The Gene Cloning And Functional Study Of Farnesyl Diphosphate Synthase In Mylabris Cichorii

Mylabris cichorii Linnaeus, one of traditional medicine, and also important veterinary chemical recorded by Chinese pharmacopeia, is commonly known as the yellow and black cantharides, belonging to Mylabris, Meloidae, Coleoptera and Insecta. The biosynthetic pathway of cantharidin, a sesquiterpenoid natural-defense substance in insects is unresolved issues so far.The demand of cantharidin, was more and more for its function of anticancer or antitumour in recent years.However, the issue of artificial rearing of blister beetles hasn’t been solved and the chemosynthesis of cantharidin is rigour, so it is of great importance for working out the source problem to make sure the biosynthetic pathway of cantharidin.The research on biosynthetic pathway of cantharidin has been for decades with the some results. However, the specific biosynthetic pathway is still obscure so far. And the study on genes or enzymes of biosynthetic pathway is very few, so it will lay the foundation for researching on the pathway to know them.In this paper, it was studied that the function of farnesyl phosphate synthase(FPPS) gene in biosynthetic pathway of cantharidin. The main results for this research are displayed as follows: 1. The cloning of the c DNA full-length and analysis of sequenceThe c DNA full-length was obtained by applying SMART RACE technology and named it Mc FPPS whose Genbank number is KM514312. The c DNA consisted of 1507 bp. The bioinformatics analysis results by softwares of ORF finder and Ex PASy showed us that it contained a 1278 bp open reading frame(ORF) which encoding 425 amino acid residues. Calculated molecular mass of the protein is 49.425 KDa, the p I is 8.91. Mc FPPS protein is a transmembrane protein with relevant transmembrane domains. The subcellular localization indicated that the protein was confined to mitochondria. It has no signal peptides. The comparison analysis of Mc FPPS protein with other FPPS proteins showed us that the Mc FPPS protein contained the seven conserved domains named I to VII in all FPPSs, including the two aspartate-rich motifs DDXXD. Phylogenetic tree analysis showed us all FPPSs of Coleoptera including Mc FPPS got together to form an independent branch, the highest sequence homology with Epicauta gorhami and far away from plants and microorganism. 2. Stage-specific expression profiles analysis of the Mc FPPS and downstream gene STE24 named Mc STE24Stage expression pattern analysis of Mc FPPS gene in both sex adults was determined by RT-q PCR. And the result revealed that the m RNA of the Mc FPPS gene was detected during all selected stages, including 5 d, 10 d, 15 d, 20 d, 25 d, 30 d post eclosion, and the expression of Mc FPPS gene continued to rise from 5 d to 20 d, and subsequently declined from 25 d to 30 d with the highest expression in 20 d in males, which is the same to the pattern of Mc STE24. In females, Mc FPPS gene is also expressed in all stages, but its expression was essentially unchanged from 5 d to 15 d, and afterwards weakened from 20 d to 25 d, interestingly rose again in 30 d. The results above are almost in line with cantharidin content. 3. Tissue-specific expression profile of Mc FPPSThe following five tissues in abdomen of 20-day-old males were selected for tissue-specific expression profile of Mc FPPS by RT-q PCR: accessory glands, testis, frontgut, midgut and hindgut. The results showed that the expression levels of Mc FPPS were universal in all tissues with higher levels in alimentary canal and with the highest level in hindgut. 4. RNA interference of gene Mc FPPSThe Mc FPPS gene in 17(~18)-day-old male adults after eclosion and the 5th instar larvae was silenced by injecting with in vitro synthetic Mc FPPS-ds RNA. The data demonstrated that the efficiency of 24 h in adults or larvae was obviously higher than 48 h compared to control groups. For this reason, the samples at 24 h post injection were chosen for cantharidin assay and the expression levels analysis of downstream genes. And the result indicated that the expression levels of Mc Men A and Mc STE24 extremely markedly declined when Mc FPPS gene was silencing successfully. At the same time, the content of cantharidin in the ds RNA-treated group was obviously lowered comparing with the controls. 5. The prokaryotic expression analysis of Mc FPPS proteinThe recombinant fusion Mc FPPS protein was obtained by construction of prokaryotic expression vector p ET30a-Mc FPPS. For the recombinant fusion protein expressed in the form of inclusion body, so the conditions were optimized. The results revealed that molecular mass of recombinant fusion protein was consistent with calculated molecular mass by software. Conclusion: FPPS gene was firstly cloned from Mylabris cichorii and named Mc FPPS. The gene was continuously and unevenly expressed in each stage which was almost in line with cantharidin content. Combining with the results of RNAi and stage-specific expression profile, we inferred that the Mc FPPS gene might play a very vital role in the biosynthesis of cantharidin. The gene was expressed in each issue of abdomen in Mylabris cichorii, particularly highest in hindgut, so it was inferred that cantharidin or its precursor might be synthesized in intestinal canal.