浙贝母角鲨烯合酶全长cDNA的克隆及功能分析

鲨烯是甾醇和其他三萜类化合物的关键代谢中间体，其生物合成由鲨烯合酶（squalene synthase，SQS）催化，该酶将2分子法呢基焦磷酸转化为鲨烯。浙贝母异甾体生物碱的生物合成途径与三萜类化合物类似。在本研究中，基于cDNA末端的快速扩增（RACE）技术克隆了浙贝母鲨烯合酶 (FtSQS）基因的全长cDNA，GenBank登录号为KF551097.2。通过生物信息学方法对FtSQS进行详细表征，包括保守区检测、序列同源分析、二级和三级结构预测及系统发育树分析。结果表明，其开放阅读框（ORF）为1 230 bp，编码409个氨基酸，FtSQS氨基酸序列与印度甘松、截形苜蓿、紫衫、马铃薯、柴胡、金铁锁和拟南芥的SQS氨基酸同源性分别达到73.84%、73.23%、72.24%、70.66%、70.66%、69.44%和68.14%。启动子分析表明，FtSQS的5′上游区域具有与生理和环境因素相关的各种潜在因素。为了获得可溶性FtSQS表达，从羧基末端截断24个疏水氨基酸，构建了原核表达载体pGEX-2T-FtSQSΔTM，并在大肠杆菌BL21(DE3)中表达。SDS-PAGE检测到约66 kD的重组FtSQSΔTM蛋白。体外酶促反应证明，FtSQS可以催化FPP转化成鲨烯。qRT-PCR分析FtSQS mRNA在叶中的表达量最高，茎、根次之，而在鳞茎中表达水平最低。这提示，叶子是浙贝母碱生物合成的主要活性器官。FtSQS的鉴定及功能研究为浙贝母次生代谢产物的研究提供了重要依据。

Cloning and Functional Characterization of Full length cDNA Encoding Squalene Synthase from Fritillaria thunbergii Miq

Squalene is a key metabolic intermediate for sterols and various other triterpenoids. Its biosynthesis is catalyzed by squalene synthase (SQS), which converts two molecules of farnesyl pyrophosphate to squalene. The biosynthetic pathway of Fritillaria thunbergii Miq isosteroid alkaloids is similar to that of triterpenoids. In this study, a full-length cDNA of squalene synthase from Fritillaria thunbergii Mig (FtSQS) was cloned using rapid amplification from cDNA ends (RACE) technology. GenBank accession number was KF551097.2. Bioinformatics methods were used to characterize the FtSQS in detail, including the detection of conserved regions, sequence homology analysis, secondary and tertiary structure prediction, and phylogenetic tree analysis. The results showed that its open reading frame (ORF) was 1 230 bp and encoded 409 amino acids. Protein-Blast alignment found that amino acid homology with SQS of Indian pine, Truncate alfalfa, Purple shirt, Potato, Bupleurum, Golden iron lock and Arabidopsis reached 73.84%, 73.23%, 72.24%, 70.66%, 70.66%, 69.44%, 68.14%. Promoter analysis indicated that the 5′ upstream region of FtSQS possessed various potential elements associated with physiological and environmental factors. To obtain a soluble recombinant protein, 24 hydrophobic amino acids were deleted from the carboxyl terminus, and the C-terminal truncated mutant FtSQS (FtSQSΔTM) was expressed in E. coli BL21 (DE3). SDS-PAGE analysis suggested that approximately 66 kD recombinant protein was checked. The in vitro enzymatic reaction proved that FtSQS could catalyze farnesyl pyrophosphate to generate squalene. Expression level of FtSQS mRNA in leaves was the highest, followed by stem and root, but in bulb was much lower than that in other tissues. It suggests that leaves are active organ for biosynthesis of peimine. The identification and function of FtSQS provides an important basis for the study of secondary metabolites of Fritillaria thunbergii Miq.