Cochliobolus heterostrophus ChCDC10 gene and application thereof

A kind of corn blight fungus and gene technology, which can be applied in the application field of discovery and its encoded protein, and can solve the problems of loss and the like

Active Publication Date: 2019-09-20
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the 1970s, with the promotion of corn disease-resistant varieties, the occurrence of small spot disease has been basically controlled, but due to the large-scale simplification of planting disease-resistant varieties and global warming, in some corn production areas in my country The occurrence of small spot disease has serious occurrence, causing heavy losses

Method used

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  • Cochliobolus heterostrophus ChCDC10 gene and application thereof
  • Cochliobolus heterostrophus ChCDC10 gene and application thereof
  • Cochliobolus heterostrophus ChCDC10 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Correlation analysis of the ChCDC10 gene of maize spot bacterium

[0038] The ChCDC10 gene of P. maize was obtained by comparison with the CDC gene in yeast by our team in P. maize. The open reading frame of ChCDC10 gene of maize leaf spot consists of 1197 nucleotides, including 2 introns. The encoded protein product consists of 341 amino acids, and domain analysis revealed that the ChCDC10 protein contains a conserved CDC-Septin domain (see figure 1 ).

Embodiment 2

[0039] Example 2 Knockout of the ChCDC10 gene of P. maize spot bacterium

[0040] 1) Amplification of the upstream and downstream of ChCDC10 gene and hygromycin gene

[0041] Primers F1 (5'-GCCATTCCTACGTCAAAACC-3') and R1 (5'-TCCTGTGTGAAATTGTTATCCGCTGGCAGACGGACAAGGTAAAA-3') were used to amplify the upstream 888p fragment of ChCDC10 gene using F2(5 '-GTCGTGACTGGGAAAACCCTGGCGTCGCGTCGATAGCAATACAG-3') and R2 (5'-GACACGGCAAACACTGAAGA-3') to amplify the 884bp fragment downstream of the ChCDC10 gene of P. 3'), using the vector pUCATPH as a template to amplify the 2549bp hygromycin gene. The reaction system is: 10mmol / LdNTPMixture, 1μL; 5×PCRbuffer, 10μL; each 2.5μL of upstream and downstream primers (10μmol / mL); template DNA, 2μL; Phusionpolymerase, 0.5μL (5U); ddH 2 O, 31.5 μL; amplification program: 98°C pre-denaturation for 2 minutes, then (1) 98°C, denaturation for 20 seconds; (2) 65°C, annealing for 30 seconds; (3) 72°C, extension for 30 seconds; (4) ) cycled 30 times; (5) ex...

Embodiment 3

[0049] Example 3 Genetic Complementation of ChCDC10 Gene Deletion Mutant

[0050]Primers C-F1 (5'-GCTCTAGATGAGCTGACCGAAGATGTTG-3') and C-R1 (5'-CACTGGAACAACTGGCATGTTTGAGAAGTTTGCCGCTCT-3') were used to amplify the full-length 3112bp (including upstream and downstream sequences) of the ChCDC3 gene of P. F2 (5'-CAGGTACACTTGTTTAGAGGT CGTGTTGTTTCTCCAAGCTG-3') and C-R2 (5'-GGGCAGAATCTTCTTTGGTG-3') amplify the downstream sequence 419bp downstream of the ChCDC3 gene of B. maize. Then the vector pⅡ99 was used as a template, and the nptⅡ gene was amplified with DW69 (5'-CATGCCAGTTGTTCCAGTG-3') and DW70 (5'-ACCTCTAAACAAGTGTACCTG-3') primers. The three complementary fragments were transferred into the genome of the ChCDC10 gene deletion mutant, and Geneticin was used as a selection marker to screen the genetic complementation strain ΔChCDC10-C. Primers F / R were selected for PCR verification.

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Abstract

The invention relates to a Cochliobolus heterostrophus ChCDC10 gene and application thereof and belongs to the technical field of microorganism genetic engineering. The DNA sequence of the ChCDC10 gene from Cochliobolus heterostrophus and used for controlling conidium formation, ascospore formation and virulence is as shown in SEQ ID No. 1. The amino acid sequence of protein encoded by the ChCDC10 gene is as shown in SEQ ID NO. 2. The ChCDC10 gene is applicable to the field of plant anti-Cochliobolus heterostrophus genetic engineering. By deleting, mutating or modifying the Cochliobolus heterostrophus protein ChCDC10 for controlling conidium formation, ascospore formation and virulence, conidium formation and ascospore formation are limited, and virulence is lowered; the protein can be used as the target to apply in the design and screening of anti-Cochliobolus heterostrophus chemicals, especially plants do not contain the protein, and plant safety is achieved.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and specifically relates to the discovery of new genes controlling fungal pathogenicity in the field of plant protection and the application of encoded proteins. Background technique [0002] The amorphic scientific name of corn spot bacterium is: Bipolaris maydis, which belongs to the subphylum Helminthosporium, which belongs to the half-knowledge subphylum; its morphological scientific name is Cochliobolus heterostrophus, which belongs to the subphylum Ascomycota. In 1925, it was first discovered as the causative agent of corn disease. Corn spot fungus is a filamentous fungus. When the mycelium grows to a certain stage, under the influence of external environmental conditions and its own factors, conidia grow on the top or side of the conidiophores. Conidia have 3-13 septa, most of which have 7-9 septa, dark brown, oblong, blunt at both ends, mostly curved to one side, th...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N3/00C12R1/645
CPCC07K14/37C12N3/00
Inventor 张祥辉于汇琳汪蕊王璐刘金亮潘洪玉
Owner JILIN UNIV
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