Application of brassica rapa brassica BraALA3 and BraENT1 gene families in regulation and control of absorption and accumulation of propiconazole
A technology of propiconazole and cabbage, which is applied in the field of plant genetic engineering, can solve the problems of pesticide residues, shorten the breeding years, hidden dangers and the like, and achieve the effects of increased sensitivity and great application prospects.
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[0040] Example 1 Yeast-deficient library screening for propiconazole-related transport genes
[0041] 1. Activation of yeast-deficient libraries
[0042] Transfer the yeast-deficient library from -80°C ultra-low temperature freezer to 4°C freezer to thaw. The mutant strains were transferred from standard 96 deep-well plates to YPD solid plates for activation using a 96-pin replicator and incubated at 30°C for 2 days until large colonies were formed.
[0043] 2. Screening of yeast-deficient libraries with propiconazole
[0044] Use a 96-pin replicator to inoculate the mutant strain on the activated plate into a standard 96 deep-well plate containing 200 μL of liquid YPD, shake it in a shaker at 180 rpm at 30°C for 12 h, and then transfer 25 μL of bacterial liquid from it to 96 containing 1 mL of liquid YPD. Continue to activate for 3h in deep well plate. Pipette 25 μL of the reactivated bacterial solution into a 96-deep well plate containing 1 mL of sterile water. Pipette 1...
Example Embodiment
[0050] Example 2 Propiconazole Sensitivity Test of Deficient Yeast Introduced into BraALA3 and BraENT1
[0051] 1. Cloning of Chinese cabbage BraALA3 and BraENT1
[0052] 1) Total RNA extraction and cDNA synthesis
[0053] The oil green 701 cabbage was sown in the seedling tray, and cultivated for 4 weeks under the condition of 20°C light for 12h / dark for 12h. RNA was extracted with the kit (OMEGA), and reverse transcription was performed according to PrimeScript reverse transcription kit (TaKaRa Bio Co., Ltd., Dalian, China).
[0054] 2) Construction of TA vector
[0055] Using the extracted cDNA as a template, PCR amplification of the upper and lower homology arm sequences of the BraALA3 gene was performed respectively. The cDNA amplification concentration was 5 ng / μL, and the following reaction mixture was used: 5 μL 5×GC buffer; 2 μL dNTPs; 10 μM each primer 1.25μL; 0.75μL DMSO; 0.25μL pusion DNA polymerase; 1μL gDNA (5ng); 2 0 to 20 μL.
[0056] The sequences of the ...
Example Embodiment
[0131] Example 3 Propiconazole sensitivity test of W303 strain yeast introduced with BraALA3 and BraENT1
[0132] 1. Construction of yeast overexpression lines
[0133] 1) Enzymatic digestion of pYES2 vector
[0134] The digestion reaction system was established: 3 μL BamHI (Takara); 3 μL EcoRI (Takara); 10 μL 10×Mbuffer (Takara); 3 ug complete pYES2 plasmid; deionized H2O to a final volume of 100 μL. Digested at 37°C for 3 h, purified by gel recovery kit (OMEGA).
[0135] 2) Amplify flanking sequences
[0136] The constructed A-Bra-ALA3-01, TA-Bra-ALA3-03, TA-Bra-ALA3-09, and TA-BraENT1 were extracted as templates, and the BraALA3 and BraENT1 genes were amplified by PCR at a concentration of 5 ng / μL, using the following reaction mixture: 5 μL 5×GC buffer; 2 μL dNTPs; 10 μM 1.25 μL each primer; 0.75 μL DMSO; 0.25 μL Pusion DNA polymerase; 1 μL gDNA (5 ng); The sequences of the homology arm amplification primers of the three homologous genes of BraALA3 are as follows:
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