阅读说明：本技术 一种提高植物抗病性的铜绿假单胞菌鞭毛蛋白及其编码基因和应用 (Pseudomonas aeruginosa flagellin for improving plant disease resistance and coding gene and application thereof ) 是由 蔡易 李琦 郭晋雅 李雍 周鑫琼 张丽梅 于 2019-10-15 设计创作，主要内容包括：本发明公开了一种提高植物抗病性的铜绿假单胞菌鞭毛蛋白及其编码基因和应用,该蛋白的氨基酸序列如SEQ ID NO:1所示或SEQ ID NO：1所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸,且表达相同功能蛋白质的氨基酸序列；编码该蛋白的基因的核苷酸序列如SEQ ID NO:2所示或SEQ ID NO：2所示的核苷酸序列经取代、缺失和/或添加一个或多个核苷酸,且能编码相同功能蛋白质的核苷酸序列。该蛋白可作为免疫激发子可以明显的提高植物的抗性病,其使用浓度低,起效快,可有效降低农业生产成本。(The invention discloses pseudomonas aeruginosa flagellin for improving plant disease resistance, and a coding gene and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO:1 or SEQ ID NO:1, one or more amino acids are substituted, deleted and/or added, and the amino acid sequences of the proteins with the same function are expressed; the nucleotide sequence of the gene for coding the protein is shown as SEQ ID NO:2 or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein. The protein can be used as an immune exciton, can obviously improve the resistance disease of plants, has low use concentration and quick response, and can effectively reduce the agricultural production cost.)

1. The pseudomonas aeruginosa flagellin for improving the disease resistance of plants is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:1, or SEQ ID NO:1 is substituted, deleted and/or added with one or more amino acids, and expresses the amino acid sequence of the same functional protein.

2. The gene encoding pseudomonas aeruginosa flagellin for improving plant disease resistance as claimed in claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 2, or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein.

3. A recombinant vector comprising the gene of claim 2.

4. A recombinant bacterium comprising the gene according to claim 2.

5. A host cell comprising the gene of claim 2.

6. Use of pseudomonas aeruginosa flagellin as claimed in claim 1 for enhancing disease resistance and inducing plant defense response.

7. The application of claim 6, in particular to the application of improving the resistance of plants to pseudomonas syringae infection, rice blast fungus infection and fusarium graminearum infection.

8. Use according to claim 6 or 7, wherein the plant is a crucifer, a solanaceous plant and a graminaceous plant.

9. The use according to claim 6, wherein the Pseudomonas aeruginosa flagellin is used at a concentration of 0.1-2 μ M.

10. A disease resistance drug for plants comprising the pseudomonas aeruginosa flagellin of claim 1.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to pseudomonas aeruginosa flagellin for improving plant disease resistance, and a coding gene and application thereof.

Background

At present, a plurality of biopesticide products are widely applied in the world, and the most extensive biopesticide products are agricultural antibiotics such as bacillus thuringiensis (Bt) and validamycin, zhongshengmycin, brassin and the like. The products achieve the aims of insect resistance and disease resistance through bacteriostatic and insecticidal substances of biological origin. Compared with chemical pesticide and antibiotic agent, these products have short degradation period and high environment compatibility. However, the occurrence of diseases and insect pests is the process of interaction between plants and pathogens, and in the past, the research and development of biopesticides often neglects the role of the immunity of plants in the process of resisting the diseases and insect pests of the plants. In recent years, with the research progress of plant disease resistance mechanism, the plant immunity induction technology becomes a new bright point in the development of biological pesticides. The research on microbial protein pesticides with the functions of stimulating plant immunity and disease resistance, promoting growth and increasing yield has attracted extensive attention and attention at home and abroad. As early as 2001, the american company EDEN developed the Messenger pesticide product from allergenic proteins of bacterial origin, however its nature is still a fungicide. In 2016, the subject group of professor Qiudens of plant protection institute of agricultural academy of China extracted an activator protein from alternaria tenuissima, and the protein is proved to greatly improve the resistance of plants to virus diseases, so that the first global plant immunity protein pesticide, namely the atrazine, is developed, the disease resistance and yield increase of agricultural products are realized by enhancing the autoimmunity of the plants, and the pioneer of 'plant immunity' is created.

