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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 one of the key enzymes in lignin biosynthesis pathway. Using Caragana korshinskii as material, the C3H gene was cloned by RACE technique. The full length cDNA and cDNA of C3H gene of Caragana korshinskii showed that there are 3 exons and 2 introns in this gene. The coding region is 1530bp in length and encodes 509 amino acids. The predicted isoelectric point of this protein was 7.67 and the molecular weight was about 57.61 kDa. Amino acid sequence analysis revealed a conserved P450 domain. Phylogenetic analysis showed that this protein had the highest homology with soybean C3H. The gene was named as CkC3H and GeneBank accession number was HQ829858. The plant expression vector of CkC3H gene driven by 35S promoter was constructed. The mutant of complement of Arabidopsis C3H (CYP98A3) gene ref8 was partially restored. These results suggest that Caragana korshinskii CkC3H and Arabidopsis C3H have at least partially the same function.