Isolation And Functinal Analysis Of McMenA, A Gene Encoding A1,4-dihydrox-y-2-naphthoate Octaprenyltransferase In Mylabris Cichorii

The generic name for Mylabris cichorii is the yellow and black cantharis,belonging to Arthropoda, Insecta, Coleoptera, Meloidae. It is used as one oftraditional medicines. They secreted yellow syrupy liquid when they stressed. Themajor ingredient of syrupy liquid is cantharidin. Cantharidin is known as one kind ofterpenoids. It is a defensive chemical that is a vesicant produced by most beetles ofMeloidae. Cantharidin was found for function of anticancer. It is produced in allstages of blister. The synthesis of cantharidin has a typical sexual dimorphismphenomenon. The most cantharidin synthesized by male beetle and present to femalewhen they mate as a defense mechanism. The female acquires cantharidin as a giftpass to the eggs for health and growth.As so far, the research in nosogenesis, extraction, measuring of cantharidin isquite mature. The biosynthetic pathway of cantharidin has been a hot area of researchfor several years. Some achievements have been achieved. But there hasn’t been anyconcrete answer to the biosynthetic pathway. Various labelling studies have shownthat farnesol are specific biogenetic precursors for cantharidin synthesis in the20stcentury. The O atoms of its molecules from the O2and H2O, and predicted that thecantharidin biosynthesis is relate to the Mevalonic acid pathway (MAV). But themolecular mechanism of the cantharidin biosynthesis is obscure. The discovery ofgenes related to cantharidin synthesis can offer additional information and helpful forfinding the biosynthesis pathway.It was shown by our previous experiment in our laboratory that the maleproduced toxin during5-30days after emergence and the biosynthesis amount of thecantharidin peaked during20-25days, and constructed SSH library of male andfemale adult of Mylabris cichorii. A full-length MenA (1,4-dihydroxy-2-naphthoateoctaprenyltransferase) cDNA was cloned based on the parital sequence of MenA genefrom SSH library (name it McMenA) and analyze its main function.The main results for this study are as follows:1. Molecular characteristics of McMenABased on the EST sequence of the McMenA, two specific primers weresynthesized. A full-length McMenA was cloned using RACE technology, and name itMcMenA. Genbank accession: KJ156619. The cDNA is1264bp with an open reading frame of1026nucleotides coding for a314amino acid,and including114bp5’UTR,124bp3’UTR. ExPASy analysis of the McMenA showed that calculated molecularmass of37.9KDa and a pI of8.94. Analysis of McMenA sequence showed it has anaspartate rich motif N/DDxxD. TMHMM analysis found that the McMenA is atransmembrane protein, have seven transmembrane segments. Subcellular localizationpredicted revealed that the transmembrane protein confined to mitochondria. It has nosignal peptides. Sequence alignment revealed that the length of the coding region ofthe McMenA compared with those of homologs from other insect organisms washighly conserved. Tribolium castaneum MenA, has a relatively high amino acidsequence identity of86%.2. Stage-specific expression profile of the McMenA by quantitative real time PCR(RT-qPCR)The expression profiles of McMenA in six developmental stages (5days,10days,15days,22days,25days,30days) were determined by RT-qPCR. The resultsshowed that the McMenA is expressed in various periods. The expression of McMenAof the male increased sustainedly from5days to22days, and afterwards graduallydeclined from25days to30days. In female, the McMenA is expressed in all periods.The expression level was lowest in female McMenA between20to25days. Theresults were consistent with the period and synthetic quantity of the cantharidin whichis synthesis by female.3. Tissue distribution of the McMenA gene in M.cichoriiThe quantitative results showed that the highest relative expression of McMenAmRNA was in testis, followd by foregut, midgut and small in hindgut. The expressionlevel of McMenA mRNA in accessory gland was used as standard to calculate thefold-change in other tissues.4. DsRNA synthesis and RNA interference of McMenA to study gene functionDouble-stranded RNA (dsRNA) of McMenA (McMenA-dsRNA) was synthesizedin vitro. The21-days-old male adults (n=10) which were in the period of highlycantharidin production were used for RNA interference. The dsRNA was injected intothe each body cavity by microinjiector. The results showed that transcriptional level ofMcMenA21-days-old males injected with McMenA-dsRNA was lower than thecontrol eGFP-dsRNA after24h injection. These results indicated that thetranscription of McMenA effectively knocked down by RNAi.5. The determination of cantharidin and ATP We measured cantharidin and ATP concentration in dsRNA treated and control.Results of quantitative measurement of cantharidin and ATP revealed that thecantharidin and ATP concentration was assayed to estimate the impact of RNAi. TheMcMenA silence showed greater decreased Cantharidin and ATP concentration at24hpost-injection.The main conclusions for this study are as follows: McMenA related to thecantharidin biosynthesis in M.cichorii; McMenA gene affected ATP biosynthesis. Butit need further study whether the cantharidin decreasing was caused by McMenAregulating directly or by McMenA regulating ATP production and then impacted thecantharidin synthesis.