胁迫和致病性所必需的。有助于深入阐明香蕉枯萎病菌的致病分子机制,为开发有效杀菌剂提供了靶标基因。(The invention discloses an application of a gene FoPDCD5 in regulation and control of pathogenicity of banana vascular wilt, and belongs to the field of plant genetic engineering. The gene is knocked out from banana fusarium oxysporum by using a homologous recombination method to obtain a knock-out mutant delta Fopdcd 5; introducing a gene complementation vector into a delta Fopdcd5 protoplast by constructing the vector; the gene was complemented back into the knockout mutant by random insertion to obtain the complementing mutant Δ Fopdcd 5-com. Pathogenicity assays indicate that the pathogenicity of the knockout mutant Δ Fopdcd5 is significantly reduced; the pathogenicity of the anaplerotic mutant Δ Fopdcd5-com was restored to wild-type levels. The invention proves that the FoPDCD5 is necessary for generating conidia of the fusarium oxysporum f.sp.cubense, and coping with oxidative stress and pathogenicity. Contributes to deeply clarifying the pathogenic molecular mechanism of the banana fusarium wilt and provides a target gene for developing an effective bactericide.)
1. The application of the gene FoPDCD5 in regulating and controlling pathogenicity of banana fusarium oxysporum is characterized in that:
the amino acid sequence of the banana fusarium oxysporum gene FoPDCD5 is shown as SEQ ID NO: 2, or as shown in SEQ ID NO: 2 by one or more amino acid substitutions, insertions and deletions, and the analogue still has the function of controlling the pathogenicity of the fusarium oxysporum f.sp.
2. Use according to claim 1, characterized in that:
the gene FoPDCD5 is applied to regulating and controlling the spore yield of the fusarium oxysporum f.sp.cubense.
3. Use according to claim 1, characterized in that:
the gene FoPDCD5 is applied to the regulation and control of the stress resistance of banana fusarium oxysporum.
4. Use according to claim 3, characterized in that: the stress is oxidative stress.
5. The application of the gene FoPDCD5 in preventing and treating banana wilt caused by banana fusarium wilt.
6. The application of the gene FoPDCD5 as a target for plant disease control drugs in claim 1 is characterized in that: the plant disease is banana vascular wilt caused by banana vascular wilt.
7. Use according to claim 1, 2, 3, 4, 5 or 6, characterized in that:
the nucleotide sequence of the gene FoPDCD5 is one of the following A, B, C:
A. encoding the amino acid sequence of SEQ ID NO: 2;
B. as shown in SEQ ID NO: 1;
C. the analogues obtained by the above A and B through base insertion, deletion or substitution still have the function of controlling the pathogenicity of the fusarium oxysporum f.sp.cubense.
8. A method of treating banana vascular wilt caused by banana vascular wilt, comprising: comprising blocking or inhibiting the expression of the gene FoPDCD5 of claim 1.
9. The application of a medicament for blocking or inhibiting the expression of the gene FoPDCD5 in claim 1 in the preparation of medicines is characterized in that: the medicament is antisense RNA or siRNA of the gene FoPDCD5 in claim 1, and the medicament is used for controlling banana vascular wilt caused by banana vascular wilt.
10. Use according to claim 9, characterized in that:
the nucleotide sequence of the gene FoPDCD5 is one of the following A, B, C:
A. encoding the amino acid sequence of SEQ ID NO: 2;
B. as shown in SEQ ID NO: 1;
C. the analogues obtained by the above A and B through base insertion, deletion or substitution still have the function of controlling the pathogenicity of the fusarium oxysporum f.sp.cubense.
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a gene FoPDCD5 in regulation and control of pathogenicity of banana vascular wilt.
Background
The banana wilt is one of the most important diseases in banana production in China, and seriously threatens the sustainable development of the banana industry. The pathogeny of banana vascular wilt is Fusarium oxysporum cubense (Fusarium oxysporum f.sp. cubense, Foc), and the Fusarium oxysporum cubense can be divided into 3 physiological races according to the difference of host susceptibility, wherein the 4 physiological race (Foc4) has the most serious harm and can almost harm all current cultivated varieties. At present, the prevention and control of banana vascular wilt mainly comprises breeding resistant varieties, but because most cultivated bananas are triploid, the bananas are highly sterile and have no seeds, the conventional crossbreeding of the bananas is very difficult. Fully excavates pathogenic related genes of banana fusarium wilt and develops functional research thereof, is helpful for comprehensively understanding pathogenic molecular mechanism of the banana fusarium wilt, and provides theoretical basis for prevention and control of the banana fusarium wilt.
PDCD5(Program Cell Death Protein 5) is an apoptosis accelerating Protein containing 1 DNA-binding domain and has high conservation in evolution. The current research on the PDCD5 protein is mainly focused on human cancer cells, and the main role is to regulate programmed cell death and immune regulation. No report is made about the specific function of PDCD5 in plant pathogenic fungi. In the study of proteomics of fusarium oxysporum f.sp.cubense, a predicted Protein
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of the gene FoPDCD5 in regulating and controlling the pathogenicity of banana vascular wilt.
