拟南芥基因组中注释为小檗碱桥环酶基因的分子克隆及异源表达  

Molecular Cloning and Heterologous Expression of Putative Berberine Bridge Enzymes 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]中国科学院研究生院,北京100049 [3]四川大学化工学院,成都610041

出　　处：《世界科技研究与发展》2012年第5期807-811,共5页World Sci-Tech R&D

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)资助

摘　　要：小檗碱桥环酶(BBE)催化(S)-牛心果碱((S)-reticuline)中N-CH_3与分子内苄基部分中羟基的邻位芳香碳之间C-C键的形成,该酶属于双共价黄素蛋白家族,是苄基异喹啉类生物碱向小檗碱类生物碱转化的关键酶。迄今,在拟南芥(Arabidopsis thaliana)中尚未发现复杂生物碱,但其基因组测序结果表明拟南芥含有众多可能与复杂生物碱生物合成相关的基因,其中与BBE类似的基因有12个。基于与已知功能的BBE及拟南芥中BBE序列的分析,选定拟南芥中4个注释为BBE的编码基因为目的基因,设计特异引物,从拟南芥cDNA中扩增并克隆到pGM-T载体中,筛选重组子,测序并分析,获得了4个BBE目的基因,分别为AT2G34810、AT5G44400、AT5G44410和AT5G44440。将上述基因克隆至表达载体pET-28a或pET-30a中,分别转入大肠杆菌Rosetta(DE3)中,IPTG诱导实现了上述基因的异源表达。Berberine bridge enzyme （ BBE ）, one member of bi - covalent flavoprotein family, catalyzes the conversion of （S） -reticuline to （S） -scoulerine via the formation of a new carbon-carbon bond between the carbon of N-methyl group and the intramolecular aromatic carbon ortho to the phenolic hydroxyl group in the benzyl motif. BBE is a key enzyme in the biosynthetic pathway of berberine alkaloids. No complex alkaloids have been detected in Arabidopsis thaliana. However, bioinformatics analysis of the whole genome sequences of A. thaliana indicated that many genes were annotated as enzymes for the biosynthesis of complex alkaloids. For instance, 12 genes were annotated as BBE involved in the biosynthesis of berberine alkaloids. To investigate on the possible function of these genes,4 putative AtBbes is selected out of 12 genes on the basis of bioinformaties analysis of known functional EcBBE and AtBBEs. The four putative AtBbe genes were obtained by RT-PCR u- sing total RNA as template. The plasmid containing AtBbes were screened and then confirmed by DNA sequencing. The target genes such as AT2G34810, AT5 G44400, AT5 G44410 and AT5 CA.A. A.A.9 are obtained. The expression plasmids were transformed into Escherichia coli Rosetta （DE3） for protein overexpression. Induced by IPTG,the AtBBEs were overexpressed in the prokaryotie host.

关 键 词：拟南芥 小檗碱桥环酶 生物信息学分析 RT—PCR TA克隆 异源表达 

分 类 号：Q943.2[生物学—植物学]