Cloning of UGT gene of Bupleurum chinense and construction of over expressing and RNAi transgenic vectors / 中国中药杂志

ABSTRACT

<p><b>OBJECTIVE</b>To clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study.</p><p><b>METHOD</b>RAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed.</p><p><b>RESULT</b>The full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained.</p><p><b>CONCLUSION</b>Our works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.</p>