However, from the macroscopic view, the plant immunity pesticide on the market has fewer varieties, and only has a plurality of varieties such as Tailing and Harpin protein. At a microscopic level, these products still have limitations. Firstly, receptors of alternaria tenuissima activator protein and Harpin protein are not clear, which means that the alternaria tenuissima activator protein and Harpin protein are difficult to use, scientific and effective guidance is made in theory, the application range and the use concentration of the alternaria tenuissima activator protein and the Harpin protein can be judged only through experimental experience, and a long-term continuous exploration process is needed; secondly, the sensitivity of the plants to the plant immunity pesticides is low, the final using concentration is 100 mu mol, the using amount per mu is 100g, and the cost is over 40 yuan per mu. In order to reduce the cost of agricultural production, research and development of new products is still required.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides pseudomonas aeruginosa flagellin for improving plant disease resistance, a coding gene and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

the pseudomonas aeruginosa flagellin for improving the disease resistance of the plants has an amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:1 is substituted, deleted and/or added with one or more amino acids, and expresses the amino acid sequence of the same functional protein.

The nucleotide sequence of the gene for coding the pseudomonas aeruginosa flagellin is shown as SEQ ID NO. 2, or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein.

A recombinant vector comprising the gene encoding pseudomonas aeruginosa flagellin as described above.

A recombinant bacterium comprising the gene encoding Pseudomonas aeruginosa flagellin as described above.

A host cell comprising the gene encoding Pseudomonas aeruginosa flagellin as described above.

The application of the pseudomonas aeruginosa flagellin in improving the disease resistance of plants and inducing the defense reaction of the plants comprises the following specific steps: the application of the composition in improving the resistance of plants to pseudomonas putida infection, rice blast infection and fusarium graminearum infection is further disclosed, and the plants are cruciferous plants (such as arabidopsis thaliana, rape and the like), solanaceous plants (such as tobacco, tomato and the like) and gramineous plants (such as rice, corn, wheat and the like). More specifically: the pseudomonas aeruginosa flagellin is applied to the improvement of the infection resistance of cruciferae and solanaceae plants to pseudomonas syringae, the improvement of the infection resistance of rice blast germs and the improvement of the infection resistance of corn and wheat to fusarium graminearum.

When the Pseudomonas aeruginosa flagellin is used in the above application, the concentration is 0.1-2. mu.M, preferably 1. mu.M.

In addition, pseudomonas aeruginosa flagellin can be prepared into a plant disease resistance medicament for preventing and treating diseases in agricultural production, so that the use cost in agricultural production is effectively reduced.

The pseudomonas aeruginosa flagellin for improving the disease resistance of plants, the coding gene and the application thereof provided by the invention have the following beneficial effects:

the pseudomonas aeruginosa flagellin serving as an immune exciton can obviously improve the resistance disease of plants, has low use concentration and quick response, and can effectively reduce the agricultural production cost.

Drawings

FIG. 1 shows the experimental results of the optimization of protein expression conditions.

FIG. 2 shows the results of the solubility analysis and purification of the protein.

FIG. 3 shows the callose detection results of the protein-stimulated plant immunity.

FIG. 4 shows the detection results of protein-stimulated disease-resistant genes.

FIG. 5 shows the experimental results of protein inhibition of Pseudomonas syringae infection in Arabidopsis thaliana.

FIG. 6

a is the actual measurement comparison result of the growth of corn infected by fusarium graminearum with protein inhibition

b is the experimental result of inhibiting corn infection by fusarium graminearum with protein

FIG. 7

a is the actual measurement comparison result of the growth of rice infected by rice blast germs inhibited by protein

b is the experimental result of inhibiting rice blast germs from infecting rice by protein

Detailed Description

The experimental materials used in the following examples are as follows:

1. rice material

Rice (OryzasativaL.): short grain japonica rice variety Nipponlily (NPB) (NPB is an international universal variety that has been sequenced from the whole genome).

2. Corn material:

the dredge sheet 20 is purchased by a seed company.

3. Arabidopsis thaliana material

Columbia wild type Arabidopsis thaliana col-0 was preserved by the subject group of Chua professor, institute of Life sciences, Sichuan university of agriculture.

4. Strain material:

pseudomonas syringae DC3000 is preserved by the subject group of Chua Yi professor of the institute of Life sciences of Sichuan university, rice blast ZB15 race material is given to the rice research institute of Sichuan university, and corn fusarium graminearum material is given to the college of Sichuan university.