Cloning and activity analysis of AvTPS1 promoter of terpene synthase from Amomum villousm / 中草药

ABSTRACT

Objective To clone the unknown sequence of terpene synthase (TPS) AvTPS1 promoter from Amomum villousm and analyze its activity. Methods In this research, AvTPS1 DNA sequence was amplified and cloned from genomic DNA (gDNA) of A. villousm leaves. Furthermore, the promoter of AvTPS1 was cloned by FPNI-PCR and the sequence was analyzed. The recombinant vector pCAM-AvTPS1p with AvTPS1 promoter for the expression of GUS gene was constructed. The activity of AvTPS1 promoter was verified by transient expression of Agrobacterium-mediated infiltration using the leaves of Nicotiana benthamiana. Results The gene sequence of AvTPS1 was 2 444 bp including seven exons and six introns. The 568 bp AvTPS1 promoter was successfully cloned using FPNI-PCR. Furthermore the sequence had ten kinds of cis-elements including conserved elements TATA-box, CAAT-box, MYC2 related element G-box and other elements. Finally, the GUS staining showed the tobacco leaves infiltrated by the pCAM-AvTPS1p were blue. Conclusion The AvTPS1 promoter can drive the transcription of GUS gene and then it was verified to have the promoter activity. These results give foundation for future research on the function of AvTPS1 involved in the terpenoid biosynthesis and its relationship with the transcription factors.