艾叶1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因的克隆及蛋白的结构性质分析  

Cloning and Characterization of 1-Hydroxy-2-methyl-2-( E)-butenyl-4-diphosphate Reductase (ArHDR) Gene in Artemisia argyi

作　　者：刘苗苗 吴家文[1,2] 吴生兵 杨青山[1,2] 周美启[1] 

机构地区：[1]安徽中医药大学,安徽合肥230038 [2]安徽道地药材品质提升协同创新中心,安徽合肥230038

出　　处：《中药材》2017年第8期1793-1797,共5页Journal of Chinese Medicinal Materials

基　　金：安徽高校科研创新平台团队建设项目(2015TD033);安徽省自然科学基金(1608085MH177);安徽省高校优秀中青年骨干人才国内外访学研修重点项目(gxfx ZD2016115);安徽中医药大学人才引进科研项目(2015RC002);安徽省留学人员科技活动启动项目(2015lx024)

摘　　要：目的:克隆艾叶1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因开放读码框(Ar HDR),分析其编码蛋白ArHDR的性质和结构。方法:采用逆转录PCR方法扩增ArHDR,纯化后与p MD19-T载体连接构建了重组质粒再转化至大肠杆菌中,获得的阳性克隆经菌液PCR及测序鉴定,序列与转录组测序得到的Unigene一致。利用系统的生物信息学软件对ArHDR的性质与结构进行了分析。结果:获得了艾叶ArHDR,长度为1 365 bp,编码454个氨基酸;生物信息学分析表明Ar HDR属于稳定蛋白,与黄花蒿HDR亲缘关系最近;ArHDR的三级结构富含α螺旋与β折叠,呈"苜蓿叶"形,3个保守的半胱氨酸位于中心,构成了锚定铁硫的酶的催化中心。结论:该研究体外成功克隆了ArHDR,分析了ArHDR的性质与结构,为增加艾叶萜类化合物合成提供理论依据。Objective： To clone the open reading frame（ ORF） of 1-hydroxy-2-methyl-2-（ E）-butenyl-4-diphosphate reductase（ ArHDR） from Artemisia argyi,and to analyze the characteristics and structure of the coding protein. Methods： The target gene fragments were amplified by reverse transcriptase PCR method and subcloned to p MD19-T vectors,and then the recombinant plasmids were transformed into Escherichia coli DH5α cells. The positive clones were confirmed by bacteria PCR and sequencing. The coding protein ArHDR was analyzed by systematic bioinformatics softwares. Results： The full-length ORF of the Ar HDR,which contained 1 365 bp encoding Ar HDR protein with 454 amino acids,was acquired. Bioinformatics analysis showed that Ar HDR was a stable protein and it has the closest relationship with HDR from Artemisia annua. Ar HDR showed cloverleaf structure consisting of three lobes,each containing alternating α/β structure. Three conserved cysteines located in the center of the three dimensional structure,which formed the catalytic region for anchoring iron-sulfur. Conclusion： The Ar HDR is successfully cloned in vitro,and the property and structure of Ar HDR are studied. The results provide theoretical basis for promoting the synthesis of terpenoids from Artemisia argyi.

关 键 词：艾叶 ArHDR基因 基因克隆 生物信息学分析 

分 类 号：R282.71[医药卫生—中药学]