Study on mechanism of regulating synthesis of β-sitosterol by SiSQS1 and SiSQS2 in Saussurea involucrata cells / 中草药

ABSTRACT

Objective: To explore the synthetic mechanism of β-sitosterol by comparing the locus mutation, prokaryotic expression, expression level of SiSQS1 and SiSQS2, and the content of β-sitosterol in three color types of cells. Methods: Firstly, we preformed the cloning and bioinformatic analysis of SiSQS1 and SiSQS2 which were key enzymes involved in the biosynthesis of β-sitosterol in Saussurea involucrata cells. Secondly, we compared the differences of prokaryotic expression between SiSQS1 and SiSQS2, then optimized the expression conditions. Finally, we compared the expression levels of SiSQS1 and SiSQS2 by Real-time PCR and the content of β-sitosterol by GC-MS in three color types of cells, and made the correlative analysis on the expression level and the content of β-sitosterol. Results: There was a locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2. The results of prokaryotic expression analysis and conditions optimization showed that both target proteins had been expressed successfully, but the optimal prokaryotic expression system was different. The results of expression level and quantitative analysis showed a positive correlation to the expression levels of SiSQS1 and SiSQS2 and the content of β-sitosterol, the correlation coefficients were 0.92 and 0.89, respectively. Conclusion: A locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2 may influence the expression of SiSQS, and there may exist the functional differences in catalytic activity and the accumulation of β-sitosterol. The study will provide technical support and lay a theoretical foundation for studying the accumulated mechanism of β-sitosterol regulated by SQS in S. involucrata cells.