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Corynespora cassicola CcTLS1 protein, coding gene and application thereof

A technology of Corynebacterium multimaster and coding gene, which can be used in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of less cloning and functional identification of pathogenic related genes, and cannot fully represent the pathogenic mechanism of Corynebacterium multimaster. question

Active Publication Date: 2021-10-08
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, except for the two MAPK genes CCk1 and CMP1 and the gene Cas encoded by Cassiicolin, other pathogenicity-related genes have rarely been cloned and functionally identified, which are far from fully representing the pathogenic mechanism of Corynespora polyarchaeum

Method used

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  • Corynespora cassicola CcTLS1 protein, coding gene and application thereof
  • Corynespora cassicola CcTLS1 protein, coding gene and application thereof
  • Corynespora cassicola CcTLS1 protein, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Obtaining of Corynespora polymata CcTLS1 knockout mutant

[0080] 1. Construction of pCAMBIA1300-ΔCcTLS1 knockout vector

[0081] For the amino acid sequence of CcTLS1 in Corynespora polymata, see Sequence 1 in the Sequence Listing, for the CDS sequence encoding CcTLS1 protein, see Sequence 2 in the Sequence Listing, and for the gene sequence of CcTLS1 in Corynespora polymata, see Sequence 4 2049-3270 in the Sequence Listing shown. According to the analysis of the uniprotKB database (https: / / www.uniprot.org / uniprot / ), the CcTLS1 protein does not contain a structural domain. Currently, the specific biological function of the CcTLS1 gene in Corynespora polybasicum is not clear.

[0082] The binary vector pCAMBIA1300 was cut with restriction endonuclease Xho I to remove the hygromycin resistance gene, and self-ligated with T4 ligase, and the resulting plasmid was named pCAMBIA1300-XhoI.

[0083] Using the CTAB method to extract the genomic DNA of Corynespora po...

Embodiment 2

[0120] Example 2, the acquisition of the CcTLS1 anaplerotic mutant of Corynespora polymata

[0121] 1. Construction of pCAMBIA1300-cΔCcTLS1 complementation vector

[0122] Using the CTAB method to extract the genomic DNA of Corynespora polybasicum HG14102524, using the genomic DNA as a template, using MaxDNAPolymerase, with primer pair 4 (primer pair consisting of F4 and R4), PCR amplification was carried out to obtain the CcTLS1 gene and its 5' end 3265kb fragment (containing the 6th-3270th position of sequence 4):

[0123] F4: 5'-CCATGATTAC GAATTC GCTCGGGATTTGGCACACG-3' (the sequence indicated by the underline is the EcoR I enzyme recognition site sequence);

[0124] R4: 5'-CTAGAGGATCCCCGGGTACCTTACAATGGCTGTCGTACTGGG-3' (the sequence indicated by the underline is the Kpn I enzyme recognition site sequence).

[0125] Using the above genomic DNA as a template, using MaxDNAPolymerase, PCR amplification was carried out with primer pair 5 (primer pair consisting of F5 and R...

Embodiment 3

[0159] Pathogenicity detection of embodiment 3 mutants

[0160] The mutants to be tested are the CcTLS1 knockout mutant ΔCcTLS1 constructed in Example 1 (abbreviated as the knockout strain ΔCcTLS1), and the anaplerotic mutant cΔCcTLS1 constructed in Example 2 (abbreviated as the anaplerotic strain cΔCcTLS1), and the control is the wild-type multi-rod Bacillus sp. HG14102524 (abbreviated as wild-type HG14102524).

[0161] The pathogenicity is identified by the isolated patch culture method, and the specific steps are as follows: Prepare a clean moisturizing box and spray an appropriate amount of water to keep it moisturized. Arrange the collected cucumber leaves with their leaf backs facing upwards in a humidifying box (three pieces in a box). Corynespora polybasicum (knockout strain ΔCcTLS1, complementation strain cΔCcTLS1 and wild-type HG14102524) activated on the PDA medium were punched into bacterial cakes at the edge of the colony with a 0.5 cm puncher, and sterilized Th...

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Abstract

The invention discloses a corynespora cassicola CcTLS1 protein, a coding gene and application thereof. Experiments in the invention prove that: after the corynespora cassicola CcTLS1 gene is knocked out, the scab area of the obtained corynespora cassicola knockout mutant on cucumber leaves is greatly reduced compared with that of a wild strain; the mycelial growth speed of the corynespora cassicola knockout mutant is obviously lower than that of wild corynespora cassicola; and the activity of cellobiose hydrolase of the corynespora cassicola knockout mutant is obviously lower than that of the wild corynespora cassicola. The result shows that the deletion of the corynespora cassicola knockout mutant CcTLS1 gene can cause the decrease of the infection ability of the corynespora cassicola to the cucumber. The CcTLS1 gene and application thereof provided by the invention have important significance in prevention and control of cucumber corynespora leaf spot.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the CcTLS1 protein and coding gene of Corynespora polymata and its application. Background technique [0002] Corynespora cassicola (Berk.&M.A.Curtis) C.T.Wei is a plant pathogen with a wide range of hosts, which can not only infect crops with important economic value, such as cucumber, tomato, pepper, eggplant , papaya, soybean and rubber trees, etc., can also infect horticultural flowers and plants. Corynespora polyarchaeum has a variety of lifestyles, including saprophytic, endophytic, and necrotic. Corynesporium leaf spot has become an important disease in vegetable production in recent years. [0003] Corynespora multiprimum attaches to diseased residues and seeds in the form of mycelium and conidia, survives in the soil, and can also survive on non-host plants, becoming a new source of infection. Corynespora multiprimum can survive on the sick body for 2 years. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/80C07K14/37
CPCC07K14/37Y02A50/30
Inventor 李宝聚柴阿丽王泉城武军石延霞谢学文李磊
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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