Liu Ying, Zhang Ning, Wang Xue-Yong, Liu Chun-Sheng, Chen Hong-Hao, Wen Hao
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China.
Zhongguo Zhong Yao Za Zhi. 2012 Dec;37(24):3777-83.
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.
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 enzymatic reaction in vitro. GC-MS was used to analyse the production.
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 level of amino acid, the proportion of conservative replace of SQS1 was 53.94%, and there were 2 mutational sites in structural domains. 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.
The polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.
分析甘草鲨烯合酶基因多态性，揭示鲨烯合酶（SQS）基因多态性对其编码酶催化效率的影响。
提取总RNA。采用PCR扩增鲨烯合酶基因编码序列并进行测序分析。构建包含不同SQS基因序列（SQS1C、SQS1F、SQS2A、SQS2B）的表达载体，转化至大肠杆菌BL21。用IPTG诱导融合蛋白表达，然后进行分离、纯化并用于体外酶促反应。采用GC-MS分析产物。
甘草SQS1基因存在3种基因多态性，包括单核苷酸多态性（SNPs）、插入/缺失长度多态性（InDels）和氨基酸水平，SQS1保守替换比例为53.94%，结构域中有2个突变位点。SQS2保守替换比例为60%，结构域中有1个突变位点。在所有4种酶促反应中均能通过GC-MS检测到鲨烯产物。SQS1F积累鲨烯的能力高于其他SQS基因。
甘草中SQS基因多态性丰富，这可能是优质甘草形成的分子基础。
