Molecular Structure Analysis And Prokaryotic Expression Of The Cucumis Melo Squalene Synthase

Objective: Cloning of the squalene synthase(SQS) genes and its sequence was analyzed by bioinformatics analysis,the SQS prokaryotic expression vector was constructed,which provided a new clue and experimental basis for the regulation of the biosynthesis of melon. Methods: Total RNA was extracted from leaves of cucumis melo using guanidine thiocyanate method,which was reversed into cDNA with Oligo(dT) kit as the cloning of melon SQS gene template; reversing the total RNA into cDNA according to the 3′-Full RACE Core Set with PrimeScriptTM RTase manual regarded as the template of cloning the 3’ end of SQS. The obtained PCR product was cloned into p GEM-T vector and confirmed by sequencing. We analysed the Reading Frame Finder Open of SQS on NCBI and predicted the structural domain with CD-Search. We analysed the hydrophobicity/hydrophilicity of SQS in Prot Scale and Prot Param according to the amino acid sequence derived from the nucleotide sequence.NetPhos2.0 Server analysed the phosphorylation sites, TMHMM Server v. 2.0analysed the transmembrane region. SWISS-MODEL analysed the tertiary structure.The plant SQS gene was downloaded from Gen Bank and the Brown grape algae was outgroup which constructed the phylogenetic tree with MEGA5.0 software. Transformed the cucumis melo SQS gene into the pET-28a(+) vector, enzyme digestion and sequencing, transformed into Escherichia coli BL21(DE3), IPTG was used to induce the expression of cucumis melo SQS, the recombinant fusion protein was extracted and purified by Ni2+ affinity chromatography. Results: The sequences of cucumis melo SQS gene and the 3’end with the biological information analysis showed that the enzyme was constituted of 417 amino acid residues with an isoelectric point of7.56.Hydrophobic/hydrophilic prediction results showed that there were 49 basic amino acid, 49 acidic amino acid, 159 hydrophilic amino acid, 100 hydrophobic amino acid and GRAVY value was-0.065, so, the protein was water soluble protein; Phosphorylation sites prediction and analysis of the protein showed that there were 6 serine(Ser) phosphorylation site, 6 threonine(Thr) phosphorylation site, 5 tyrosine(Tyr) phosphorylation site. The analysis of protein phosphorylation site indicated that the phosphorylation site S48 located in the active center-related motif 47VSRSF52 of the enzyme and the phosphorylation site S196 was a positive selection site for the SQS, suggesting both S48 and S196 might be the critical sites for the activity regulation of the enzyme. The secondary structure of the enzyme was mainly α helix. Domain prediction suggested that the cucumis melo SQS belonged to the isoprenoid biosynthase superfamily. Tertiary structure prediction revealed that the SQS was a monomeric enzyme with a cave-like active center formed by α helixes.Phylogenetic analysis with the open reading frame sequence of the cucumismelo SQS gene suggested that the SQS gene can be used as a candidate gene for phylogenetic classification. Double enzyme digestion and sequencing showed that the prokaryotic expression vector was successfully constructed. p ET-28a(+)-SQS vector was transformed into Escherichia coli BL21(DE3), IPTG induction,Ni2+ affinity chromatography and gel column chromatography separation and purification, SDS-PAGE electrophoresis, appeared at about 47 kD of the target protein bands. Conclusions: The SQS gene sequences were isolated and reported for the first time, and the prokaryotic expression vector was constructed.bioinformatics analysis showed that the S48 and S196 phosphorylation sites of SQS were related to the regulation of enzyme activity, and laid the foundation for further research on the structure and function of SQS, and promoted the regulation of the biosynthesis of the gourd.