Cloning、bioinformatics Analysis And Expression Analysis Of HMGR、FPS、SE、P450 Genes In Psammosilene Tunicoides

Psammosilene tunicoides W. C. Wu et C. Y. Wu(Caryophyllaceae) is a narrowly distributed and endemic monotypic herbal plant in south-western China. This species is valued highly in traditional Chinese medicine for its analgesic, hemostyptic, and immune adjustment properties, and it is one of the important raw material medicines of "yunnan baiyao" series of products. Recent research has shown that Psammosilene pentacyclic triterpenoid saponins are the main active components in P. tunicoides root.However, populations of this species have been declining rapidly due to extensive exploitations in recent years such that it is now classified as a rare and endangered species in the Chinese Plant Red Book. Nowadays, more and more researches in biosynthesis of triterpene saponins pathway using molecular biology and genetic engineering technology was becoming a heat spots. In this study, four genes which might be involved in the triterpenoid saponins synthesis were cloned from P.tunicoides and prokaryotic expressions were successfully performed to inducing protein expression, and the expression patterns of the key enzymes were detected in different organizations and different locations, which provides a basis for the further discovery of in vitro heterologous expression of Psammosilene triterpene saponins.The major findings are as follows:Based on the results of transcriptome sequencing, numerous transcripts encoding key enzymes in the triterpene saponin biosynthesis were found. According to the annotation information, all the unique sequences encoding the full-length ORFs of HMGR, SEs, CYP450 and FPS were selected and used for cloning by RT-PCR,RACE-PCR. HMGR cDNA total length is 1716 bp, the ORF length of 1641 bp coding for 546 amino acids, with a calculated molecular Weight of 58 kD; FPS cDNA total length is 1135 bp, ORF has a length of 1029 bp coding for 342 amino acids,with acalculated molecular Weight of 39 kD; SE-1 cDNA total length is 1642 bp, ORF has a length of 1578 bp coding for 525 amino acids, with a calculated molecular Weight of57.3 kD; SE2 cDNA total length is 1552 bp, ORF has a length of 1446 bp coding for481 amino acids, with a calculated molecular Weight of 52.3 kD; P450 cDNA total length is 1612 bp,ORF has a length of 1560 bp coding for 519 amino acids, with a calculated molecular Weight of 60 kD. Prokaryotic expression vector of these key enzymes were successfully constructed and induced Protein expression.Bioinformatics analysis were carried out, all these genes were highly homologous to other known plants,Further, The expression of these eight key enzymes genes were validated and measured by RT-qPCR. The expression of these genes was analyzed across leaf, stem and root tissues collected from three different populations. Based on the analyzed qRT-PCR data, all eight key enzymes genes showed significantly differential expression levels in different tissues. The seven genes(HMGR, SE1,2, SS, FPS,β-amyrin, and UGPT) showed the similar trends with their expressions levels being low in leaves and stems but more highly expressed in roots. A different behavior was observed for CYP450 gene expression, indicates that a relatively lower levels were detected in root samples than leaf and stem samples. In general, these results are consistent with that triterpenoid are mainly detected in the roots.In summary, four key enzymes genes(HMGR、FPS、SE、P450) were successfully cloned, and the stable prokaryotic expression system were established. This study is the first comprehensive analysis of the expression patterns of eight key enzymes genes for triterpene saponin biosynthesis in P. tunicoides and provides a basis to further elucidate the molecular mechanism for the biosynthesis of these medically important compounds. This study will provide a basis to further elucidate the molecular mechanism for triterpene saponin biosynthesis pathway of Psammoilene triterpene saponins.