北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

摘要

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础.方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450.结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体.结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础.%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.