| The blister beetle Mylabris cichorii(Coleoptera: Meloidae), has been widely applied to treat a variety of conditions. The dried body of M. cichorii has been used in traditional Chinese medicine for thousands of years, and the preparation was also recorded in the Chinese Pharmacopoeia. Cantharidin is a sesquiterpenoid, synthesized as a defensive substance when they are attacked. Most blister beetles demonstrate sexual dimorphism in terms of cantharidin production. Cantharidin is mostly synthesized by adult male beetles and transferred to adult females as a precopulatory gift. Cantharidin and its derivatives have recently been used to treat several cancers as well as other diseases. However, overexploitation of insect resources and habitat destruction mean that blister beetles are now considered to be scarce.Over the past few decades, studies of blister beetles have focused mainly on their biology and ecology, artificial breeding technologies, and antitumor mechanisms. Although some studies have reported on cantharidin biosynthesis, the pathways involved in this process in meloid beetles remain poorly understood. Reaserches on cantharidin biosynthesis and metabolism will have important scientific significance and can provide significant theoretical references of cantharidin in vitro biosynthesis as well as the reasonable utilization of wildlife resources.In the present study, we conducted de novo transcriptome and expression profiling analysis(RNA-seq) of M. cichorii to gain a deeper insight into the genes and pathways involved in cantharidin biosynthesis. Futhermore, we cloned some important genes and identified their function to explore the mechanism of cantharidin biosynthesis. The main results are as following:(1) De novo transcriptome and expression profiling analysis of M. cichorii to reveal relative genes and pathways potentially involved in cantharidin biosynthesisAs the lacking of genomic background of M. cichorii, we conducted de novo transcriptome using an Illumina HiSeq 2000 platform. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences(about 81.17%). To classify orthologous gene products, 8,580 non-redundant unigenes were subdivided into 25 COG classifications. Based on GO classifications, 12,951 sequences were classified into three major functional categories and 58 subcategories. Using KEGG, 16,660 unigenes were assigned to six specific pathways and 258 subcategories. Finally, 24,358 protein coding regions were predicted. We also constructed two expression profile libraries(20–25 day-old adult males and 20–25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Compared with females, 1,468 were up-regulated and 997 genes were down-regulated, respectively in males. Besides, a total of 1,506 differentially expression genes(DEGs) were assigned to 249 KEGG pathways, all of which were significant enrichment pathways. We examined the specific KEGG pathways and identified 14 genes potentially involved in cantharidin biosynthesis, discovered that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate(MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate(MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone(JH) biosynthesis or degradation. Futhermore, 13 unigenes were selected for the RT-qPCR, which validated the expression profiles of the genes and also verified the reliability and accuracy of our transcriptome analysis.(2) Cloning full-length cDNA of McSTE24?McCYP305a1?McJHEH genes and the bioinformatics analysis3'RACE and genome walking were performed to obtain the full-length cDNA of STE24 endopeptidase(STE24), cytochrome P450 gene CYP305a1 and juvenile hormone epoxide hydrolase(JHEH). The full-lengh cDNA of three genes contain ORF of 1,237 bp, 1,601 bp and 1,497 bp respectively and encode 433, 492, 459 amino acid residues separately. Three genes were then named with McSTE24(GenBank accession number KU941585), McCYP305a1(GenBank accession number KU941587) and McJHEH(GenBank accession number KU941586). Amino acid sequence multiple alignment of three genes showed highly conservation with their homologous protein sequences-the McSTE24 protein contain the HEXXH Zn2+-metalloprotease signature; the McCYP305a1 protein exist the motifs Hinge, I helix, Helix K, Heme binding loop; the McJHEH protein have the specific catalytic triad of epoxide hydrolase and the residues consisting of the potential oxyanion hole.(3) Stage-specific expression pattern of McSTE24?McCYP305a1?McJHEH genes in male and female adultsStage-specific expression pattern analysis of McSTE24?McCYP305a1?McJHEH genes in both sex were determined by RT-qPCR. The results showed the expression of three genes was detected during all selected stages in male and female adults. In 5-10 day-old after eclosion, three genes were highly expressed or showed a gradually increased tendency in male group, and subsequently declined at 15 d. The expression levels of three genes significantly went up again in 20-25 d and then weakened in 30 d. The expression patterns of three genes were consistent with the change trend of cantharidin content from the early to the terminal stage of cantharidin mass biosynthesis in male group. Moreover, the highly expression in 5-10 d of these three genes indicated they might participate in the gonad development after the beginning of emergence. In female group, the expression levels of three genes were low or showed decreasing trend in 5-10 d, but suddenly increased in 15 d, and then declined in 20-25 d, finally went up again in 30 d. The expression patterns of three genes were not consistent with the change trend of cantharidin content in female group, suggested the three genes might participate in some other physiological activites such as the development of ovary, vitellogenesis and oviposition. The differentially expressed pattern analysis during early and peak stage of cantharidin mass biosynthesis showed that the expression levels of three genes were low during early stage of cantharidin mass biosynthesis(15 d) in male group, even lower than that in female(such as McSTE24 and McCYP305a1). However, the expression levels of three genes were significantly increased during the peak stage of cantharidin biosynthesis in male group and much more than the expression levels at 25 d in female. By and large the male adults reached sexual maturity at this stage, for the reason of sexual dimorphism and the ability of cantharidin biosynthesis in male, it can be speculated that the functions of three genes during this period might related to cantharidin biosynthesis.(4) RNA interference(RNAi) knockdown of McSTE24?McCYP305a1?McJHEH genesThe 19 d male and female adults after emergence were chosen and injected with synthetic gene specific dsRNA in vitro at inter-segmental membrane by using a microsyringe. The results showed that the RNAi knockdown of all three genes had a positive impact on cantharidin biosynthesis. The cantharidin contents in experimental groups were obviously lower compared with control groups, suggested that three genes can directly take part in cantharidin biosynthesis. Besides, the results indicated the female beetle could synthesize a small amount of cantharidin. However, the detection of cantharidin array in all male and female groups found the cantharidin levels in male adult were well above than the amounts in female, which complying with the sexual dimorphism, but the reason of cantharidin biosynthesis ability in female needs further researches. McJHEH might be involved in juvenile hormone metabolism, the declined cantharidin level after RNAi knockdown of this gene indicated cantharidin biosynthesis might be related to juvenile hormone metabolism, which should be confirmed by further studies.(5) Influence on the expression levels of the corresponding downstream genes after the RNAi knockdownThe expression levels of the corresponding downstream genes were analyzed after injecting McSTE24-dsRNA and McCYP305a1-dsRNA by RT-qPCR. RNAi knockdown of McSTE24 gene has a direct impact on the expression level of the downstream gene McCYP305a1, while it play a negative regulation role in gene McJHEH possibly because of the continuous precursor blockade. When it comes to McCYP305a1 gene, RNAi knockdown might not directly influence the expression of McJHEH gene, but by affecting the juvenile hormone biosynthesis and then result in an imbalanced juvenile hormone titre, which in turn influenced the expression of gene McJHEH.Cantharidin biosynthesis is a complex process, which need plenty of enzymes to be participated in and exists complicated mechanisms of hormonal regulation. In the present study, we constructed the first adult transcriptome and expression profiles of M. cichorii, cloned three genes involved in cantharidin biosynthesis and studied their functions related to cantharidin biosynthesis. The results gave preliminary insight into the mechanisms of cantharidin biosynthesis and established the foundation of the molecular genetics of meloidae insects. |
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