阅读说明：本技术 一种负调控稻曲病菌产孢的致病因子、基因及应用 (Pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production, gene and application ) 是由 寇艳君 熊萌 时焕斌 邱结华 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种负调控稻曲病菌产孢的致病因子、基因及应用。氨基酸序列如SEQ ID No.1所示,基因序列如SEQ ID No.2所示。本发明经研究发现源于稻曲病菌的真菌致病性基因稻曲病UvPSR1基因在真菌营养生长、孢子产量、侵染菌丝形成和致病过程中具有重要作用,该基因或其编码的蛋白质可以作为用于设计和筛选抗真菌药物的靶标。(The invention discloses a pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production, a gene and application. The amino acid sequence is shown as SEQ ID No.1, and the gene sequence is shown as SEQ ID No. 2. The invention discovers that UvPSR1 gene derived from ustilaginoidea virens and used as a fungal pathogenic gene plays an important role in the processes of fungal vegetative growth, spore yield, infected hypha formation and pathogenesis, and the gene or the protein coded by the gene can be used as a target for designing and screening antifungal medicaments.)

1. A pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

2. A gene encoding the pathogenic agent according to claim 1.

3. The gene of claim 2, wherein the gene sequence is as shown in SEQ ID No. 2.

4. Use of the pathogenic agent of claim 1 or the gene of claim 2 or 3 as a drug target for screening drugs against ustilaginoidea virens.

5. The anti-ustilaginoidea virens drug is characterized in that the active ingredient is at least one of the following substances:

(1) an inhibitor capable of inhibiting the pathogenic agent of claim 1;

(2) a DNA or RNA sequence capable of reducing the expression level of the gene of claim 2 or 3;

(3) a sequence capable of knocking out the gene of claim 2 or 3.

6. An anti-ustilaginoidea virens pharmaceutical according to claim 5, wherein the sequence capable of knocking out the gene according to claim 3 is: recombinant plasmids obtained by inserting flanking sequences Uvpsr1-5 'and Uvpsr 1-3' having the sequences shown in SEQ ID Nos. 3 and 4, respectively, into the vector plasmid pFGL 821.

7. The Ustilaginoidea virens drug of claim 6, wherein the plasmid is introduced into Ustilaginoidea virens cells using Agrobacterium transformation.

8. Use of the Ustilaginoidea virens medicament as claimed in claim 5 for inhibiting Ustilaginoidea virens.

9. Use of the Ustilaginoidea virens medicament as claimed in claim 5 in the prevention or treatment of Ustilaginoidea virens infection in rice.

Technical Field

The invention relates to the technical field of prevention and control of rice false smut, in particular to a pathogenic factor for negatively regulating and controlling rice false smut bacteria spore production, a gene and application.

Background

False smut is a rice disease whose incidence area has been gradually increased in recent years, and has an important influence on the yield and quality of rice. The disease is widely distributed in major rice producing areas, and is more severe in Asian countries such as China, Japan, India, and Philippines. The disease is influenced by climate change, fertilizer application, single variety and other factors in China, and the disease is gradually increased year by year and gradually expanded. The disease is caused by infection of ascomycete subgenus ergomycetaceae sclerotinia (Ustilaginoidea virens), rice flower organs without heading are infected, a dark green rice aspergillus is formed on the ear in the later period, on one hand, the yield of rice is influenced by Ustilaginoidea virens, and on the other hand, the rice quality is influenced by secreting fungaltoxin ustilaginoides and ustilaginomycin. The ustilaginoidea virens wraps grains, produced ustilaginoidea virens pollutes rice, and after people and livestock eat the rice polluted by the ustilaginoidea virens, the ustilaginoidea virens can strongly inhibit human and livestock tubulin assembly and cell skeleton formation, seriously inhibit normal growth of human and livestock cells and greatly threaten human and livestock health. Therefore, in order to ensure the production safety of grains, a target for effectively preventing and treating ustilaginoidea virens is searched, and the research and development of high-efficiency prevention and treatment medicaments are urgent scientific research subjects.

The life history of ustilaginoidea virens comprises sexual stages and asexual stages, wherein the sexual stages can generate sclerotium, the sclerotium germinates to generate stroma, an ascospore is formed in the ascospore, and the ascospore can infect rice. Chlamydospores are generated in the asexual stage, and conidiospores generated by chlamydospore germination also have important functions in infection. Pathogenic bacteria invade from glume flowers to obtain nutrition in rice cells for hypha growth and rice false smut formation. The rice koji ball is wrapped by a film at the early stage, the surface is smooth, the faint yellow is changed into dark green at the later stage, and finally, the rice koji ball cracks to release dark green powder. The early stage of the rice false smut has no obvious symptoms, and rice false smut can be observed on rice ears after 10-15 days of shallow breeding. The ustilaginoidea virens is a living body nutritional pathogenic fungus, does not kill host cells after infecting floral organs, and absorbs nutrients from a living body. In addition to infecting flowers, coleoptiles and seedling parts may also be infected. The biological process of infection of Ustilaginoidea virens is disclosed, which has important significance for effectively preventing and treating the disease.

