Verticillium dahlia secreted protein elicitor VdCP1 and application thereof

A technology of Verticillium dahliae and secreted protein, which is applied in the field of Verticillium dahliae secreted protein elicitor, can solve problems such as few reports of protein sequences and gene sequences, and achieves the enhancement of plant resistance to pathogenic bacteria infection and the improvement of Disease resistance, effect of low concentration

Inactive Publication Date: 2017-06-13
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] All the published literature shows that Verticillium dahliae can indeed produce elicitors that cause plant defense responses and improve plant resistance. The properties and types of elicitors are different, but there are few reports

Method used

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  • Verticillium dahlia secreted protein elicitor VdCP1 and application thereof
  • Verticillium dahlia secreted protein elicitor VdCP1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0031] Example 1 Knockout and pathogenicity detection of vdcp1 gene in Verticillium dahliae

[0032] Verticillium dahliae belongs to the genus Verticillium dahliae. Verticillium dahliae can grow at 10~30℃. The optimum temperature for the growth of pathogens is 20~25℃. Most strains do not grow at 33℃, but some strains are more resistant to high temperature. Can grow slowly. The micro sclerotia has strong resistance to bad environment and can withstand high temperature of 80℃ and low temperature of -30℃. The suitable temperature for germination of microsclerotia is 25~30℃. The colony is white, the primary mycelium is colorless, and then becomes olive brown with compartments; the diameter is 2-4μm; the mycelium is often swollen, and can bud from a single or several roots into micro sclerotia; conidia ellipse Shape, single cell, size 4.0-11.0×1.7-4.2μm; split one by one from the end of conidiophore.

[0033] (1) Knockout of vdcp1 gene

[0034] Extract the Verticillium dahliae genome...

Example Embodiment

[0042] Example 2 Cloning of the gene encoding protein elicitor VdCP1

[0043] According to the VdCP1 gene sequence, the primers are finally selected after screening and verification:

[0044] VdCP1-forward 5'-ATGCAGCTGTCCAACCTCCT-3 (shown in SEQ ID NO: 11) and VdCP1-reverse 5'-TCAGAGGCCGCACTTGCTCT-3 (shown in SEQ ID NO: 12), extract the Verticillium dahlia strain mRNA, and reverse transcribed into cDNA, using cDNA as template, select HiFi polymerase for PCR amplification (94℃5min; 94℃30s, 55℃30s, 72℃30s, 35cycles; 72℃5min) to amplify 417bp The DNA fragment, which includes a complete open reading frame of 417bp, was named vdcp1 gene. The sequence of this gene is shown in SEQ ID NO:1.

Example Embodiment

[0045] Example 3 Eukaryotic expression and purification of VdCP1 protein

[0046] (1) Construction of expression vector

[0047] Design a specific primer for the protein elicitor gene vdcp1, and introduce a restriction site with a protection base for restriction. Forward primer: 5’- GAATTC GCCTCTGTCTCCTACGACAA-3', (underlined is the digestion sequence of EcoRI) (shown in SEQ ID NO: 13), reverse primer: 5'- TCTAGA CCGAGGCCGCACTTGCTCTTGT-3', (underlined sequence of XbaI) (as shown in SEQ ID NO: 14), amplify the full-length vdcp1 gene (94°C 5min; 94°C 30s, 55°C 30s, 72°C 30s, 35 cycles; 72°C for 5min). First, clone the vdcp1 gene target fragment into the pMD18-T simple vector. After sequencing verification, the vdcp1 fragment was cloned into the EcoRI / XbaI site of the pPICZαA vector (Invitrogen) after digestion with EcoRI and XbaI.

[0048] (2) Induced expression

[0049] The recombinant vector pPICZαA-vdcp1 obtained in step (1) is heat shocked into the E. coli TransT1 strain, a larg...

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Abstract

The invention discloses a verticillium dahlia secreted protein elicitor VdCP1, and discloses application of the protein elicitor VdCP1 in verticillium dahlia infected cotton and application for improving plant resistance and inducing plant defense reaction. The protein elicitor VdCP1 can obviously improve the plant resistance and is rapid in effect and long in action time. The verticillium dahlia secreted protein elicitor VdCP1 provides a novel way for improving the plant disease resistance, and the wide application prospect is achieved on agricultural production.

Description

technical field [0001] The invention relates to the technical field of encoding microbial proteins, in particular to a secreted protein elicitor of Verticillium dahliae. Background technique [0002] Cotton verticillium wilt is a fungal disease caused by Verticillium dahliae infection, which can harm 660 species of plants in 38 families, including 184 species of crops and 153 species of weeds. The main harmed field crops include cotton, eggplant, tomato, Tobacco, potatoes, watermelons, cucumbers, peanuts, soybeans, etc., resulting in huge economic losses. Verticillium dahliae belongs to the genus Deuteromycotina, Leuchromosporaceae, and the genus Verticillium in terms of taxonomic status. At present, the control of plant diseases mainly adopts chemical control and breeding of disease-resistant varieties. However, they often have disadvantages that cannot be eliminated, especially after the crops are infected by pathogens, there is no effective fungicide to deal with them. ...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19A01N47/44A01P21/00C12R1/84
CPCA01N47/44C07K14/37
Inventor 邱德文杨秀芬曾洪梅郭立华袁京京张易
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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