中国中药杂志

2020, v.45(06) 1334-1341

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山楂鲨烯合酶CpSQS1,CpSQS2的基因克隆及原核表达分析
Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida

单婷玉;于大庆;韩晓静;徐睿;查良平;袁媛;
SHAN Ting-yu;YU Da-qing;HAN Xiao-jing;XU Rui;ZHA Liang-ping;YUAN Yuan;School of Pharmacy, Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;

摘要(Abstract):

为认识山楂三萜成分生物合成途径中鲨烯合酶基因结构特征,克隆获得山楂鲨烯合酶基因并进行生物信息学分析和原核表达分析。通过RT-PCR方法从山楂果实中克隆获得2条鲨烯合酶CpSQS1,CpSQS2的基因,其ORF长度分别为1 239,1 233 bp,分别编码412,410 aa。使用在线工具预测CpSQS1,CpSQS2均为稳定的酸性蛋白,二级结构主要由α-螺旋结构组成,利用同源建模对三级结构进行预测;结构功能域分析表明,CpSQS1和CpSQS2的35~367 aa都具有反式异戊烯基焦磷酸合酶的保守结构域;跨膜结构域分析预测CpSQS1,CpSQS2均具有2个跨膜结构域;序列分析表明山楂、丹参、甘草SQS基因编码的氨基酸序列具有较高的同源性;系统进化树分析显示CpSQS1,CpSQS2与蔷薇科的苹果MdSQS1,MdSQS2聚为一支,与系统进化规律一致;构建原核表达载体pGEX-4T-1-CpSQS1,pGEX-4T-1-CpSQS2转化至大肠杆菌Transetta (DE3)进行诱导表达,在65 kDa处均能成功表达出目的蛋白;基因表达分析结果显示,CpSQS2的基因表达水平在山楂的3个不同发育时期均明显高于CpSQS1。该研究首次克隆并分析了山楂SQS1,SQS2基因,为进一步研究山楂三萜生物合成途径奠定基础。
In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

关键词(KeyWords): 山楂;鲨烯合酶;基因克隆;原核表达
Crataegus pinnatifida;squalene synthase;cloning;prokaryotic expression

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81703633,81803675);; 安徽省自然科学基金青年科学基金项目(1808085QH290);; 安徽省中央引导地方科技发展专项资金项目(YDZX20183400004233);; 中央本级重大增减支项目(2060302);; 安徽高校自然科学研究重点项目(KJ2018A0380)

作者(Author): 单婷玉;于大庆;韩晓静;徐睿;查良平;袁媛;
SHAN Ting-yu;YU Da-qing;HAN Xiao-jing;XU Rui;ZHA Liang-ping;YUAN Yuan;School of Pharmacy, Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;

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