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Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof

A technology of shell mold and biocontrol bacteria, applied in the fields of application, fungi, genetic engineering, etc.

Inactive Publication Date: 2010-12-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because sclerotinia occurs frequently and is popular in the middle and late stages of rapeseed growth, specific pesticide dosage and labor intensity are required to make the pesticides contact the damaged parts (stems and branches of rapeseed); and another problem that cannot be ignored is the sclerotinia Antibiotic resistance of bacteria, long-term large-scale use of single-type pesticides has a very high risk

Method used

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  • Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof
  • Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof
  • Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Isolation of CmPex2, a sporulation-related gene of C. shieldiae

[0028] 1.1 Screening and identification of mutants

[0029] 1.1.1 Construction of T-DNA marker insertion mutant library of C. shield ZS-1 strain

[0030] According to the mature Agrobacterium-mediated genetic transformation method of C. shieldiae (Li Moxiao et al., 2005), C. shieldiae ZS-1 strain was transformed to obtain a C. shieldiae insertion mutant library containing more than 45,000 T-DNA markers.

[0031] 1.1.2 Screening of mutants

[0032] Use PDA medium (PDA adopts the conventional production method: 200g peeled potatoes, after adding appropriate amount of distilled water to cook, add 20g of glucose, 13g of agar powder, and supplement distilled water to 1000ml) at 20°C to activate and cultivate the ZS-1 bacterial strain of C. shieldum and For the transformant of Shell mold, use a hole puncher (5mm in diameter) to get the mycelial block on the edge of activation, and transfer it to the...

Embodiment 2

[0059] Example 2 Bioinformatics analysis of CMPEX2 protein and its homologous proteins in other fungi

[0060] The amino acid sequence putatively encoded by the CmPex2 gene was analyzed through the NCBI BLASTP database, and it was found that the sequence contained a conserved region, namely the PEX2-PEX12 domain ( Figure 6 A), PEX2 and PEX12 are two important proteins in the peroxisome of the organism. And the amino acid carbon-terminal contains a cysteine-rich zinc finger structure, and the peroxisome synthesis factor 2 (PEX2) in other fungi such as Aspergillus nidulans has the same structure ( Figure 6 B). At the same time, it was found that some plant pathogens such as wheat yellow leaf blight (Pyrenophora tritici-repentis), wheat glume blight (Phaeosphaeria nodorum), gray mold (Botryotinia fuckeliana), sclerotinia (Sclerotinia sclerotiorum), rice blast fungus (Magnaporthe grisea ) and so on, all have homologous proteins, and their amino acid sequence identities with CM...

Embodiment 3

[0061] Demonstration of the role of embodiment 3 CmPex2 gene in the production of shell mold conidia

[0062] By constructing the knockout vector and complementation vector of the CmPex2 gene, the Agrobacterium mediated transformation of the ZS-1 strain and the sporulation-deficient mutant ZS-1T16229 strain were carried out to achieve gene knockout and gene complementation. Phenotypic changes validate the gene's role in conidia production of C. shield. The gene knockout vector refers to dividing the gene of CmPex2 into two fragments and connecting them into one vector, and separating them with hygromycin resistance gene (hph) between the two DNA fragments (700bp and 740bp respectively). A good vector was transferred into strain ZS-1, and according to the principle of homologous recombination, the partial sequence of the corresponding CmPex2 gene in the genome was replaced with the hygromycin gene, resulting in the deletion of the CmPex2 gene (see Figure 7 ). The constructio...

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Abstract

The invention relates to a sporulation-related gene clone in biocontrol fungus coniothyrium minitans and application thereof. The invention provides a gene sequence of peroxisome synthesis related gene CmPex2 in the coniothyrium minitans and an encoded protein sequence thereof. The invention also provides application of the peroxisome related gene CmPex2 in the coniothyrium minitans. The expression analysis of the peroxisome related gene CmPex2 in the coniothyrium minitans indicates that the expression level of the gene remarkably increases at the conidium generation stage, and a gene targeting knockout and gene complementation test indicates that the gene is closely related to the conidium generation of the biocontrol fungus coniothyrium minitans. The gene can be taken as a candidate gene for controlling the conidium output of the coniothyrium minitans and is used for increasing the conidium output of the coniothyrium minitans and reducing the scale production cost of the coniothyrium minitans.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to the isolation, cloning, functional verification and application of a gene CmPex2 related to sporulation of Shell mold. Background technique [0002] Sclerotinia sclerotiorum (Lib.) de Bary] is a necrotrophic fungus that is widely distributed throughout the world and can infect more than 400 species of plants in 75 families, including major oil crops such as rapeseed, soybean, sunflower, and lettuce , Chinese cabbage, celery, carrots and other important vegetable crops, causing sclerotinia of plants. Because the temperature and humidity in vegetable protected areas are conducive to the prevalence of sclerotinia, with the large-scale promotion of vegetable production in protected areas, the occurrence of vegetable sclerotinia is becoming more and more serious, and it has risen to become the main disease of various vegetable crops. Since no germpla...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N1/15C07K14/37C12R1/645
Inventor 付艳苹谢甲涛姜道宏郭育林程家森
Owner HUAZHONG AGRI UNIV
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