柠条锦鸡儿香豆酸-3-羟化酶基因克隆及其功能初步研究  

Cloning and Sequence Analysis of the CkC3H Gene from Caragana korshinskii Kom. and Preliminary Studies of Its Function

作　　者：李高[1,2] 杨杞[1] 张烨[1,3] 王瑞刚[1] 李国婧[1] 

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018 [2]北京市颐和园管理处,北京100091 [3]鄂尔多斯市食品药品检验研究中心,鄂尔多斯017000

出　　处：《中国生物工程杂志》2013年第4期61-67,共7页China Biotechnology

基　　金：国家自然科学基金(31060105);教育部新世纪优秀人才支持计划(ECNT-11-1020);教育部博士学科点基金博导类联合资助项目(20111515110001);内蒙古自然科学基金重点项目(2010Zd13)资助项目

摘　　要：香豆酸-3-羟化酶(Coumarate 3-Hydroxylase,C3H)是木质素生物合成途径中的关键酶之一。以柠条锦鸡儿为材料,利用RACE技术克隆了C3H基因。对柠条锦鸡儿C3H基因的gDNA和cDNA全长分析显示该基因具有3个外显子,2个内含子,编码框长度为1530bp,编码509个氨基酸。预测该基因编码蛋白的等电点位7.67,分子量约57.61kDa。氨基酸序列分析显示具有一个保守的P450结构域。系统进化分析表明该蛋白与大豆C3H具有最高的同源性,将该基因命名为CkC3H,GeneBank登录号为HQ829858。构建了35S启动子驱动的CkC3H基因植物表达载体,互补拟南芥C3H(CYP98A3)基因突变体ref8,转基因植物表型得以部分恢复。这些结果说明柠条锦鸡儿CkC3H与拟南芥C3H至少具有部分相同的功能。Coumarate 3-Hydroxylase （C3H） is a key enzyme in lignin biosynthesis pathway. A C3H encoding gene was cloned by rapid amplification of cDNA end technique from Caragana korshinskii Kom. The full length gDNA of CkC3H was 4235bp, contained three exons and two introns. The full length of ORF was 1530bp, and the protein deduced from this cDNA comprised 509 amino acids with a calculated molecular weight of 57. 61kDa and an isoelectric point of 7.67. The deduced protein contains a P450 domain and was named as CkC3H （GenBank accession no. KC309408）. Phylogenetic analysis indicated that CkC3H shared the closest homology with the C3H from Glycine max. Binary vector carried the cDNA sequence of CkC3H driven by 35S promoter was constructed and transformed into the ref8 mutant of Arabidopsis thaliana. The dwarf and sterile phenotype of the ref8 mutant were partially restored by the transgene. These showed that CkC3H might have at least partially similar function with AtC3H.

关 键 词：C3H 木质素 序列分析 ref8 

分 类 号：Q786[生物学—分子生物学]