苹果己糖激酶基因MdHXK1的克隆与表达分析

Molecular Cloning and Expression Analysis of a Hexokinase Gene MdHXK1 in Apple

从‘嘎啦’苹果中克隆了己糖激酶基因MdHXK1(基因序列号:MDP0000309677)全长,测序发现其包含长为1 497 bp完整的开放阅读框,编码499个氨基酸,预测其编码蛋白质分子量为54.05k D,等电点为5.76。系统进化树分析表明,HXK1在不同物种间具有高度的序列保守性;其中,苹果MdHXK1与中国白梨Pb HXK1亲缘关系最近。功能域分析表明,MdHXK1蛋白含有两个保守的激酶域。Southern blotting结果表明,MdHXK1在苹果基因组中有一个拷贝。亚细胞定位预测表明,MdHXK1主要定位于细胞质。分析MdHXK1基因启动子序列发现含有与抗逆、糖信号及激素信号相关的顺式作用元件。荧光定量PCR分析表明,MdHXK1在苹果的茎和花中表达量较高;在苹果果实发育期间,MdHXK1基因的表达、MdHXK1葡萄糖磷酸化相对酶活性和葡萄糖积累水平都呈现先升高后降低的趋势,MdHXK1基因的表达明显受盐、低温和ABA的诱导。原核诱导并纯化了MdHXK1蛋白,为后续MdHXK1蛋白的功能鉴定奠定基础。

A hexokinase gene named MdHXK1（MDP0000309677）was cloned from‘Gala＇apple（Malus × domestica Borkh.）. Sequence analysis showed that the length of MdHXK1 gene was 1 497 bp,which encoded 499 amino acids. It was predicted that the molecular mass of this protein was 54.05 k D,and p I was 5.76. A phylogenetic tree indicated that the HXK1 had a higher sequence conservation among different species,while apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri Pb HXK1. Analysis of functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that MdHXK1 protein was mainly localized in cytoplasm. There was an indication that MdHXK1 existed as one copy in apple genome by Southern blotting. In silico analysis suggested that the promoter sequence contained several typical cis-acting elements,including defense responsive elements,sugar signaling responsive elements and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower. During the development of apple fruits,the expression of MdHXK1 gene increased at first and then decreased. The changes on Glc phosphorylation activity（relative）and glucose concentration showed the same trend. Besides,the expression of this gene was induced by salt stress,low temperature and ABA. Finally,we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression,which established the foundation for the further study on the functions of MdHXK1 protein.