拟南芥基因组中注释为(S)-去甲乌药碱合成酶的分子克隆与异源表达(英文)  

Molecular Cloning and Heterologous Expression of Putative (S)-norcoclaurine Synthases from Arabidopsis thaliana

作　　者：张翔[1,2] 马磊[3] 田永强[3] 张国林[1] 罗应刚[1] 

机构地区：[1]中国科学院成都生物研究所,成都610041 [2]中国科学院大学,北京100049 [3]四川大学化学工程学院,成都610065

出　　处：《应用与环境生物学报》2013年第1期61-68,共8页Chinese Journal of Applied and Environmental Biology

基　　金：中国科学院知识创新工程领域前沿项目;“西部之光”联合学者项目;生命科学领域优秀青年科技专项(KSCX2-EW-Q-6);国家自然科学基金面上项目(21172216)联合资助~~

摘　　要：至今在拟南芥中尚未发现复杂生物碱,但是其基因组测序则表明有35个以上基因可编码(S)-去甲乌药碱合成酶(NCS).本研究首先以经过生化表征、机理清楚的来自罂粟、日本黄连、黄唐松草和花菱草的NCS为参考,与拟南芥中注释的NCS进行了广泛的生物信息学分析与比对,从中选择5个AtNCS为目的基因,设计特异性引物;从拟南芥中提取总RNA,一步法RT-PCR克隆得到上述基因;通过TA克隆将上述基因转入pGM-T载体,筛选、酶切及PCR鉴定,DNA测序验证它们的核酸序列;将测序验证的AtNCS基因经过PCR扩增、质粒构建、转入Escherichia coli BL21(DE3)中进行异源表达和蛋白纯化;在整体细胞、细胞裂解液、细胞裂解液上清和纯化蛋白共4个层次进行了AtNCS酶功能表征,未发现目标产物或新产物的生成;上述基因的具体功能还有待进一步研究.No complex alkaloid was detected in Arabidopsis thaliana till today. However, 〉 35 genes were annotated as NCSs [（S）-norcoclaurine synthases]-encoding genes involved in the biosynthesis of benzylisoquinoline alkaloids. Based on bioinformatics alignment and phylogenetic analysis between putative A. thaliana NCSs （AtNCSs） and biochemically characterized plant NCSs, five putative AtNCSs were chosen to be biochemically characterized in this work. The cDNAs encoding the five putative AtNCSs were obtained by RT-PCR and cloned into pET28a or pET30a vector. The nucleotide sequences of the five AtNCS genes were confirmed by DNA sequencing. Induced by IPTG, AtNCS-1 - 5 were heterologously overexpressed in Escherichia coli BL21 （DE3）. The fusion proteins were soluble and purified by nickel-chelate affinity chromatography. The putative catalytic activity of the recombinant AtNCSs was assayed at four levels, including whole cell, cell lysate, supernatant, and purified enzyme, by adding the native substrates dopamine and 4-HPAA to the reaction mixture. No desired or new products were detected in the reaction mixture by HPLC-DAD detection, which suggested that these putative AtNCSs did not display the Pictet-Spengler condensation activity. Fig 7, Tab 2, Ref20

关 键 词：分子克隆 RT-PCR 异源表达 (S)-去甲乌药碱合成酶 苄基异喹啉 

分 类 号：Q946[生物学—植物学] Q78