红肉火龙果HpHXK基因克隆、表达及酶学特性分析

Cloning,expression and enzymatic characteristics of HpHXK gene in red pitaya(Hylocereus polyrhizus)

【目的】分析红肉火龙果催化己糖磷酸化糖酵解途径关键酶—己糖激酶(Hexokinase,HXK)基因的克隆、表达及酶学特性,为研究HXK基因在果实可溶性糖积累过程中的作用机制及提高红肉火龙果果实品质提供理论依据。【方法】以红肉火龙果紫红龙的成熟茎组织及花后20、23、25、27和30 d的果实为研究对象,通过转录组测序获得HXK蛋白序列,反转录PCR克隆HXK基因,使用ExPASy、Plant-PLoc、Phobius和SMART等在线工具对HpHXK蛋白进行生物信息学分析,利用Clustal W进行氨基酸序列多重比对,采用MEGA 7.0邻接法(Neighbor-joining,NJ)构建系统发育进化树;构建pGEX-4T-HpHXK载体并转化至烟草叶片;分析HpHXK基因在不同组织中的表达模式并利用比色法测定重组蛋白的酶活性。【结果】HpHXK基因开放阅读框(ORF)长度为1410 bp,编码469个氨基酸残基。生物信息学分析表明,HpHXK蛋白相对分子量为52.47 kD,理论等电点(pI)为5.42,该蛋白具有HXK特征结构域。系统发育进化树结果显示,HpHXK序列与水稻、拟南芥、苹果、梨、葡萄和枇杷的HXKs聚为一类。HpHXK基因在花后20 d果实中的相对表达量最高,显著高于花后25、27和30 d果实及成熟茎(P<0.05)。亚细胞定位结果显示,HpHXK蛋白定位于细胞质和细胞膜。聚丙烯酰胺凝胶电泳(SDS-PAGE)检测结果显示,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后,重组质粒表达产物与预期相对分子质量一致,表明成功获得HpHXK重组蛋白。HpHXK蛋白对葡萄糖[米氏常数(Km)=0.61 mmol/L]的亲和力高于果糖(K_(m)=2.92 mmol/L)。【结论】HpHXK蛋白具有HXK家族保守结构域,在红肉火龙果果实发育期间,HpHXK基因负向调控果实可溶性糖积累,在细胞质中催化以葡萄糖为主的己糖磷酸化。

【Objective】To analyze the cloning,expression and enzymatic characteristics of hexokinase(HXK)gene,a key enzyme in the hexose phosphorylation glycolysis pathway of Hylocereus polyrhizus,and to provide a theoretical re-ference for studying the mechanism of HXK gene in the accumulation of soluble sugar in fruits and improving the quality of H.polyrhizus.【Method】The mature stem tissues of H.polyrhizus cv.‘Zihonglong’and its fruits at 20,23,25,27 and 30 d postanthesis were used as the research objects.The HXK protein sequence was obtained by transcriptome sequen-cing,and the HXK gene was cloned by reverse transcription PCR.The bioinformatics analysis of HpHXK protein was carried out by online tools such as ExPASy,Plant-PLoc,Phobius and SMART.The amino acid sequence was multiple aligned by Clustal W,and the phylogenetic tree was constructed by Neighbor-joining(NJ)method of MEGA 7.0.The pGEX-4T-HpHXK vector was constructed and transformed into tobacco leaves.The expression pattern of HpHXK gene in different tissues was analyzed and the enzyme activity of the recombinant protein was measured by colorimetry.【Result】The open reading frame(ORF)of HpHXK gene was 1410 bp in length,encoding 469 amino acid residues.Bioinforma-tics analysis showed that the relative molecular weight of HpHXK protein was 52.47 kD,the theoretical isoelectric point(pI)was 5.42,and the protein had the characteristic domain of HXK.The results of phylogenetic tree showed that HpHXK was clustered with HXKs of rice,Arabidopsis,apple,pear,grape and loquat.The results of real-time fluore-scence quantitative PCR showed that the relative expression of HpHXK gene was the highest in fruit at 20 d postanthesis,which was significantly higher than that in fruit and mature stem at 25,27 and 30 d postanthesis(P<0.05).Subcellular localization results showed that HpHXK protein was localized in the cytoplasm and cell membrane.The results of SDS-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the expression product of the recombinant plasmid was consistent with the expected relative molecular weight after induction with isopropylthio-β-D-galactoside(IPTG),indicating that HpHXK recombinant protein was successfully obtained.The affinity of HpHXK protein for glucose Michaelis constant(K_(m)=0.61 mmol/L),was higher than that of fructose(K_(m)=2.92 mmol/L).【Conclusion】HpHXK protein has a conserved domain of HXK family.During the fruit development of H.polyrhizus,HpHXK gene negatively regulates the accumulation of soluble sugar in fruit and catalyzes the phosphorylation of hexose dominated by glucose in cytoplasm.