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.
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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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