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Method for screening anti-plant nematode infection compound by taking 2-Cys Prx as target

A technology for plant nematodes and screening methods, applied in the fields of botanical equipment and methods, biochemical equipment and methods, nematicides, etc.

Active Publication Date: 2015-04-29
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of using Prx as antigen, there are reports of relevant patents abroad. For example, Brugia malayi (Brugia malayi) Prx6 has applied for a U.S. patent (U.S. Patent 6,352,836) as a vaccine antigen; Leishmania donovani (L. .donovani) Prx1a has applied for a US patent (U.S. Patent 7795406) ​​as a vaccine antigen candidate, but there are still relatively few reports on the development of drugs for the prevention and control of plant nematodes with Prx as the target

Method used

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  • Method for screening anti-plant nematode infection compound by taking 2-Cys Prx as target
  • Method for screening anti-plant nematode infection compound by taking 2-Cys Prx as target
  • Method for screening anti-plant nematode infection compound by taking 2-Cys Prx as target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of D. destructor potato 2-Cys Prx gene

[0028] Specific primers were designed according to the full-length sequence of D. destructor cDNA in GeneBank, and the upstream primer N-PRXF: 5'- CCATGG TACCCAAGTTTGAGACCATC-3' (SEQ ID No.1), the underline is the NcoI restriction site; downstream primer X-PRXR: 3'-ATAAAGCCGTTCGTCTTC GAGCTC- 5' (SEQ ID No.2), the underline is the XhoI restriction site. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0029] Using the cDNA of D. destructor potato prepared by reverse transcription method as a template, PCR amplification was carried out with the above primers. The reaction system was 5×GXL PCR Buffer 10 μl, dNTP Mixture 4 μl, N-PRXF 1 μl, X-PRXR 1 μl, Prime STAR GXL 0.5μl, cDNA 1μl, and H 2 O 32.5 μl. The reaction conditions are: 94°C for 5 min; 35 cycles of 94°C for 30s, 53°C for 30s, 72°C for 1 min; 72°C for 10 min. Embodiment 2 Construction of recombinant expression plasmid

Embodiment 2

[0029] Using the cDNA of D. destructor potato prepared by reverse transcription method as a template, PCR amplification was carried out with the above primers. The reaction system was 5×GXL PCR Buffer 10 μl, dNTP Mixture 4 μl, N-PRXF 1 μl, X-PRXR 1 μl, Prime STAR GXL 0.5μl, cDNA 1μl, and H 2 O 32.5 μl. The reaction conditions are: 94°C for 5 min; 35 cycles of 94°C for 30s, 53°C for 30s, 72°C for 1 min; 72°C for 10 min. Embodiment 2 Construction of recombinant expression plasmid

[0030]The PCR product obtained in Example 1 was ligated into the pEASY-Blunt cloning vector and transformed into Trans1-T1, and 200 μl of bacterial liquid was cultured on an LB plate containing 50 ug / ml kanamycin overnight at 37°C. The next day, a single colony was picked for bacterial culture, and the positive clones verified by bacterial liquid PCR were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the known D. destructor Prx cDNA usin...

Embodiment 3

[0032] Embodiment 3 recombinant DdPrx protein expression

[0033] The expression vector constructed in Example 2 was optimized at different temperatures (18-37° C.) and different IPTG concentrations (1.4-0.2 mmol / L) to see if the recombinant DdPrx protein was expressed. Depend on figure 2 It can be seen that compared with the low molecular weight protein Marker, it can be seen that the recombinant DdPrx protein has an obvious thick band at 55kDa, which is consistent with the expectation. It shows that when the concentration of IPTG is 1.4-0.2mmol / L at 18-37°C, the stable expression of recombinant DdPrx protein can be induced.

[0034] The BL21 (DE3) bacterial liquid introduced with the recombinant DdPrx plasmid is induced and cultured, and the specific steps are as follows: Take 2 μl of the preserved BL21 (DE3) bacterial liquid containing the recombinant DdPrx plasmid and add 5 ml of LB liquid medium containing 50 μg / ml kanamycin Incubate overnight at 37°C. The next day, t...

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Abstract

The invention discloses a method for screening an anti-plant nematode infection compound by taking 2-Cys Prx type peroxiredoxin as a target. The method comprises the following steps: constructing recombinant expression plasmids pET41b-DdPrx containing Ditylenchus destructor 2-Cys Prx genes; transferring the recombinant expression plasmids into BL21 (DE3) competent cells, inducing the culture by virtue of IPTG so as to stably express recombinant Dd-PRX proteins; and co-incubating a candidate compound and the recombinant DdPrx proteins, and detecting whether the candidate compound has in-vitro inhibitory activity on the recombinant DdPrx proteins according to a ferric thiocyanate method. The method has the advantages of directness, simplicity, convenience and rapidness, can be used for manual screening and also can be used for screening large-scale high flux inhibitors. The method can be used for researching, developing and screening medicines for resisting plant nematode infection.

Description

technical field [0001] The invention relates to the field of plant parasitic nematode insecticides, in particular to a screening method for compounds targeting 2-Cys type peroxiredoxins (Prx), ie, 2-Cys Prx, against plant nematode infestation. Background technique [0002] Plant parasitic nematodes are one of the important pathogens that cause plant diseases. On a global scale, the occurrence and damage of plant pathogenic nematodes have become increasingly serious with the development of agriculture and forestry and the evolution of farming systems, and have become the third largest disaster in agricultural biological disasters after insects and plant diseases. According to reports, there are more than 100 kinds of nematodes that seriously harm my country's agriculture, forestry, and economic crops. Plant-parasitic nematodes cost an estimated $125 billion in annual crop yield losses worldwide. The current method of controlling nematodes is mainly through cultivation techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12Q1/28A01N43/42A01P5/00
Inventor 丁中刘洋刘建喜
Owner HUNAN AGRICULTURAL UNIV
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