甜玉米2个β-胡萝卜素羟化酶等位基因(ZsBCH2-a和ZsBCH2-b)的cDNA克隆与分析

Isolation and Characterization of Beta-Carotene Hydroxylase Alleles (ZsBCH2-a and ZsBCH2-b) cDNA in Sweet Corn

β-胡萝卜素羟化酶(beta-carotene hydroxylase, BCH)是植物类胡萝卜素合成代谢中的关键酶,在植物抵抗逆境环境中发挥重要作用。本研究利用普通玉米BCH2基因序列,采用同源克隆方法,从2个甜玉米自交系材料中分离了BCH2等位基因(ZsBCH2-a和ZsBCH2-b) cDNA序列。序列分析结果显示,ZsBCH2-a和ZsBCH2-b基因全长序列分别为1 246 bp和1 217 bp,分别编码325和314个氨基酸,存在4个SNP和1个连续33个碱基插入/缺失突变。蛋白特性分析表明,这2个基因编码的蛋白分子量分别为35 371.84 Da和34 151.40 Da,Zs BCH2-a的等电点略高于Zs BCH2-b,且均为疏水性蛋白。保守结构域与跨膜结构分析表明,这2个蛋白具有脂肪酸羟化酶家族保守结构域,均有2组由10个高度保守的组氨酸组成的"HXXXXH"和"HXXHH"结构,是典型的脂肪酸羟化酶的结构,属于脂肪酸羟化酶超家族;且膜上分别具有4个和3个明显的跨膜结构域,属于跨膜蛋白。进化树分析表明,甜玉米Zs BCH2蛋白的进化高度保守,与同科的高粱BCH蛋白的进化关系最为相近。本研究结果为进一步了解ZsBCH2基因的等位变异在甜玉米类胡萝卜素生物合成及非生物胁迫中的功能提供参考依据。

Beta-carotene hydroxylase is a key enzyme in plant carotenoid biosynthesis and metabolism and plays an important role in plant resistance to adverse environment. In this study, the cDNA sequences of BCH2 alleles(ZsBCH2-a and ZsBCH2-b) were isolated from two sweet corn inbred lines by homologous cloning method using BCH2 sequences of the maize. Sequence analysis results revealed that the full-length sequences of ZsBCH2-a and ZsBCH2-b genes were 1 246 bp and 1 217 bp respectively, encoding 325 and 314 amino acids respectively, with four SNPs and a continuous 33 base insertion/deletion mutation. Analysis of protein characteristics showed that the molecular weights of the proteins encoded by the two genes were 35 371.84 Da and 34 151.40 Da respectively. The isoelectric point of Zs BCH2-a was slightly higher than that of Zs BCH2-b, and both were hydrophobic proteins.The analysis of conserved domains and transmembrane structures showed that the two proteins have conserved domains of the fatty acid hydroxylase family, and both have two groups of "HXXXXH" and "HXXHH" structures composed of 10 highly conserved histidine, which are typical structures of fatty acid hydroxylase and belong to the fatty acid hydroxylase superfamily. There are 4 and 3 obvious transmembrane domains on the membrane, which belong to transmembrane proteins. Phylogenetic tree analysis indicated that the evolution of Zs BCH2 proteins in sweet corn were highly conservative, which had high homology with sorghum of the same family. The research results provide reference basis for further understanding the allelic variation of ZmBCH2 gene in carotenoid biosynthesis and abiotic stress in sweet corn.