目的 通过比较天山雪莲Saussurea involucrata细胞中鲨烯合酶SiSQS1和SiSQS2的位点变异、蛋白原核表达及表达水平差异以及下游产物β-谷甾醇量的高低，探索天山雪莲细胞SiSQS1和SiSQS2催化β-谷甾醇合成的机制。方法 从天山雪莲细胞中克隆出β-谷甾醇生物合成途径上关键酶SiSQS1与SiSQS2基因，并对其进行生物信息学分析；分别对SiSQS1和SiSQS2进行原核表达与条件优化，比较二者在天山雪莲细胞中的蛋白原核表达差异；采用RT-PCR技术对二者在天山雪莲三色系细胞中的表达水平进行比较，采用GC-MS对雪莲三色系细胞中β-谷甾醇进行定量分析，并对β-谷甾醇量与SiSQS1、SiSQS2表达水平做相关性分析。结果 SiSQS1和SiSQS2在242E/D存在残基变异，原核表达及条件优化实验表明二者都有目的蛋白条带出现，但二者之间的原核表达最适体系不同；化学定量与基因表达水平的相关性分析表明β-谷甾醇量与SiSQS1、SiSQS2基因表达水平呈正相关，相关系数分别为0.92和0.89。结论 SiSQS1与SiSQS2二者242E/D残基变异可能影响SiSQS蛋白的表达，二者在催化活性以及对β-谷甾醇积累调控作用上可能存在一定的功能差异，为研究鲨烯合酶SQS调控天山雪莲细胞β-谷甾醇积累的机制提供了技术支持和奠定了理论基础。

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.

国家高技术研究发展计划（SS2014AA022201）