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目的:分析甘草鲨烯合酶(squalene synthase,SQS)基因多态性及其对编码酶催化效率的影响,为揭示优质甘草形成的分子机制奠定基础。方法:提取甘草总RNA;PCR扩增其SQS基因编码序列;测序后分析SQS序列的多态性;将具有明显差异的4个甘草SQS序列(SQS1C,SQS1F,SQS2A,SQS2B)连入表达载体pET-32a(+),转化大肠杆菌BL21;IPTG诱导蛋白表达,纯化后用于体外酶促反应;GC-MS鉴定产物并比较相对含量。结果:甘草SQS1基因存在单核苷酸多态性(single nuclearpolymorphisms,SNPs)、插入与缺失多态性(InDel)、选择性剪接(AS)多态性;SQS2基因只存在SNPs。氨基酸序列分析显示,SQS1非保守替换占53.94%,结构域中有2个位点突变;SQS2非保守替换占60%,结构域中有1个位点突变。在4种编码酶的体外酶促反应中,利用GC-MS均能检测到产物生成;SQS1F编码酶积累鲨烯的能力高于其他序列。结论:甘草SQS基因具有丰富的多态性,其编码酶催化效率差异显著,这可能是优质甘草形成的分子基础。
OBJECTIVE: To analyze the polymorphism of squalene synthase (SQS) gene and its effect on the enzyme-catalyzing efficiency, and lay a foundation for revealing the molecular mechanism of high-quality licorice formation. Methods: The total RNA of Glycyrrhiza uralensis Fisch. Was extracted. The coding sequence of SQS gene was amplified by PCR. The sequence of the SQS gene was sequenced and sequenced. Four SQS1C, SQS1F, SQS2A and SQS2B with significant differences were inserted into the expression vector pET -32a (+) was transformed into E. coli BL21; IPTG induced protein expression, purified for in vitro enzymatic reaction; GC-MS identified the product and compared the relative content. Results: Single nucleotide polymorphisms (SNPs), insertion and deletion polymorphisms (InDel) and alternative splicing (AS) polymorphisms were found in the SQS1 gene of Glycyrrhiza uralensis. Only SNPs existed in SQS2 gene. Amino acid sequence analysis showed that SQS1 non-conservative substitution accounted for 53.94%, domain 2 mutations; SQS2 non-conservative substitution accounted for 60% of the domain has a site mutation. Product production was detected by GC-MS in four in vitro enzymatic reactions encoding enzymes; SQS1F encoding enzymes accumulated higher capacity for squalene than other sequences. Conclusion: The SQS gene of Glycyrrhiza uralensis has rich polymorphism, and the enzyme-catalyzed efficiency of SQS gene is significantly different, which may be the molecular basis for the formation of high-quality licorice.