关键词: 锯角豆芫菁 , FPPS , RACE技术 , 生物信息学 , 原核表达

Abstract: 【Objective】 Farnesyl pyrophosphate synthase (FPPS) plays an important role in deciding the biosynthetic pathway of monoterpene and sesquiterpene, obtaining FPPS from Meloidae insects is the basis for studying the relationship between FPPS and the biosynthetic pathway of cantharidin. The main purpose of this study is to clone cDNA sequence of FPPS from Epicauta gorhami Marseul, to predict the structures, properties and functions of this gene and its encoding protein, and to successfully prokaryotic express its encoding protein. 【Method】 First, according to the conservative sequences from those known insects FPPS, degenerate primers were designed by using CODEHOP software and RT-PCR to gain cDNA template, nested PCR was used to amplificate E. gorhami FPPS conserved region. Next, specific primers were designed according to the conserved fragment of E. gorhami FPPS, RACE technique was used to clone E. gorhami FPPS 3′ and 5′ sequences, then the open reading frame (ORF) region specific primers were designed according to the prediction of the ORF and nested PCR to amplificate encoding area. Finally, splicing different fragments by DNAMAN to obtain the full-length cDNA of FPPS from E. gorham. The sequences of E. gorham FPPS, phylogenetic relationship and properties of Fps, FPPS protein subcellular localization were analyzed by a variety of bioinformatics softwares such as DNAMAN, MEGA 5.05, ProtParam and TargetP 1.1 Server et al. Using pET-28a (+) as a fused expression vector, a recombinant plasmid pET-28a-EgFPPS containing the coding sequence of E. gorhami was constructed. Then inducing its expression in Escherichia coli BL21 (DE3) with IPTG. Next, samples induced at different times were collected and SDS-PAGE was used to analyze the protein expressed by E. coli BL21 (DE3). Using ultrasonic wave to broke the efficiently expressed bacterium liquid, then the supernatant and precipitate were centrifuged and boiled respectively, finally SDS-PAGE was used to analyze the solubility of EgFPPS. Western Blot confirmed the successful expression of EgFPPS in E. coli BL21 (DE3). 【Result】 The full-length cDNA of FPPS from E. gorhami was obtained. It was 1 857 bp in length, containing a 5′-untranslated region (5′-UTR) of 146 bp and a 3′-untranslated region (3′-UTR) of 436 bp. The ORF of 1 275 bp encodes 424 amino acid residues with a predicted molecular weight of 49.2 kD and an isoeletric point value of 8.89, its predicted molecular formula was C2192H3457N605O632S25. Bionformatical analysis showed that its instability index was 38.62 and the GRAVY was -0.429, suggesting that EgFPPS was a stable hydrophobic protein. Homology comparison found that FPPS from E. gorhami and other six species insects had 72.56% homology at amino acid level and all of them had one R**S tetrapeptide which was a consensus cleavage motif in mitochondrial targeting peptide. Besides, seven conserved regions and two aspartate-rich regions (DD**D) could also be identified in EgFPPS. Phylogenetic tree showed that E. gorhami had the closest evolutionary relationship with Tribolium castaneum which belongs to Coleoptera, Tenebrionidae. A mitochondrial targeting peptide was found in the N-terminal of EgFPPS by subcellular localization software, TargetP 1.1 Server. Prokaryotic expression results showed that efficient expression of FPPS protein could be realized after induced with 1 mmol•L-1 IPTG in E. coil BL21 (DE3) for 4 h at 37℃. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western Blot confirmed that the molecular weight of the recombinant EgFPPS was about 49 kD, consistent with the predicted result.【Conclusion】The cDNA sequence of FPPS was successfully cloned from E. gorhami, and the prokaryotic expression of its encoding protein was achieved, which lay a foundation for further functional research on the role of FPPS in the biosynthetic pathway of cantharidin in blister beetles.

Key words: Epicauta gorhami , FPPS , RACE technique , bioinfomatics , prokaryotic expression