西尔斯山羊草(Aegilops searsii)α-醇溶蛋白编码基因的克隆及原核表达  

Cloning and Prokaryotic Expression of α-Gliadin Genes from Aegilops searsii

作　　者：刘国娟[1] 马信[1] 尹华燕 孙鑫[1] 杜旭烨[1] 王宏伟[1] 李安飞[1] 陈呈涛 孔令让[1] 

机构地区：[1]山东农业大学农学院/作物生物学国家重点实验室,山东泰安271018 [2]山东省新泰市天宝镇农业技术推广站,山东新泰271200

出　　处：《山东农业大学学报（自然科学版）》2015年第3期321-325,共5页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：国家转基因专项"优质转基因小麦新品种培育(2011ZX08002004-003)

摘　　要：醇溶蛋白是小麦加工品质的重要影响因素,主要决定面团的粘性和延展性。根据酸性条件下迁移率的不同,可将醇溶蛋白分为α-,β-,γ-,ω-醇溶蛋白。α-醇溶蛋白占贮藏蛋白的15%～30%,是含量最丰富的一类贮藏蛋白,同时,α-醇溶蛋白中含有一些致敏性的毒性多肽。克隆西尔斯山羊草α-醇溶蛋白基因,并进行序列分析,通过构建原核表达载体,使其在大肠杆菌中诱导表达融合蛋白,并通过切胶纯化法,获得纯度较高的目的蛋白。根据α-醇溶蛋白基因编码区保守序列设计引物,通过PCR扩增克隆得到α-醇溶蛋白基因并进行序列分析。将目的基因KC421089连到表达载体p EASY-E1上,在大肠杆菌BL21（DE3）中经IPTG诱导表达,获得表达的融合蛋白。通过切胶纯化法,获得纯度较高的目的蛋白。从小麦近缘植物西尔斯山羊草（Aegilops searsii）中首次克隆了7个α-醇溶蛋白编码基因,它们的编码区长度分布在849～954 bp之间,可编码282～317个氨基酸。通过与已发表的其它物种的α-醇溶蛋白氨基酸序列进行多重比对分析,发现这些基因都具有α-醇溶蛋白编码基因典型的结构特点,同时还存在着碱基的缺失、插入以及SNPs。在克隆目的基因的基础上,本研究还通过大肠杆菌体外表达和切胶纯化方法,获得了纯度较高的目的蛋白,实现了该基因的表达。克隆了7个α-醇溶蛋白基因序列,登录号为KC421089的基因可在原核系统中成功表达,并通过切胶纯化法获得目的蛋白,为进一步利用西尔斯山羊草改良小麦加工品质奠定了基础。Gliadins are the major influence factors of wheat processing quality, which determine dough extensibility. On the basis of the electrophoretic mobility by acidic polyacrylamide gel electrophoresis, gliadin can be separated into α-,β-,γ- andω-gliadin. Among them, α-gliadins are the most abundant and accounted for 15%~30% of the wheat storage proteins. On the other hand, α-gliadins can cause different diseases. The present study aimed at cloning and analyzing the α-gliadin genes from Aegilops searsii, expressing it in E.coli and obtaining high purified proteins. The α-gliadin genes were amplified by PCR and then inserted the gene KC421089 into p EASY-E1. The recombinant plasmids were expressed in the E.coli BL21（DE3） and then the purified proteins were obtained by cutting the gel slices. Seven novel α-gliadin genes were cloned from Aegilops searsii. Their length of the open reading frames ranged from 849-954 bp, encoding the putative proteins with282-317 amino acid residues, respectively. A BLAST search showed that these sequences have the typical structure ofα-gliadin genes and SNPs and In/Dels. Moreover, the target proteins were expressed by E. coli and highly purified proteins were obtained by cutting the gel slices. Seven novel α-gliadin genes were cloned from Aegilops searsii, and the gene KC421089 were expressed by E. coli and highly purified proteins were obtained by cutting the gel slices. This study laid a good foundation for wheat quality improvement.

关 键 词：α-醇溶蛋白 西尔斯山羊草 序列分析 原核表达 

分 类 号：S5[农业科学—作物学]