Molecular Cloning And Expression Of Squalene Epoxidase In Panax Notoginseng

Object: To further study the mechanism of saponins biosynthesis at the molecular level and application in the other medicinal plants,a cDNA encoding squalene epoxidase (SE) that is one of key enzymes in the synthesis of triterpene saponins in the root of Panax notoginseng (Burkill) F. H. Chen was cloned and expressed. Methods:①The panax notoginseng total RNA was extracted with the modificated single-step guanidinium isothiocyanate. The reverse transcription reaction was performed using the Fermentas RevertAidTM First-Strand cDNA Synthesis kit. The panax notoginseng's SE cDNA was amplified by PCR. The amplified products was ligated into pMD18-T plasmid, a prokaryotic cloning vector. Then the plasmid was transformed into E.coli JM109. The Plasmids with correct cDNA fragment inserts were identified by digestion with two restriction enzymes and by DNA sequencing.②The primers were designed to place the NcoI site upstream of the native initiation codon and the BamHI site downstream of the native stop codon. The SE cDNA with integrated open reading frame (ORF) was amplified by PCR. Then the gene was ligated into the NcoI and BamHI sites of the pET30a(+) plasmid,a prokaryotic expressing vector.The recombinant plasmids were transformed into E.coli. JM109, The Plasmids with correct cDNA fragment inserts were identified by digestion with two restriction enzymes and by DNA sequencing.Then the plasmids were transformed into E.coli. BL21(DE3) plys. Expression of the gene was made by isopropyl-β-D-thiogalactopyranoside (IPTG) induction. Result:①The notoginseng SE cDNA was amplified by RT-PCR. The fragment of SE was...