Cloning and expression of squalene epoxidase from Paris polyphylla var. yunnanensis / 中草药

ABSTRACT

Objective: To clone and express a full-length cDNA encoding squalene epoxidase which is key to the biosynthetic pathway of triterpenoid saponins in Paris polyphylla var. yunnanensis. Using real-time PCR method to detect the relative content of SE1 gene and SE2 gene from the cDNA sample. Methods: Total RNA was extracted from the roots of P. polyphylla var. yunnanensis and transcription was reversed. Using the reversed transcription of cDNA as template, specific primer was designed according to the transcriptome data about two groups of SE gene sequence from the HiSeq2500 sequencing platform, and then SE gene was cloned. The production was inserted to pEASY-T1 Simple Cloning Vector, and pEASY-E1-SE expression vector was built after sequencing appraisal right. The ArtMedia protein Expression/Amp+ medium was used to induce expression automatically. Using real-time PCR method to detect the relative contents of SE1 gene and SE2 gene from the cDNA sample. Results: Two SE genes of P. polyphylla var. yunnanensis were obtained, which were named as ppSE1 and ppSE2, repectively. cDNA was 1 598 bp of ppSE1 and 1 509 bp of ppSE2, respectively. The full length for ppSE1 was 1 932 bp, length of ORF was 1 578 bp, coding 525 AA; The full length for ppSE2 was 1 828 bp, length of ORF was 1 548 bp, coding 515 AA. Fluorescence quantitative PCR results showed that the expression of ppSE1 and ppSE2 genes had significant differences in stem and leaf, and the expression of ppSE1 was most pronounced in the leaf. Enzyme digestion and sequencing results showed that the prokaryotic expression vector pEASY-E1-SE was built successfully. SDS-PAGE analysis showed that two fusion proteins of SE genes were induced expression successfully in BL21 (DE3) express competent cells. Conclusion: The SE gene of P. polyphylla var. yunnanensis has been cloned, and the SE protein with biological activity in vitro has been obtained. The ppSE1 and ppSE2 genes have different expression patterns in P. polyphylla var. yunnanensis, and play a different role in the synthesis of secondary metabolites.