小麦avenin-like的克隆、原核表达与品质效应研究

doi:10.3864/j.issn.0578-1752.2012.04.001

摘要/Abstract

摘要： 【目的】研究普通小麦不同品种的类燕麦贮藏蛋白（avenin-like）基因序列的多样性及其表达产物的加工品质效应。【方法】利用特异性引物对来源于不同地区的12个小麦品种（系）进行PCR扩增、克隆、测序及序列分析；构建JN542444原核表达载体后转入表达菌株Escherichia coli Rosetta gami B(DE3)，IPTG诱导融合蛋白表达，并通过His-Trap HP柱纯化蛋白后进行微量掺粉试验。【结果】克隆获得22条avenin-like新基因序列（GenBank登录号JN542443—JN542464），长度均为855 bp，且均具有可编码284个氨基酸残基的完整的开放阅读框，无内含子；半胱氨酸数目及位置相对保守，除JN542452含有17个Cys外，其余avenin-like所编码的蛋白质序列均含有18个Cys，且位置相同，预测可能形成7个分子内二硫键和4个分子间二硫键；发现3条罕见的avenin-like假基因（JN542448、JN542455和JN542456），均因终止密码子的提前出现引起基因沉默；7份小麦品种共享一条avenin-like亚基，并与GenBank中来自普通小麦的HM027636完全一致，暂将该亚基命名为“Avenin-likeⅠ”；系统演化分析表明avenin-like编码蛋白与LMW-GS关系最近，与ω-gliadin的亲缘关系最远；SDS-PAGE检测融合蛋白成功表达，对纯化蛋白进行微量掺粉试验，发现Avenin-likeⅠ型蛋白亚基对小麦加工品质具有显著的正向效应。【结论】avenin-like在供试小麦品种中具有很低的多态性，且Avenin-likeⅠ型亚基能提高面粉加工品质。

关键词: 小麦, avenin-like蛋白, 原核表达, 品质效应

Abstract: 【Objective】The present study aimed at cloning, prokaryotic expression and functional analysis of avenin-like genes from common wheat.【Method】The avenin-like genes were cloned by gene specific primers, which designed according to conserved domains of published genes, followed by sequencing and bioinformatic anayzing. Hereafter, JN542444 was cloned into the expression vector, and transformed into the host bacteria Escherichia coli Rosetta-gami B(DE3). After induced by IPTG, the fusion protein was purified by His-Trap affinity chromatography, and then integrated into the control flour to determine the quality effect utilization of 10 g Farinograph. 【Result】 Twenty-two novel avenin-like genes (GenBank accession No. were from JN542443 to JN542464) with 855 bp were isolated by gene-specific primers from 12 wheat cultivars, and all of them encoded 284 amino acid residues. The number and the positions of Cys residues were strictly conservative in 21 genes, which possibly could form 7 intra- and 4 inter-disulfide bonds, except JN542452 with 17 Cys residues. Three pseudogenes, including JN542448, JN542455 and JN542456, were resulted from a single base substitution. An unique type of the avenin-like gene, which was identical to 7 different cultivars in this study and event the other cultivars in GenBank, was named “avenin-like I”. Phylogenetic and evolutionary analysis revealed that avenin-like subunits were closely related to LMW-GS and least homology with ω-gliadin in wheat storage proteins. SDS-PAGE resuslts showed that the gene was successfully expressed in E. coli system. The farinograph data of purified protein revealed that avenin-like I has a significant positive effect on wheat dough quality.【Conclusion】Although lack of polymorphism in the tested cultivars, the Avenin-like I subunit could improve wheat flour processing quality.

Key words: Triticum aestivum L., avenin-like protein, prokaryotic expression, quality effect

魏慧, 董剑, 陈其皎, 高翔, 王蕾, 李晓燕, 赵万春, 吴丹. 小麦avenin-like的克隆、原核表达与品质效应研究[J]. 中国农业科学, 2012, 45(4): 607-616.

WEI Hui, DONG Jian, CHEN Qi-Jiao, GAO Xiang, WANG Lei, LI Xiao-Yan, ZHAO Wan-Chun, WU Dan. Cloning, Prokaryotic Expression and Quality Effective Analysis of avenin-like Genes from Wheat Cultivars[J]. Scientia Agricultura Sinica, 2012, 45(4): 607-616.

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