Cloning Of Squalene Synthase CNDA From Trichosanthes Rubriflos And Momodica Cochinchinensis, Prokaryotic Expression Of Recombinant Squalene Synthase From Momodicacochinchinensis

In this paper, cDNAs of squalene synthase genes of Trichosanthes rubriflos and Momordica cochinchinensis were cloned via RT-PCR and RACE. Prokaryotic expression of squalene synthase of Momordica cochinchinensis was conducted. Bioinformatic analysis of this gene was carried out. The major findings are listed as follows:(1) cDNA sequence of squalene synthase gene of Trichosanthes rubriflos was obtained. The obtained sequence consisted of 1466 nucleotides, and an open reading frame of 1254 bp, encoding 417 amino acids. Blast analysis indicated that amino acid sequence encoded by SS gene of Trichosanthes rubriflos shared 78%～94% similarity to that encoded by SS gene in plants, and their similarity in nucleotide was 74%-93%. Phylogenetic analysis showed that Trichosanthes rubriflos had the highest genetic relationship with Momordica grosvenori and Gynostemma pentaphylla. Therefore, it has verified that the similarity of SS gene of Cucurbitaceae was agreed with the prediction. Also it was found that amino acid sequences of SS in Cucurbitaceae plants had genetic similarity with that cloned from tuniclike psammosilene root, but the latter is a member of Caryophyllaceae family according to NCBI-adopted plant classification. In light of morphological classification, Cucurbitaceae and Caryophyllaceae are two different branches. Therefore, the genetic relationship of SS of the two families is needed to be further explored.(2) cDNA sequence of SS gene of Momordica cochinchinensis was obtained. The obtained sequence consisted of 1449 nucleotides, and an open reading frame of 1254 bp, encoding a protein of 417 amino acids (relative molecular mass is 47.8 kDa). Blast analysis indicated that amino acid sequence encoded by SS gene of Momordica cochinchinensis shared 80%～94% similarity to that encoded by SS gene in plants, and their similarity in nucleotide was 73%-92%. Phylogenetics analysis indicated that SS of Momordica cochinchinensis showed the high genetic similarity with that of Trichosanthes rubriflos, Momordica grosvenori and Gynostemma pentaphylla. The result was agreed with the analysis result of SS of Trichosanthes rubriflos.(3) Squalene synthase of Momordica cochinchinensis was expressed in prokaryotic cells. After cDNA sequence of SS gene of Momordica cochinchinensis was obtained,, and its ORF was cloned using primers with restriction enzyme cutting site. The recombinant plasmid was successfully constructed via connecting with expression vector pET32a(+). Furthermore, the target fusion protein of 66 kDa was successfully expressed in Escherichia coli BL21(DE3), in which the expression tag of pET32a(+) was about 18.3 kDa. In this study, cDNAs of SS genes of Trichosanthes rubriflos and Momordica cochinchinensis were successfully cloned via RT-PCR and RACE, laying a foundation for the study on structure, expression and mutation of SS gene of Trichosanthes rubriflos and Momordica cochinchinensis, and providing data support for correlation analysis of positive selected site and the function of SS, a key enzyme related to triterpenoid synthesis in Cucurbitaceae family.