Clone And Functional Analysis Of Farnesyl Pyrophosphate Synthase From Epicauta Chinensis Laporte(Coleoptera:meloidae)

Cantharidin known as cantharides camphor,is a valuable defensive terpene secreted by Coleoptera Meloidae insects.Figuring out its biosynthesis pathway in meloidae bettles can be helpful in exploiting cantharidin on a large scale.Farnesyl pyrophosphate synthase is a key enzyme in downstream branch point of the mevalonic acid(MVA)pathway,exploring the relationship between this enzyme and the biosynthesis of canthridin will greatly promote our understanding about the biosynthesis pathway of cantharidin in beetles.Researches in this paper mainly consist of two parts:firstly,the full-length cDNA sequence of Epicauta chinensis Laporte FPPS was cloned by RT-PCR and RACE technology,afterward characters of this gene was analysized by a range of bioinformatics software;Secondly,the transcript level of FPPS mRNA in E.chinensis was suppressed by RNAi and then RNAi effect in different experimental groups was evaluated by RT-qPCR,meanwhile the cantharidin content in all experimental insects was detected by gas chromatography(GC)method,the relationship between the transcript levels of FPPS mRNA and the cantharidin content were deduced.The main results of this research are as follows:1.The full-length cDNA of FPPS from E.chinensis was obtained,named as EcFPPS(GenBank accession number:KC758147).It was 1 842 bp in length,containing a 5'-untranslated region(5'-UTR)of 144 bp and a 3'-untranslated region(3'-UTR)of 423 bp.The open reading frame(ORF)of 1 275 bp encodes 424 amino acid residues with a predicted molecular weight of 49.2 kD and an isoeletric point value of 8.89,its predicted molecular formula was C2188H6604N34520635S24.Bioinformatical analysis showed that its instability index was 38.68 and the GRAVY was-0.449,suggesting that EcFPPS was a stable hydrophobic protein.Homology comparison found that FPPS from E.chinensis and other six insects species had 72.56%similarity in amino acid level and all of them had one R**S tetrapeptide which was a consensus cleavage motif in mitochondrial targeting peptide.Besides,seven conserved regions and two aspartate-rich regions(DD**D)could also be identified in EcFPPS.Phylogenetic tree showed that of all known insects' FPPS,E.chinensis had the closest evolutionary relationship with Tribolium castaneum which belongs to Coleoptera Tenebrionidae.A mitochondrial targeting peptide was found in the N-terminal of EcFPPS by subcellular localization software TargetP 1.1 Server.2.The cantharidin content and the expression of FPPS mRNA in male E.chinensis were detected at 0,2,4,6,8 and 10 days respectively after mating.Results showed that the cantharidin content was very low immediately after mating,but it had rised 4 fold at 2 days after mating,after that it reached its peak level at the 4th day after mating,which is 17 folds of that in immediately treated mating beetles.That means the male E.Chinensis synthesized cantharidin after mating.During the same time,the expression level of FPPS mRNA increased slightly only at 2 days after mating,and then kept stable.In addition,the correlation of these two effectors was analyzed and the result showed that there was no significant correlation.We can infer that the transcription level of FPPS may not have a directly influence on the cantharidin contents.3.The transcription abundance of FPPS mRNA in different treatment groups of male E.chinensis were detected by RT-qPCR at 2,4,6,8 and 10 days after mated.Results showed that RNAi technology succeeded in inhibiting FPPS mRNA transcript level in dsFPPS injection group.Results from cantharidin content detection indicated that if FPPS mRNA transcript level was normal,cantharidin content would reach its peak level at 4 days after mated,then it would drop down slightly and remains stable;when the FPPS transcript level was suppressed,the peak level of cantharidin would be advanced to 2 days after mated and then keep stable.Conclusion from the research is that when the FPPS transcript level was suppressed,E.chinensis still could synthesize cantharidin,what's more time needed to reach the maximum cantharidin content was earlier.The research cloned the full-length cDNA sequence of FPPS from E.chinensis,bioinformatics of this gene was also analyzed.Besides,the expression level of FPPS mRNA was successfully suppressed by RNAi technology.From the research results,we concluded that when the transcript level was suppressed,E.chinensis still can synthesize cantharidin and the final content has no significant difference between dsFPPS injection group and the control groups.It is noteworthy that the cantharidin contents in dsFPPS injection group reached its stable level in advance.This means that the transcription level of FPPS may not play an important role in determining cantharidin biosynthesis.