The invention discloses a gene FoPDCD5 of banana fusarium wilt and a new function of a coding protein PDCD5 thereof. The gene FoPDCD5 is SEQ ID NO: 1, and the encoded protein PDCD5 is SEQ ID NO: 2; the PDCD5 protein contains 1 DNA-binding domain. The invention constructs a gene knockout vector and introduces the gene knockout vector into a protoplast of the fusarium oxysporum f.sp.cubense; knocking out the gene from fusarium oxysporum f.sp.cubense by using a homologous recombination method to obtain a knock-out
The purpose of the invention is realized by the following technical scheme:
the invention provides application of a gene FoPDCD5 in regulation and control of pathogenicity of banana vascular wilt.
Further, the gene FoPDCD5 is applied to regulating and controlling the spore yield of the fusarium oxysporum f.sp.cubense.
Further, the gene FoPDCD5 is applied to the regulation of the stress resistance of banana fusarium oxysporum;
preferably, the stress is oxidative stress.
The invention provides application of a gene FoPDCD5 in preventing and treating banana wilt caused by banana fusarium wilt.
The invention provides application of a gene FoPDCD5 as a target for a plant disease control drug, wherein the plant disease is banana vascular wilt caused by banana vascular wilt.
The invention further provides a method for treating banana vascular wilt caused by banana vascular wilt, which comprises blocking or inhibiting the expression of the gene FoPDCD5 in the banana vascular wilt (for example, by using antisense RNA or siRNA of the gene).
An application of a medicament (for example, antisense RNA or siRNA and the like using the gene) for blocking or inhibiting the expression of the gene FoPDCD5 in the fusarium oxysporum f.sp.cubense in preparing a medicament for controlling the fusarium oxysporum f.sp.cubense caused by the fusarium oxysporum f.sp.cubense.
Wherein, the amino acid sequence of the banana fusarium oxysporum gene FoPDCD5 is shown as SEQ ID NO: 2, or as shown in SEQ ID NO: 2 through one or more amino acid substitutions, insertions and deletions, and the obtained analogue still has the function of controlling the pathogenicity of the fusarium oxysporum;
the nucleotide sequence of the gene FoPDCD5 is one of the following A, B, C:
A. encoding the amino acid sequence of SEQ ID NO: 2;
B. as shown in SEQ ID NO: 1;
C. the analogues obtained by the above A and B through base insertion, deletion or substitution still have the function of controlling the pathogenicity of the fusarium oxysporum f.sp.cubense;
further, the banana fusarium oxysporum is a No. 4 physiological race of the banana fusarium oxysporum (Foc 4).
The application of the knock-out vector and the recombinant bacteria containing the gene FoPDCD5 in the aspects also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following advantages and effects:
the gene FoPDCD5 of the banana fusarium oxysporum provided by the invention contains 1 DNA-binding domain, but the biological function of the gene in the banana fusarium oxysporum is not clear. Replacing a coding gene FoPDCD5 of protein PDCD5 with a hygromycin phosphotransferase gene (hph) and a fluorescent protein gene (SGFP) to obtain a Foc4 knockout mutant delta Fopdcd 5; experiments prove that compared with Foc4 wild type, the yield of the delta Fopdcd5 spores is obviously reduced, and the spores are more sensitive to oxidative stress; pathogenicity tests show that the deletion of FoPDCD5 obviously reduces the pathogenicity of banana vascular wilt; after the gene is complemented back, the pathogenicity of the gene is recovered. The invention proves that the FoPDCD5 is necessary for generating conidia of the fusarium oxysporum f.sp.cubense, and coping with oxidative stress and pathogenicity. The research of the people is helpful to deeply elucidate the pathogenic molecular mechanism of the banana fusarium oxysporum and provides a target gene for developing an effective bactericide.
Drawings
FIG. 1 is a schematic diagram of construction of a banana fusarium oxysporum gene FoPDCD5 knockout vector.
FIG. 2 is a schematic diagram of the construction of a banana fusarium oxysporum gene FoPDCD5 complementation vector.
FIG. 3 is a PCR amplification of part of the hygromycin-resistant transformant A-hph gene; m: marker 5000; lane 1: the plasmid pCT 74; lanes 2-6:
FIG. 4 is a PCR amplification of the partial hygromycin-resistant transformant gene of
FIG. 5 is a Southern blot analysis of Foc4 knock-out transformants probed with FoPDCD5 fragment;
FIG. 6 is a Southern blot analysis of Foc4 knockout transformants probed with the hph fragment;
FIG. 7 is an analysis of knockout mutant △ Fopdcd5 under different stress conditions.
FIG. 8 is a PCR analysis of the FoPDCD5 gene of a portion of bleomycin resistant transformants; m: marker 5000; lane 1: foc4 wild type; lanes 2-5:
FIG. 9 is a pathogenicity analysis of the knockout mutant Δ Fopdcd5 and the anaplerotic mutant Δ Fopdcd 5-com.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.