Study On The Spatiotemporal Expression Of Cantharidin And Farnesyl Pyrophosphate Synthase FPPS Gene In Ceratonia Ovat

Cantharidin(CTD)is a sesquiterpene compound synthesized by insects such as blister beetles.It has been widely used in clinical treatment of skin diseases,edema,rabies and tumors for a long time,and has important medicinal value.In addition,CTD has strong insecticidal and bactericidal activities,which can be used as a potential new bio-source pesticide.At present,the biosynthetic pathway and biological significance of CTD in insects are still unclear,and the related research has become a hot spot in current research.Epicauta impressicornis is a dominant species in southwest China.It has high CTD content and has been subcultured under laboratory conditions.It is an ideal natural CTD source insect.In this paper,the expression of CTD and its possible synthesis-related gene,farnesic pyrophosphate synthase gene(FPPS),were studied in different development stages,before and after mating,and in different parts(tissues).The results are of great significance for further understanding the biosynthesis pathway,synthesis site,transfer inside and outside the insect body,and the biological significance of CTD in E.impressicornis.The main results are as follows:1.Spatio–temporal expression of CTD in E.impressicornisThe contents of CTD in different developmental stages(egg,larva,pupa,adult)and different parts(tissues)of Pre–Mating and Post–Mating female and male were determined.The results showed that except for the 1st to 3rd instar larvae,there was a certain amount of CTD in the developmental stage of E.impressicornis.Among them,the CTD content in eggs and the 4th instar larvae was relatively high.Therefore,it is speculated that the CTD of eggs mainly comes from female adults.The 1st to 3rd instar larvae do not synthesize CTD,and new CTD can be synthesized from the 4th instar larvae.There was no significant difference in the content of CTD between males and females at first emergence.Both males and females could synthesize a small amount of CTD at 0–5 d after emergence.Males could synthesize a large amount of CTD at 5–10 d after emergence,while females did not synthesize CTD.Post–Mating between females and males,the CTD content increased significantly compared with that Pre–Mating,indicating that females obtained a large amount of CTD from males through mating.Since then,with the increase of time,the CTD content had no significant change.Under isolated feeding,the CTD content of male adults increased significantly from 0–10 d after emergence,indicating that male adults could continuously synthesize CTD during development.The synthesis of CTD in male mating treatment was significantly higher than that in non-mating treatment,indicating that mating could promote the mass synthesis of CTD in males.The CTD content of male reproductive system decreased significantly Post–Mating,while that of female reproductive system increased significantly Post–Mating,but the increase was much higher than that of male reproductive system.It is speculated that males continue to synthesize and transfer CTD to females during mating.The synthesis of CTD is most likely to occur in the hemolymph and eventually accumulate in the reproductive system.2.Cloning and analysis of FPPS gene from E.impressicornisIn order to further clarify the CTD synthesis pathway,this study studied farnesic pyrophosphate synthase gene(FPPS),a possible functional gene in CTD synthesis pathway of E.impressicornis.The FPPS gene sequence was obtained by PCR cloning,and Gen Bank accession number was MZ502667,which was named EIFPPS.The full–length c DNA sequence of EIFPPS was 1,905 bp,with an open reading frame(ORF)of 1275 bp,and a total of 630 bp in the non–coding region,including 247 bp the 5’ end and 383 bp at the 3’ end.The coding region encoded a total of 424 amino acids.The amino acid sequence homology analysis showed that EIFPPS had high similarity with FPPS of E.gorhami,E.chinensis and Mylabris cichorii,which were99.06 %,98.11 % and 80.42 %,respectively.The phylogenetic tree analysis also showed that EIFPPS clustered together with FPPS of E.gorhami,E.chinensis and M.cichorii,and the support rate was 100 %.It was confirmed that the cloned gene sequence was the FPPS gene sequence of E.gorhami.3.Spatio–temporal expression of FPPS gene in E.impressicornisIn this study,farnesic pyrophosphate synthase gene(FPPS),a possible functional gene in CTD synthesis pathway,was studied to study its expression profile in different developmental stages of CTD and different tissues of female and male Pre–Mating and Post–Mating.The results showed that the expression of EIFPPS m RNA was different in different developmental stages and tissues of E.impressicornis.The expression level was significantly high in eggs,and gradually decreased from E–L2.The expression level was significantly increased in the L3.When the larvae developed to L4,the expression level gradually decreased with the occurrence of pupation and eclosion.In pre–mating females and males,the expression of EIFPPS m RNA was significantly decreased as the development time went on compared with that in the newly emerged females.After mating with female adults,the expression of EIFPPS m RNA in male adults was significantly increased.However,the expression levels of female adults after mating did not change significantly compared with those Pre–Mating.4.Preliminary identification of FPPS gene function in E.impressicornisThe synthesized exogenous ds RNA(double–stranded RNA,ds RNA)was injected into the male after mating,and the samples were taken 24 h and 48 h after injection,respectively.The expression of FPPS m RNA in different treatment groups was detected by quantitative real–time fluorescent PCR(q RT–PCR),and the content of cantharidin in different test insects was detected by high performance liquid chromatography(HPLC)with ds GFP injection group and WT control group.The results showed that the relative expression of FPPS m RNA in ds FPPS group was significantly decreased compared with ds GFP and WT groups,indicating that RNAi interference effect was significant.Moreover,the content of cantharidin in the test insects injected with ds FPPS was significantly lower than that in the ds GFP and WT groups,indicating that the interference with the FPPS gene of E.impressicornis could significantly inhibit the synthesis of CTD.Therefore,FPPS played an important role in the biosynthesis of CTD in E.impressicornis.Combined with the changes of CTD content and the expression of EIFPPS m RNA in different developmental stages of E.impressicornis,it is concluded that FPPS plays an important role in the biosynthesis of CTD in E.impressicornis at the macro and molecular levels.In addition,the EIFPPS gene was highly expressed in the fat body Pre–Mating and Post–Mating male,suggesting that the fat body may be the synthesis site of CTD synthesis catalytic enzymes.