摘要
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 analyse the polymorphism of squalene synthase gene and reveal the influence of squalene synthase （SQS） gene polymorphism on the catalytic efficiency of its encode enzyme in Glycyrrhiza uralensi. Method： The total RNA was extracted. PCR was used to amplify the coding sequences of squalene synthase gene, which were sequenced and analysed. The expression vectors containing different SQS gene sequences, including SQS1C, SQS1F, SQS2A, SQS2B, were constructed and transformed into Escherichia coli BL21. The fusion protein was induced to express by IPTG, then was isolated, purified and used to carry out the enzy- matic reaction in vitro. GC-MS was used to analyse the production. Result： There were three kinds of gene polymorphism existing in SQS1 gene of G. uralensis, including single nucleotide polymorphism （ SNPs）, insertion/deletion length polymorphism （InDels） and aternative splicing（AS）. But there was only one kind of gene polymorphism, SNPs, existing in SQS2 sequences of G. uralensis. In the level of amino acid, the proportion of conservative replace of SQS1 was 53.94% , and there were 2 mutational sites in structural do- mains. The proportion of conservative replace of SQS2 was 60%, and there was 1 mutational site in structural domains. The production squalene could be detected by GC-MS in all the 4 kinds of enzymatic reactions. The capacity of accumulating squalene of SQS1F was higher than other SQS genes. Conclusion ： The polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2012年第24期3777-3783,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81072988)
关键词
甘草
SQS
基因多态性
单核苷酸多态性
插入与缺失多态性
选择性剪接
原核表达
GC—MS
鲨烯
Glycyrrhiza uralensis
squalene sythase （SQS）
gene polymorphism
single nuclear polymorphisms
insertion/de-letion length polymorphism
aternative splicing
prokaryotic expression
GC-MS
squalene
