肥城桃果实己糖激酶Ⅱ基因克隆、生物信息学分析及原核表达

摘要

为了深入研究己糖激酶(HK)的同工酶HKⅡ调控线粒体通透性转换孔(MPTP)的开放机理,通过RT-PCR技术和RACE技术,以克隆得到的肥城桃果实cDNA为模板,成功克隆得到HKⅡ基因全长,经原核表达和SDS-PAGE检测,体外构建原核表达载体pET-30a-HKⅡ,成功地在大肠杆菌BL21(DE3)中进行表达;使用镍柱纯化法,得到纯化的重组蛋白.克隆得到HKⅡ基因共1835 bp,其中编码区1496 bp,共编码497个氨基酸.HKⅡ蛋白预测分子式为C2379 H3846 N652 O717 S17,原子总数为7611个,分子量为53.6 kDa,理论等电点为6.17,总平均疏水指数为0.023,表明HKⅡ蛋白为疏水性蛋白.TMHMM跨膜结构预测HKⅡ为跨膜蛋白,有1次跨膜,绝大部分在膜外.从二级结构预测结果来看,HKⅡ蛋白由15段α螺旋和11段β折叠和无规则卷曲构成.肥城桃HKⅡ蛋白预测的三级结构呈V字型,折叠结构较多.系统进化树和氨基酸相似性分析发现,桃果实HKⅡ与枇杷的相似度最高,达到了96.98％,桃果实HKⅡ与其他植物HK的氨基酸的同源性较高,说明HKⅡ具有较高的保守性.为进一步解析HKⅡ蛋白的结构和功能从而探讨HKⅡ调控线粒体MPTP的机理奠定了基础.%In order to study the regulation mechanism of hexokinase Ⅱ ( HKⅡ) to mitochondrial permeability transition pore ( MPTP) opening,a cDNA encoding HKⅡ protein was isolated from Feicheng peach fruits by RT-PCR and rapid amplification of cDNA ends (RACE). Induced by IPTG and determinated by SDS-PAGE,the ex-pression vector pET-30 a-HKⅡwas expressed by BL21 . The HKⅡ protein was purified through nickel chelating af-finity chromatography. The full-length cDNA of HKⅡwas 1835 bp including 1496 bp of coding sequence encoding a polypeptide of 497 amino acids. The predicted protein molecular formula of HKⅡ was C2379 H3846 N652 O717 S17 ,con-taining 7611 atoms. For HKⅡ,the molecular weight was 53. 6 kDa,the theoretical isoelectric point was about 6. 17, and the total average hydrophobicity index was 0 . 023 , which indicated that HKⅡ protein was a hydrophobic pro-tein. TMHMM transmembrane structure predicted that HKⅡ was a transmembrane protein with 1 transmembrane, most of which were outside the membrane. According to the results of the two level prediction,the HKⅡprotein was composed of 15 segments ofαhelix and 11 segments ofβfolding and the irregular curl. The three level structure of peach HKⅡ protein in Feicheng showed a V font,there were a large number of folded structure. It showed the high-est homology with the loquat (96. 98％) through the phylogenetic tree and multiple sequences alignment analysis. The high homology of amino acids of HK in peach fruit and other plants,indicating that HKⅡis more conservative.The results would lay a foundation for further analysis of the structure and function of HKⅡ protein to explore the mechanism of HKⅡ regulation of mitochondrial MPTP.