目的克隆黄连Coptischinensis N-甲基乌药碱-3′-羟化酶[(S)-N-methylcoclaurine-3′-hydroxylase]基因并做序列分析。方法采用RT-PCR方法,以新鲜的黄连嫩叶cDNA为模板,克隆出黄连N-甲基乌药碱-3′-羟化酶基因的全部序列。结果克隆到的基因(命名为CYP80B3)全长为1680bp,编码一个488个氨基酸残基组成的多肽。其氨基酸序列与日本黄连、唐松草、罂粟和花菱草的N-甲基乌药碱-3′-羟化酶的氨基酸序列同源性分别达95%、82%、70%、68%。将得到的序列提交Genbank,序列号为EF492879。通过与日本黄连的氨基酸序列比对,具有相同功能区域,因而可以参与N-甲基乌药碱的3′位羟基化反应。结论黄连N-甲基乌药碱-3′-羟化酶基因的成功克隆为黄连植物中原小檗碱型苄基异喹啉类生物碱的基因工程等研究打下了良好的基础。

ObjectiveTo clone and sequence the cDNA encoding (S)-N -methylco claurine-3′-hy droxylase from Copt is chinensis. MethodsThe cDNA, encoding(S )-N-methylcoclaurine-3′-hydrox ylase, was amplified by RT-PCR with cDNA library of tender leaf as the template. ResultsThe full-length cDN A of(S )-N -methylco claurine-3′-hydrox ylase (named as CYP80B3) had 1680bp with an open reading frame encoding 488 aminoacids of protein.The CYP80B3 had 95% ,82% ,70% ,and 68% aminoacid sequence homology to the sequence of (S )-N -methylcoclaurine-3′-hydroxylase from C.japonica ,Thalictrum flavum , Eschscholziacal ifornica and Papaver somniferum,respectively.The sequence was reported to the GenBank and coded as EF492879. Comparison of sequence with (S )-N -methy lcoclaurine-3′-hydrox ylase from C. japonica showed CYP80B3 possessed the same functional regions involved with 3′-hydroxylation of (S ) -N-methy lcoclaurine. Conclusion The cDN A encoding CYP80B3 f rom C.chinensis was cloned and reported. This work underlays the first step for exploring the pathway of benzyli soquinoline alkaloid biosynthesis and for improving the content of benzy lisoquinoline alkaloidin C.chinensis.

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