| Cantharidin(C10H12O4),a kind of natural defenses toxin,produced by insects mostly in Meloidae and some Oedemeridae and Fulgoridae insects as well.Cantharidin has been used as a antitumor medicine over hundreds of years ago.Nevertheless,it has also been reported that cantharidin had toxicity activities against some insect pests,plant pathogens and weeds,which indicates its potential value in the field of medicine and agriculture.It can not be put into use in mass production because of the limited wild resource.In order to solve this problem,we focused on the biosynthesis pathway of cantharidin from the meloide insects.In this study,we cloned the key enzyme gene,3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)involving in mevalonate pathway,from Epicauta chinensis Laporte by RT-PCR and RACE technology and analyzed this gene with bioinformatic method.We also observed cantharidin content changing after injecting inhibitors(mevastatin)and gene expression activators(juvenile hormone III),and we can then based on this to infer whether cantharidin synthesis depending on mevalonate pathway in blister beetle.The main results are as follows:1.The full-length cDNA of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)gene from Epicauta chinensis was cloned and named as EcHMGR.It is 3167 bp in length with an ORF of 2595 bp,containing a 5’-untraslated region(5’-UTR)of 228 bp and a 3’UTR of 344 bp.It encodes a deduced protein of 864 amino acids residues.It shares 74%nucleotide identity with that from Phaedon cochleariae while 60%with other insects’ HMGR gene.These results indicate that this gene is HMGR gene of Epicauta chinensis(EcHMGR gene,GeneBank accession number:JX096638).2.EcHMGR gene encodes a deduced protein with a predicted molecular mass of 95.1 kDa and isoeletric point(pI)of 6.11.Its predicted molecular formula is C4234H6765N1127O1247S53.Bioinformatic analysis showed that its instability index is 45.59,indicating that it is an unstable protein.It has 7 transmembrane domains,proving that it is a transmembrane protein.This HMGR protein has 2 O-Glyc,4 N-Glyc and 2 O-GlcNAc.However no signal peptide was detected from the deduced protein sequence.From above we hypothesized that it should be a non secretory glycoprotein playing the role anchored on the biofilm.Secondary structure analysis indicated that EcHMGR has 42.94%random coil,37.38%alpha helix and 19.68%extended strand.3D structure predicted EcHMGR into "V"in the space and contained N-structure domain,L-structure domain with two HMG-CoA bind motifs and a NADP(H)bind motif and S-structure domain with a NADP(H)bind motif.Amino acid homology comparison indicated that EcHMGR had 74.5%amino acid identity with Phaedon cochleariae and 46%～48%indentity with Lepidoptera insects.3.Mevastatin(1.6 mM)injection test result showed the content of cantharidin in the test group was 1.02%and that of the control was 2.05%.Statistical analysis showed the two groups had significant difference(P<0.01).The result indicated that the cantharidin content in mevastatin injection group was significantly lower than that in the control.Mevastatin is a HMGR inhibitor,combined analysis with this result,we can preliminary speculate that cantharidin biosynthesis is relative to HMGR.4.Juvenile hormone Ⅲ(JH Ⅲ,10μ g/μL)injection test result showed that the content of cantharidin in test group was 2.48%and that of the control was 1.24%.Statistical analysis showed the two groups had significant difference(P<0.01).The result indicated that the cantharidin content in JH Ⅲ injection group was significantly higher than that in the control.We speculated that JH Ⅲ had promoting function to cantharidin synthesis in blister beetle.Meanwhile,in test group,the cantharidin content was increased from 6h to 12h and decreased at 22h,but it was always much higher than that in the control.This result showed that the effect of JH Ⅲ is very short,which may be due to its action as a hormone in the body.5.The result of HMGR gene expression in JH Ⅲ injection test detected by real-time quantitative RT-PCR showed that,the average relative expression level of test group was 1.56 and that of the control was 0.68.It is indicated that juvenile hormone Ⅲ can also improve the HMGR gene expression.Moreover,HMGR gene expression was increased from 6h to 12h and decreased at 22h,which is similar to the content of cantharidin in the JHⅢ injection group.6.Analyzed the correlativity between cantharidin content and HMGR gene expression we found significant positive correlativity between them and the correlation coefficient R2=0.873.All the results indicated that the biosynthesis of cantharidin in the blister beetles may be relate to the mevalonate pathway.In this study,we firstly cloned the full-length cDNA of HMGR gene from Epicauta chinensis and analyzed it by bioinformatics method.We found that the biosynthesis of cantharidin in E.chinensis was correlated with mevalonate pathway by injecting the HMGR inhibitor(mevastatin)and the activator of HMGR gene expression(JH Ⅲ).The real-time quantitative PCR detection result indicated there is significant positive correlativity between the content of cantharidin and the gene expresson of HMGR in E.chinensis.This study laid a foundation for further study of cantharidin biosynthesis. |