The size of the Ustilaginoidea virens genome is about 39.4Mb, the size is similar to that of the Magnaporthe grisea, 8426 encoding genes are contained, and the gene density is lower than that of the Magnaporthe grisea. The genetic sequence evolution analysis shows that the genetic relationship between ustilaginoidea virens and the metarhizium is recent. The annotation of genome data lays a good foundation for the development of later functional genomics. The genome information analysis of Ustilaginoidea virens shows that the number of proteins formed by the Ustilaginoidea virens in nutrition absorption, energy metabolism and attachment cells is small, which explains the genetic basis that Ustilaginoidea virens can only form diseases by being infected from rice floral organs to a certain extent.

The molecular mechanism of the rice green smut pathogen is not known. Recently, the biological functions of UvSpo76, Uvt-726, UvSUN2, UvT-B1464R, Uvt-1241 UvHOG1, UvPRO1, Uvt3277 and UvSLT2 genes are determined by a forward genetics method of random insertion of T-DNA and a reverse genetics method of knocking out a target gene by homologous recombination and the like, and the genes are proved to be positively involved in spore differentiation, vegetative hypha growth and pathogenicity of ustics but are reported to negatively regulate and control pathogenic factors of spore production.

Disclosure of Invention

The invention provides a pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production, a gene of the pathogenic factor and application.

A pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production has an amino acid sequence shown as SEQ ID No. 1.

The invention also discloses a gene for coding the pathogenic factor. The gene has a gene sequence shown in SEQ ID No. 2.

The invention also discloses application of the pathogenic factor or the gene as a drug target in screening of anti-ustilaginoidea virens drugs.

The invention also discloses an anti-ustilaginoidea virens drug, the active ingredient is at least one of the following substances:

(1) an inhibitor capable of inhibiting the pathogenic agent;

(2) a DNA or RNA sequence capable of reducing the expression level of said gene;

(3) a sequence capable of knocking out said gene.

The Ustilaginoidea virens resistant drug can knock out the gene with a sequence as follows: recombinant plasmids obtained by inserting flanking sequences Uvpsr1-5 'and Uvpsr 1-3' having the sequences shown in SEQ ID Nos. 3 and 4, respectively, into the vector plasmid pFGL 821. The anti-ustilaginoidea virens drug uses an agrobacterium transformation method to introduce the plasmid into ustilaginoidea virens cells.

The invention also provides application of the anti-ustilaginoidea virens medicament in inhibiting ustilaginoidea virens.

The invention also provides application of the ustilaginoidea virens resistant medicament in preventing or treating ustilaginoidea virens infection.

The invention discovers that UvPSR1 gene derived from ustilaginoidea virens and used as a fungal pathogenic gene plays an important role in the processes of fungal vegetative growth, spore yield, infected hypha formation and pathogenesis, and the gene or the protein coded by the gene can be used as a target for designing and screening antifungal medicaments.

Drawings

FIG. 1 is a schematic diagram of UvPSR1 gene knockout strategy for ustilaginoidea virens.

FIG. 2 is a graph showing the results of detecting the expression level of UvPSR1 gene using QRT-PCR.

FIG. 3 is a graph showing the results of tests on the involvement of UvPSR1 gene in the regulation of hyphal growth, wherein A is a colony map, and B is a colony diameter statistical result.

FIG. 4 is a spore production detection result of UvPSR1 gene negative regulation Ustilaginoidea virens, wherein A is a spore production amount micrograph, and B is a spore production amount statistical result.

FIG. 5 is a graph showing the results of the test of the involvement of the UvPSR1 gene in oxidative stress response, wherein A is a graph of colonies after cultivation, and B is an inhibition rate calculated by the colony diameter.

Fig. 6 is a graph of the results of the detection of UvPSR1 gene participating in the synthesis of ustilaginoidea virens toxin, wherein A is a photograph of late indica 98 seeds, and B is a statistical result of the root length of the late indica 98 seeds.

FIG. 7 is a graph showing the results of detecting the pathogenicity of UvPSR1 gene knockout on rice false smut, wherein A is a photograph of rice false smut on rice ears, and B is the statistical result of the number of rice false smut.

Detailed Description