Cloning And Expression Of SE,HMGR And FPS From Paris Polyphylla Var. Yunnanensis

Objective To clone and make prokaryotic expression the genes of SE、HMGR and FPS, which were key enzymes in the biosynthetic pathway of steroid saponin in Paris polyphylla var. yunnanensis. And the Real-time PCR was used to detect the relative expression of SE1、SE2、HMGR and FPS.Method Taking the root of Paris polyphylla var. yunnanensis, which were collected from the Liang Wang mountain, Kunming, as the primal material to extract the total RNA, and subsequently doing a reverse transcription to obtain the c DNA. Using the c DNA as temple and the specific primers were designed based on two all-length SE genes、HMGR gene and FPS gene, which were obtained by Hi Seq2500 sequencing platform from the root of Paris polyphylla var. yunnanensis, then clone the SE genes、HMGR gene and FPS gene. Transferred the cloned SE genes、HMGR gene and FPS gene into p EASY-T1 Simple Cloning Vector and then sequenced. The p EASY-E1-SE、p EASY-E1-HMGR and p EASY-E1-FPS expression vectors were biult after sequenced rightly. The Art Media protein Expression/Amp+ medium was used to induce an automatic expression. The Real- time PCR method was used to detect the relative content of SE1 gene, SE2 gene HMGR gene and FPS gene in the c DNA sample.Results Two SE genes of Paris polyphylla var. yunnanensis were obtained, and named SE1,SE2 repectively. The full-length of SE1 was1932 bp, and the full-length of SE2 was 1828 bp. The length of ORF of SE1 was 1578 bp, coded 525 amnio acid, and the length of ORF of SE2 was 1548 bp, coded 515 amnio acid. Fluorescence quantitative PCR results showed the expression of SE1 in stem and leaves had significant differences compaired with SE2 genes, SE1 expresses mostly obvious in leaves. A HMGR gene of Paris polyphylla var. yunnanensis was obtained, and the full-length was 1731 bp. The length of ORF of HMGR was 1593 bp, coded 531 amnio acid. Fluorescence quantitative PCR results showed the expression of HMGR in root was higher than the stem and leaves and leaves. A FPS gene of Paris polyphylla var.yunnanensis was obtained, and the full-length was 1206 bp. The length of ORF of HMGR was 1050 bp, coded 350 amnio acid. Fluorescence quantitative PCR results showed the expression of FPS was higher in leaves. Sequenced results show that the prokaryotic expression vector p EASY-E1-SE 、 p EASY-E1-HMGR and p EASY-E1-FPS was built successfully. SDS-PAGE analysis showed the inducible expression of this two SE genes, HMGR gene and FPS gene in competent cell BL21(DE3) were successful and four relative fusion proteins were obtained.Conclusions SE1 and SE2 genes had different expression patterns and maybe play different roles in the synthesis of secondary metabolites in Paris polyphylla var. yunnanensis. The expression of HMGR in root was higher and the expression of FPS was higher in leaves,the result laid the foundation of methodology for the expression of functional genes of Paris polyphylla var. yunnanensis.