Cloning and Plant Over Expression Vectors Construction of a Cytochrome P450 Enzyme Gene from Bupleurum chinense DC.
Objective: To clone a cytochrome P450 enzyme gene which may be involved in saikosaponin biosynthesis from Bupleurum chinense DC, and to construct the transgenic vectors for over expression of the cloned cytochrome P450 enzyme. These works will provide foundation for further analysis on its function by transgenic study. Methods: LD-PCR was used to clone the full-length cDNA of the cytochrome P450 enzyme gene, on the basis of its 5' and 3' partial cDNA sequence obtained from our previous 454-sequenced dataset. With the full-length cDNA obtained, primers with corresponding restriction enzymes cutting sites were synthesized. And then, the ORF of the cytochrome P450 enzyme gene was PCR cloned with high fidelity polymerase and the reverse transcriptase products of RNA as template. The PCR products were inserted into pEASY-T1 Simple vector and transformed into the competent cells of E.coli DH5α. The recombinant pTl-P450 was sequenced after verification by PCR amplification of the bacterium liquid and enzyme digestion. Using NCBI online Blastx, DNAman and MECA4, the sequences were bioinformatieally analyzed. After that, pTl-P450 was digested with corresponding restriction enzymes and then was inserted into a binary vector pCAMB1A-SUPER 1300. Finally, PCR amplification of the bacterium liquid and enzyme digestion were used to verify the recombinant p1300-P450. Results: The cytochrome P450 enzyme gene, BcCYP87E, was cloned from B.chinense. The transgenic vectors for over expression of BcCYP87E were constructed. Conclusion: Our works on gene cloning and transgenic vectors construction provide a substantial foundation for follow-up bio-function analysis of the cytochrome P450 enzyme through transgenic research.
NETL
NSTL
万方数据
维普
