Cloning Of The Gene Family. Salvia Cps Rnai On Tanshinone Class Of Secondary Metabolites

Salvia Miltiorrhiza Bunge is a kind of famous Traditional Chinese Medicine. It was first recorded in "Shen Nong's Materia Medica" a nd classified as top grade medicine. It has many medicinal effects such as promoting blood circulation and regulating menstruation, eli minating the blood stasis and relieving pain, cooling blood and eli minating carbuncle, clearing heart and relieving annoyance as well as nourishing blood and soothing the nerves. Fat-soluble diterpenoid s compounds in Salvia especially tanshinones are the hot topic of c urrent research。Our group clone a full-length gene of SmCPS, the n get SmCPS protein via heterologous expression, and established the vitro enzymatic reaction system, after identificated the gene fun ctions by analyzing the enzymatic reaction products, the result sugg est that SmCPS is the key enzyme of plant diterpenoid biosynthetic pathway genes, which catalytic diterpenoid common precursor GGP P formed the CPP that participate in the tanshinone biosynthesis, h owever the function of SmCPS with vivo regulatory mechanism re mains unclear, in addition, the result of recent deep sequencing of Salvia transcriptome sequencing that there might be more than one CPS genes, after blasting in NCBI, our group found that there was an gene fragment similar to entCPS gene in plants, which might c atalyze GGPP into entCPP that involved in the gibberelliin biosynth esis。Based on the above understanding, this experient trying to clo ne the full-length entCPS and built the SmCPS and entCPS body i nterference expression vector to explore diterpene synthase RNAi im pact on the accumulation of tanshinone。The introduction of exogen ous elicitor to promote SmCPS and entCPS overexpression, to valid ate the effect of CPS gene family to the secondary metabolites of t anshinone。The results of research are as follows:1the cloning of full-length ent-CPSAfter getting an ent-CPS gene fragment by deep sequencing, we use the method of Rapid amplification of cDNA ends to get the fu11-length of entCPS, that is design the prime of5`and3`, and extr act the RNA from the hairy root, and then reverse transcript into t he first chain cDNA, with the prime of the ent of5`and3`, we got the product of5`RACE and3`RACE fragment, then masaic t he5`ends and the3`ends by the software of contigl, we got two gene fragments, blast the two gene in the NCBI,finally we got the full-length of the ent-CPS gene.which is a length of2379bp gene and coding region encoding792amino acid protein, predicted the mature protein of molecular weight is89.9kDa, and the isoelectric point is6.47. Analyzing the ent-CPS conserved domain we found t hat ent-CPS has the N-terminal domain.the phylogenetic tree of ent-CPS nucleotide showed that the entCPS is far from the CPS that c loned from salvia miltiorrhiza, so it may be the diterpene synthase that involved in the secondary metabolite biosynthesis of gibberellin and other diterpenoid.2SmCPS and ent-CPS gene silencing on the effect of tanshinone compounds This experiment designed about2Int primers on the basis of the fu11length SmCPS and ent-CPS fragment by Gateway technology, an d then joined attB sequences in SmCPS and ent-CPS primers, respe ctively; built SmCPS and ent-CPS expression vectors through BP a nd LR reaction; obtained the transgenic lines of Salvia miltiorrhiza hairy root by agrobacterium mediated method. All of above method s are to check after diterpene synthase expressed in silence in Salvi a miltiorrhiza hairy root, the change of Salvia miltiorrhiza hairy roo t apparent,the contents of tanshinone and the expression of relate d genes in the biosynthese of tanshinones and gibberellins, thereby further validate the gene function and regulatory mechanism of ent-CPS and SmCPS. 3the effect of Elicitor induced expression in vitro on the diterpeno id synthase and the accumulation of tanshinoneThis experiment studied the effect of three elicitors Ag+,YE,MJ on the SmCPS transient expression and tanshinone components in d ifferent induction time, draws the following conclusion:after induce d12h, the strongest effect induced by MJ, SmCPS expression was slightly upward, and mainly content of crytoshinone upregulation o f24h after induction, the expression of SmCPS were raised, the in duction groups of tanshinone content were raised, and the Ag and YE combined to induce the strongest effect, Ag,YE alone induced effect of cryptotanshinone and hydroxyl, tanshinone I increase the volume of more than of tanshinone IIA and tanshinone I.36h after induction, the content of cryptotanshinone increased obviously and the YA and YAM induced the strongest effect.the expression of Sm CPS induced byYA and YAM were strongest too.Diterpene synthase is a key enzyme in the tanshinone biosynthesis, whose family members division functions had a direct impact on t he synthesis of secondary metabolites。This study analysis systemati cally the vitro elicitors impact on the accumulation of tanshinones a nd diterpene synthase,at the same time, the cloning of ent-CPS and the building of SmCPS and ent-CPS RNAi vectors, genetic transfor mation in order to further explore how the diterpene synthase regul ate the tanshinone synthesis and laid a good foundation for Salvia growth and development。...