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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 on protein sequences and gene sequences, so looking for The new protein elicitor is very important for revealing the interaction mechanism between Verticillium dahliae and plants and for eliciting elicitors to improve plant immune resistance

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

Embodiment 1

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

[0032] Verticillium dahliae belongs to the fungus of the genus Verticillium of the subphylum Verticillium. Verticillium dahliae can grow at 10-30°C, the optimum temperature for the growth of the bacteria is 20-25°C, and most of the strains do not grow at 33°C, but some strains are more resistant to high temperature and can still survive at 33°C. Can grow slowly. Microsclerotia has strong resistance to adverse environments, and can withstand high temperatures of 80°C and low temperatures of -30°C. Microsclerotia germination optimum temperature is 25 ~ 30 ℃. Colony white, primary mycelium colorless, later turning olive brown, separated; diameter 2-4μm; mycelium often swollen, single or several mycelium can germinate into microsclerotia; conidia ellipsoid Shaped, unicellular, 4.0-11.0×1.7-4.2μm in size; divided one by one from the end of the conidiophores.

[0033] (1) Knockout of vd...

Embodiment 2

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

[0043] According to the VdCP1 gene sequence, after screening and verification, the final selection of primers:

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

Embodiment 3

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

[0046] (1) Construction of expression vector

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

[0048] (2) Induced expression

[0049] The recombinant vector pPICZαA-vdcp1 obta...

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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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