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Cochliobolus heterostrophus ChCDC3 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: 2019-10-08
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 ChCDC3 gene and application thereof
  • Cochliobolus heterostrophus ChCDC3 gene and application thereof
  • Cochliobolus heterostrophus ChCDC3 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Correlation analysis of ChCDC3 gene

[0035] The ChCDC3 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 the ChCDC3 gene of maize leaf spot consists of 1403 nucleotides and contains 2 introns. The encoded protein product is composed of 432 amino acids, and domain analysis revealed that the protein encoded by ChCDC3 contains a conserved CDC-Septin domain (see figure 1 ).

Embodiment 2

[0036] Example 2 The Knockout of the ChCDC3 Gene of Corn Leaf Spot Bacteria

[0037] 1) Amplification of the upstream and downstream of the ChCDC3 gene and the hygromycin gene

[0038] Primers F1 (5'-T C G T G C C T C C T C A T T T A A C C-3') and R1 (5'-TCCTGTGTGAAATTGTTATCCGCTGGAGAAGTAGCACCGTTGGA-3') were used to amplify the upstream 942p fragment of ChCDC3 gene using the genomic DNA of wild-type strain C4 of P. (5'-GTCGTGACTGGGAAAACCCTGGCGGCTATCGGTAAACGGGACAA-3') and R2 (5'-CCCGCTCATGATACTCTTCG-3') to amplify the downstream 805bp fragment of ChCDC3 gene of P. AGCGGATAACAATTTCACACAGGA-3'), using the vector pUCATPH as a template to amplify the 2549bp hygromycin gene. The reaction system is: 10mmol / L dNTP Mixture, 1μL; 5×PCRbuffer, 10μL; each 2.5μL of upstream and downstream primers (10μmol / mL); template DNA, 2μL; Phusion polymerase, 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,...

Embodiment 3

[0046] Example 3 Genetic Complementation of ChCDC3 Gene Deletion Mutants

[0047] Primers C-F1 (5'-TCGTGCCTCCTCATTTAACC-3') and C-R1 (5'-CACTGGAACAACTGGCATGCACTCCTCGCTCCGAACTAC-3') were used to amplify the full-length 2755bp (including upstream and downstream sequences) of the ChCDC3 gene of P. F2 (5'-CAGGTACACTTGTTTAGAGGTCTCAGATATGGCACCCATGA-3') and C-R2 (5'-TTGTAGACGACGATGCCGTA-3') amplify 827 bp of the downstream sequence 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 ChCDC3 gene deletion mutant, and Geneticin was used as a selection marker to screen the genetic complementation strain ΔChCDC3-C. Primers F / R were selected for PCR verification.

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Abstract

The invention relates to a cochliobolus heterostrophus ChCDC3 gene and an application thereof, and belongs to the technical field of microorganism genetic engineering. The DNA sequence of the ChCDC3 gene which is from cochliobolus heterostrophus and used for controlling the growth speed of colonies, conidium formation and ascospore formation provided by the invention is as shown in SEQID No:1, andthe amino acid sequence of protein coded by the provided ChCDC3 gene is as shown in SEQID No:2. The ChCDC3 gene can be applied to the genetic engineering field of plants for resisting corn southern leaf blight. The protein ChCDC3 for controlling the growth speed of colonies of the cochliobolus heterostrophus, conidium formation and ascospore formation is subjected to deletion, mutation or modification, the growth of the cochliobolus heterostrophus is slow, the conidium formation and the ascospore formation are limited, lifecycle of the cochliobolus heterostrophus is obstructed, the ChCDC3 gene can be used as a target to be applied to designing and screening corn southern leaf blight resisting medicaments, especially the protein does not exist in plants, and the ChCDC3 gene is safe to theplants.

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 disease is: Bipolaris maydis, which belongs to the half-knowledge fungus subphylum Helminthosporium, and its sexual morphological name is Cochliobolus heterostrophus, which belongs to the Ascomycota subphylum Heterostrophus. 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, thick in the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37
CPCC07K14/37
Inventor 张祥辉孙玥李乐王璐刘金亮潘洪玉
Owner JILIN UNIV
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