Application Of RNA Interference Technology On Gene Functional Researching Of Squalene Synthase In Gynostemma Pentaphyllum

Squalene Synthase is one of the key enzymes in the Gynostemma pentaphyllum triterpenoid saponin biosynthetic pathway. In this study, two plant expression vectors had been designed with RNA interference (RNAi) technology ,which can block down the Gynostemma squalene synthase (SS) gene transcription function. In further experiments those vectors were transformed into Gynostemma successfully by Agrobacterium tumefaciens. The stady could lay a foundation for further study in the molecular mechanism of triterpenoid saponins biosynthesis.The main results are as follows:1. The RNA were extracted successfully from fresh leaves of Gynostemma pentaphyllum with a modified CTAB method. The total RNA can feed the requirement for the subsequent RT-PCR.2. Analysising the Squalene Synthase gene of Gynostemma pentaphyllum , considering homologous fragments, hairpin, introns, promoter and all the other respects to design the intermediate vector pMD-190 and SS RNA interference vector pBIISS-1\pBIISS-s1-s2.3. Abtained the callus of Gynostemma pentaphyllum by changing factors like composition of phytohormone and illumination. It is a pity that the callus redifferentiation was failed. In this stady we had also explored the conditions about micro injection the Agrobacterium-mediated foreign gene into Gynostemma pentaphylla.4. According to the GAPDH sequence homology comparison results of known plant, we had designed a pair of degenerate primer,useing cDNA as template, amplified,cloned by PCR, we obtained a GAPDH fragment in glyceraldehyde-3-phosphate dehydrogenase of Gynostemma pentaphylla.It can be used for fluorescence quantitative PCR analysissing in the future research.It's the first time to apply the RNA interference technique to analyze the relevant enzyme genes of triterpenoid saponin biosynthetic pathway in Gynostemma pentaphyllum. The results of this research could supply a molecular biology basis for gaining a wide knowledge about the molecular mechanisms of triterpenoid biosynthetic pathway and the structural characteristics . There is a possibility in the future that we can use artificial control in triterpenoid biosynthesis at the molecular level, and increase triterpenoid saponin content in the Gynostemma pentaphyllum.