Molecular Cloning And Functional Characterization Of Squalene Synthase SS Gene From Cucurbitaceae Trichosanthes Plant

Squalene synthase (squalene synthase, SS, EC2.5.1.21) is a key enzyme of phytosterol and triterpenoid saponins biosynthetic pathway in the process of catalytic with two molecules farnesyl pyrophosphate (FPP) condensation generated squalene (SQ) and the squalene is catalyzed by 2,3-oxidosqualene cyclase to generate triterpene, sterol, cholesterol and other important plant secondary metabolic product. Squalene synthase play an important role in the subsequent biosynthesis pathway of the squalene, and its content and activity determines the synthesis of the subsequent products.Therefore, the study focused on squalene synthase has caused wide concern in recent years.In this study, with the rapid amplification of cDNA ends (RACE) and terminal deoxynucleotidyl transferase (TdT) method, the first, cDNA sequences of squalene synthase were isolated from Trichosanthes truncata, Trichosanthes kirilowii; the second, the prokaryotic expression and bioinformatics analysis of SS gene from Trichosanthes truncata, Trichosanthes kirilowii and Trichosanthes rubriflos were performed, and the third, the deduced protein characteristics were mutually compared among the three SSs. In the genus trichosanthes of family cucurbitaceae, SS is one of the key enzymes in the biosynthetic pathway of plant triterpene. The main research results of this paper as follow:1. Cloning of SS gene:(1) using RACE and TdT method to obtained two full length cDNA clones of Trichosanthes truncata.The full length cDNA sequences are 1983 bp and 1902 bp, respectively, the difference between the two cDNAs presents at outside the coding region of 3’UTR. Both the cDNAs contain a 1254 bp open reading frame sequence encoding 417 amino acid residues, which the relative molecular weight is 47.6 kD. (2) with the method of 3’RACE and 5’RACE, Trichosanthes kirilowii SS gene cDNA sequence was cloned, the full length of the sequence length of 1522 bp, which includes 1254 bp open reading code sequence, encoding 417 amino acid residues, and the molecular weight size is about 47.6 kD.2. Sequence analysis of SS gene:through the NCBI Blast, we found that the nucleotide sequence of Trichosanthes truncata SS gene shown the nucleotide sequence homology of 78%-91%, and amino acid sequence homology 79%-94% when compered with the known plant SS; The nucleotide sequence ofTrichosanthes kirilowii SS gene shown nucleotide sequence homology of 78%-93%, amino acid sequence homology 79%-95%. By constructing phylogenetic tree analysis, the SSs from Trichosanthes truncata, Trichosanthes kirilowii and Siraitia grosvenorii, Gynostemma pentaphyllum, Trichosanthes rubriflos, Momodica cochinchinensis show the closest genetic relationship. The homology of the SS sequences of Trichosanthes truncata, Trichosanthes kirilowii and other Cucurbitaceae plants squalene synthase gene is the highest, and consistent with predicted results.3. Prokaryotic expression analysis:squalene synthase ORFs of Trichosanthes truncata, Trichosanthes kirilowii and Trichosanthes rubriflos were respectively inserted into the prokaryotic expression vector pET32a (+), then, the recombinant prokaryotic expression vector pET32a(+)-SS was transformed into E.coli BL21(DE3). After inducing with IPTQ SS was detected through SDS-PAGE protein electrophoresis. The electrophoresis results showed that the recombinant fusion SS was successfully expressed in BL21(DE3), the molecular weigh was approximately 66 kD, which pET32a(+) expression label size was about 18.3 kD.This study, the squalene synthase full-length cDNA sequences were successfully cloned from Trichosanthes truncate and Trichosanthes kirilowii, and Trichosanthes truncate. The ORFs of squalene synthase were recombined with the prokaryotic expression vectors pET32a (+), which lays the foundation for the further study of key enzymes of SS positive selection sites in Cucurbitaceae plants triterpene synthesis pathway and functional relevance analysis. For further understanding of family Cucurbitaceae plants of the genus Trichosanthes triterpenoid saponins biosynthetic pathway and its molecular mechanism of regulation, it is important to uncover triterpenoid saponin synthesis route. The structure characteristics and function of key enzymes play important roles during the biosynthesis of triterpenoid. Therefore, at the molecular level, the triterpenoid saponin biosynthesis would be artificially regulated and improving the output of effective components of triterpenoid saponin biosynthesis has the vital significance.