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目的通过比较天山雪莲Saussurea involucrata细胞中鲨烯合酶Si SQS1和Si SQS2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Si SQS1和Si SQS2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Si SQS1与Si SQS2基因,并对其进行生物信息学分析;分别对Si SQS1和Si SQS2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Si SQS1、Si SQS2表达水平做相关性分析。结果 Si SQS1和Si SQS2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Si SQS1、Si SQS2基因表达水平呈正相关,相关系数分别为0.92和0.89。结论 Si SQS1与Si SQS2二者242E/D残基变异可能影响Si SQS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 compare the site variation of squalene synthase Si SQS1 and Si SQS2, the difference of prokaryotic expression and expression level and the content of β-sitosterol in downstream Saussurea involucrata cells, and to explore the relationship between Si SQS1 and Si SQS2 Catalytic mechanism of β-sitosterol synthesis. Methods The key enzymes, Si SQS1 and Si SQS2, of β-sitosterol biosynthesis pathway were cloned from Tianshan lotus cells and analyzed by bioinformatics. The prokaryotic expression and conditions of Si SQS1 and Si SQS2 were optimized respectively. Were analyzed by RT-PCR. The expression of both proteins in Tianshan Saussurea tricolor cells was analyzed by GC-MS. The results showed that β-sitosterol in Saussurea tricolor cells Quantitative analysis, and β-sitosterol and Si SQS1, Si SQS2 expression levels do correlation analysis. Results Residues of Si SQS1 and Si SQS2 at 242E / D were polymorphic. Prokaryotic expression and conditional optimization experiments showed that both of them had the target protein bands appearing, but the optimal system for prokaryotic expression was different. The relationship between chemical quantitative and gene expression The level of correlation analysis showed that the amount of β-sitosterol and Si SQS1, Si SQS2 gene expression levels were positively correlated, the correlation coefficients were 0.92 and 0.89. Conclusion The variation of 242E / D residues in both Si SQS1 and Si SQS2 may affect the expression of Si SQS protein. There may be some functional differences between the two on the catalytic activity and the regulation of β-sitosterol accumulation. In order to study the effects of squalene synthase SQS regulation of Tianshan snowdrop cells β-sitosterol accumulation mechanism provides the technical support and laid the theoretical foundation.