大麦Amy32b的遗传多样性及其对α-淀粉酶活性的影响

doi:10.3864/j.issn.0578-1752.2012.05.001

摘要/Abstract

摘要： 【目的】通过对Amy32b遗传多样性研究，发掘与α-淀粉酶活性相关的等位变异。【方法】设计能够覆盖Amy32b序列的特异引物，研究该基因在中国大麦的等位变异类型：单核苷酸多态性位点（SNP）及插入（insert）、缺失（delete）等。利用RT-PCR获得Amy32b的全长cDNA序列，分别将携带等位变异的全长cDNA和截短cDNA连入表达载体pET-28a(+)，并对表达产物进行纯化、活力测定及比较研究。【结果】根据对已公布的Amy32b（x05166）的扩增，发现在基因编码区有一多态性位点G/A（cSNP）和1个A碱基的插入/缺失突变。分别位于Amy32b的碱基序列2 269 bp和2 403 bp，其中，G/A转换导致第355位氨基酸由谷氨酸（E）替换为赖氨酸（K）；A碱基插入导致终止密码的形成，引发了碱基片段缺失以及编码氨基酸序列和α-淀粉酶蛋白C端的提前终止。克隆出Amy32b全长和截短cDNA，长度分别为1 314 bp和1 266 bp。酶活性测定结果表明，两种全长重组酶（rAmy32b_A和rAmy32b_G）均具有正常且相同的淀粉酶活性，而截短的突变酶（rΔAmy32b）未能测出淀粉酶活性。【结论】Amy32b的碱基插入/缺失突变形成终止子，引发碱基片段缺失和编码α-淀粉酶C端截短，造成酶活性的丧失；而G/A转换造成的氨基酸替换则对该酶活性没有影响。

关键词: 大麦, Amy32b, 编码区单核苷酸多态性, &alpha, -淀粉酶活性

Abstract: 【Objective】 The objective of the experiment is to study the polymorphism of the Amy32b gene in Chinese barley and dissect its relevance to the α-amylase activity 【Method】 Three pairs of specific primers covering the whole sequence of the gene were designed to locate single nucleotide polymorphism (cSNP) and other In/Del mutations. The complete cDNA of Amy32b was cloned from barley via RT-PCR. Full-length cDNA carrying alleles（G/A）and truncated cDNA of Amy32b were cloned into expression vector pET-28a(+). Their expression products were purified, characterized, and compared. 【Result】 According to the result of amplified DNA sequences of Amy32b (x05166), there was a single nucleotide polymorphism cSNP(G/A) and an insertion/deletion of A were found in the coding region of the gene, located in 2 269 bp and 2 403 bp, respectively. The SNP(G/A) caused an E355K amino acid substitution in the enzyme. And the In/Del variation of A insertion formed an in-frame stop codon, resulting in the deletion of a 48 bp fragment in cDNA as well as a β5 sheet in carboxyl terminal of the α-amylase. The full-length of Amy32b cDNA is 1 314 bp, while the truncated cDNA is 1 266 bp in length. The enzymatic activity assay showed that rAmy32b_A and rAmy32b_G exhibited normal and similar amylase activities. But for the truncated Amy32b gene, the mutant enzyme (rΔAmy32b) did not produce detectable actvity. 【Conclusion】 The In/Del mutation formed an early translation stop codon, resulting in the losses of 48 bp sequence fragment and a β-strand in C-terminal end of α-amylase, and seriously affected enzymatic activity, while the G/A transition only resulted in amino acid substitution and did not alter enzymatic activity.

Key words: barley, Amy32b, coding region single nucleotide polymorphism, α-amylase activity

姜晓东, 张京, 郭刚刚. 大麦Amy32b的遗传多样性及其对α-淀粉酶活性的影响[J]. 中国农业科学, 2012, 45(5): 823-831.

JIANG Xiao-Dong, ZHANG Jing, GUO Gang-Gang. Polymorphism of the Amy32b Gene and Its Effect on the α-Amylase Activity in Barley[J]. Scientia Agricultura Sinica, 2012, 45(5): 823-831.

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