Discovery And Functional Verification Of Genes Involved In Cantharidin Biosynthesis In Epicauta Chinensis

Cantharidin is a natural defensive compound major produced by Meloidae and other insects.It not only exhibit good therapeutic effect upon antitumor and treating various dermatoses,but also show excellent control efficiency upon agricultural pests as a new potential insecticide.Due to the limited natural biological resources and too expensive to mass production by chemical synthesis,the biosynthesis will show a new way.To reveal the pathway and mechanism of cantharidin biosynthesiswillprovide a theoretical basis for the artificial synthesis of cantharidin.In order to verify the biosynthesis pathway of cantharidin in blister beetles,we performed de novo transcriptomicanalysis of Epicauta chinensis and verified several genes involved in cantharidin biosynthesis.Furthermore,we detected the content of cantharidin and transcription level of the related genes in different tissues of E.chinensis on various days after mating and proposed the potential organ involved in cantharidin production.The results are as follows:1.Analysis of RNA-seq on male and female E.chinensisThe gene expression in E.chinensis was studied based on the transcriptomic samples?just mated male?R1?,?males 7 days after mating?R2?and?females on the 3^rd day after eclosion?R3?.We obtained 130,150 and 150 million clean reads from these three samples respectively,and 46696 unigenes,which were classified to cellular component,molecular function and biological process.There were 627 differential expressed genes?DEGs?detected between the R1 and R2 groups.2481 DEGs were found between the R1and R3 groups,and the chemorepellent molecular function was specific enriched.Moreover,6 DEGs of this group were found in metabolism of xenobiotics by cytochrome P450 and drug metabolism pathways.2989 DEGs were discovered in the R2 vs R3 groups and showed the same results with the R1 vs R3 groups on the Gene Ontology analysis.There were 5 DEGs between male and female E.chinensis were found in the MVA pathway which belongs to the terpenoids biosynthesis pathway,and 3 DEGs were found in the downstream branches.2.The DEGs primary screen based on the MVA pathwayTemplates with different cantharidin content for DEGs screening were successfully constructed and 14 unigenes were screened out from 73 candidate DEGs with these templates using semi-quantitative RT PCR.We verified the correlation between cantharidin production and the expression of EcUn7,EcUn14 and EcCYP4C genes from the 14unigenes by RNAi.The results showed that interferences of EcUn7 and EcCYP4C significantly inhibited the biosynthesis of cantharidin in male E.chinensis after mating.We amplified and sequenced the cDNA full-length of these two genes by 5'&3'RACE.EcUn7and EcCYP4C were identified as a peritrophic matrix protein and a cytochrome P450monooxygenases CYP4BM1 using bioinformatics analysis.3.The correlation between cantharidin biosynthesis and genes at the downstream branches of the MVA pathwayEcFDRs,EcPFTb and EcGGPS cDNA full-length sequences were cloned from E.chinensisand the temporal and spatial expression patterns of the three genes tested.These genes exhibited different expression trend in males and females on different days after mating but specific high transcript levels were found in eggs and first instar larvae.Cantharidin content accumulated in male E.chinensis after mating and showed the highest level on the 6^th and 7^th day,whereas,females exhibited a similar high level after mating.The significantly highercantharidin content was in eggs and the lowest in the 2^nd and 3^rd instar larvae.We have proved that interfering the expression of EcPFTb and EcGGPS didn't seem to affect the cantharidin production in male E.chinensis after mating,but interferingexpression of EcFDRs can significantly inhibit the biosynthesis of cantharidin.4.The effect of cantharidin biosynthesis by genes involved in JH synthesis and metabolismWe first cloned the cDNA full-length of EcMFE,EcJHEH and EcJHAMT genes from E.chinensis and detected the temporal and spatial expression patterns of the three genes.The mRNA of the three genes showed the similar tendency with the cantharidin content in males after mating.The highest transcript level of EcMFE showed in the 1^st instar larvae whereas the EcJHAMT in the 2^nd and 3^rd instar larvae and EcJHEH in the 1^st and 5^th instar larvae.We found that interfering the expression of EcMFE and EcJHEH could significantly reduce the content of cantharidin in male E.chinensis after mating.However,interferingthe expression of EcJHAMT showed no obvious influence on cantharidin production.We therefore proposed that JH may not be the direct precursor for cantharidin biosynthesis.5.The distribution and variation of the genes related to cantharidin biosynthesis in tissues of E.chinensisWe compared and analyzed the temporal and spatial expression patterns of EcHMGRwhich can significantly regulate the cantharidin biosynthesis in male E.chinensis after matingIn order to eli minate the differences of physiological reactions between genders in E.chinensis,another Coleoptera insect,Tenebrio molitor,which does not synthesize cantharidin,was selected as a reference.We also compared and analyzed the HMGR expression level at different developmental stages and in different tissues from males between E.chinensis and T.molitor?Meloidae?.The results showed that EcHMGR exhibited a specific high transcript level in the fat body of male E.chinensis.Cantharidin accumulated in accessory gland and fat body of male E.chinensis from just mated to the 9^th day after mating,and the cantharidin contentwas the highest in the accessory gland and the second in fat body.The expression levels of genes involved in cantharidin biosynthesis in different tissues of male and female E.chinensis after matingwere detected and revealed that EcHMGR and EcJHEH showed significantly high transcript level in the fat body of males and the expression level of EcHMGRwas increasing from just mated to the 9^th day after mating;the highest transcript level of EcMFE was found in the head of both males and females;the significantly higher expression levels of EcFDRs gene were found in the head and accessory glands from males and the head and midgut from females;EcCYP4BM1showed the highest expression level in the midgut of both sexesand also high level in malpighian tubules and accessory gland of male E.chinensis;the significantly higher transcript levels of EcPMP gene were found in the head of just mated malesand the midgut of male E.chinensison the 9^th day after mating.Based on all these,we speculated that fat body may be involved in the biosynthesis of cantharidin in male E.chinensis after mating.