Cloning Of Genes For Relevant Enzymes Of Triterpenoid Saponin Biosynthetic Pathway In Gynostemma Pentaphyllum

The RACE method was applied to identify the relevant enzyme genes from medicinal plant Gynostemma pentaphyllum which participate in triterpenoid saponin biosynthetic pathway. Two relevant enzyme genes have been cloned successfully, and the results would set up a basis for the research on the triterpenoid biosynthesis and the regulation of the enzynme gene expression in Gynostemma pentaphyllum. The main results of this project were presented below:First, RNA were extracted successfully from fresh leaves of Gynost- emma pentaphyllum with a modified CTAB method. The OD260 /OD280 ratio of RNA extracted were 1.9-2.0 and the quality of RNA can meet the requirement for the subsequent RT-PCR and RACE reaction.Secondly, degenerate primers were designed according to the homogenous region of some plant SS or SE sequences registered in GenBank, respectively, then to isolate the middle conserved fragment of SS or SE cDNA by PCR and gene cloning, resulting in 443bp fragments of SS and 535bp of SE genes in Gynostemma pentaphyllum; then special primers based on these sequences were designed to amplify respectively the full length cDNA of SS (1496bp) or SE (1818bp) genes from Gynostemma pentaphyllum with RACE method. SS gene contained a 1254bp open reading frame (ORF) encoding 417 amino acids of protein, including 231bp 3'untranslated regions, molecular weight of SS peptide deduced were 47.9 Kda and isoelectric point 8.34. SE gene contained a 1578bp open reading frame (ORF) encoding 525 amino acids of protein, including 56bp 5'untranslating region and 125bp 3'untranslating region, molecular weight of SE peptide deduced were 57.8 Kda and isoelectric point 9.04, respectively.Thirdly, protein-protein BLAST analysis of SS or SE showed that deduced amino acid sequence of Gynostemma pentaphyllum SS gene shared 84.3%,83.3%,82.2%,82.2%,81.0% with those of Glycine max,Ra- dix Glycyrrhizae,Panax ginseng and Panax notoginseng, respectively; and the deduced amina acid sequence of Gynostemma pentaphyllum SE gene shared 80.2%,79.2%,78.6%,78.4%,77.3%,76.7% with those of Dat- ura Stramonium L,Oryza sativa Japonica Group, Medicago truncatula,P- anax ginseng, and Radix Notoginseng, respectively. The above SS and SE sequence information cloned from Gynostemma pentaphyllum have been submitted to GenBank and coded as FJ906799, FJ906798, respectively.It's the first time to apply the RACE technique to analyze the relevant enzyme genes of triterpenoid saponin biosynthetic pathway in Gynostemma pentaphyllum. The research result could supply molecular biology basis for gaining more insight into the molecular mechanisms of triterpenoid biosynthetic pathway and the structural characteristics of the key enzymes in it as well as their functions. It should be possible to realize artificial control to triterpenoid biosynthesis at the molecular level, and to improve effective components of triterpenoid saponin in Gynostemma pentaphyllum.