小麦淀粉酶的生物信息学、基因表达与活性分析

Bioinformatics,gene expression,and activity analysis of wheat amylase

试验旨在探究小麦α-淀粉酶和β-淀粉酶的结构特征及萌发过程中的基因表达与酶活力变化。试验对小麦α-淀粉酶和β-淀粉酶的结构特征进行了生物信息学预测,通过荧光定量PCR检测小麦种子萌发过程中α-淀粉酶和β-淀粉酶基因的表达量,并测定其酶活性。结果显示:小麦α-淀粉酶是碱性蛋白,β-淀粉酶是酸性蛋白,α-淀粉酶有信号肽和1个跨膜区,而β-淀粉酶均没有,二者二级和三级结构及磷酸化位点数目相似;小麦萌发时,α-淀粉酶基因表达量呈升高后降低的趋势,β-淀粉酶基因表达量处于低水平,α-淀粉酶基因表达受到低浓度盐碱诱导和高浓度盐碱抑制,β-淀粉酶基因表达量随盐碱浓度的升高而增加,α-淀粉酶和β-淀粉酶活性分别在萌发后期和早期明显增加。研究表明,小麦α-淀粉酶为分泌型膜蛋白,β-淀粉酶为基质游离蛋白,α-淀粉酶和β-淀粉酶对盐碱的应激反应不同,分别在小麦萌发后期和早期发挥主要作用。

The purpose of this experiment was to explore the structural characteristics,expression and enzymatic activity changes during germination of wheatα-amylase andβ-amylase.The structural characteristics ofα-amylase andβ-amylase in wheat were predicted by bioinformatics,fluorescence quantitative PCR was used to detect the expression level ofα-amylase andβ-amylase gene in wheat seeds during the germination process,and their enzymatic activity was measured.The results showed that wheatα-amylase was an alkaline protein,β-amylase was an acidic protein,α-amylase had a signal peptide and a transmembrane region,whileβ-amylase did not,and the secondary and tertiary structures and number of phosphorylation sites of both amylases were similar.When wheat sprouted,the expression level ofα-amylase gene first increased and then decreased,while the expression level ofβ-amylase gene was at a low level,the expression ofα-amylase gene was induced by low concentration of salt alkali and inhibited by high concentration of salt alkali,the expression level ofβ-amylase gene increased with increasing salt alkali concentration,the activity ofα-amylase andβ-amylase significantly increased in the late stage and early stage of germination,respectively.The results indicated that wheatα-amylase was a secreted membrane protein,β-amylase was a stroma free protein,the stress responses ofα-amylase andβ-amylase to salt alkali were different,and they played a major role in the late and early stages of wheat germination respectively.