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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。
Gliadin is an important factor in the processing quality of wheat and mainly determines the stickiness and ductility of the dough. According to the different mobility under acidic conditions, the prolamin can be divided into α-, β-, γ-, ω-gliadin. α-gliadin is 15% -30% of the storage protein, which is the most abundant type of storage protein. At the same time, α-gliadin contains some allergenic toxic peptides. Cloning Seq sheep α-gliadin gene and sequence analysis, through the construction of prokaryotic expression vector, make it expressed in E. coli fusion protein, and through the gel purification method to obtain the desired protein of higher purity. Primers were designed based on the conserved sequence of the coding region of α-gliadin gene. The α-gliadin gene was amplified by PCR and sequenced. The target gene KC421089 was linked to the expression vector pEASY-E1 and induced by IPTG in E. coli BL21 (DE3) to obtain the expressed fusion protein. Through cut gel purification method, to obtain a higher purity of the target protein. Seven α-gliadin genes were cloned from Aegilops searsii. Their coding regions were between 849 and 954 bp in length and encoded 282 to 317 amino acids. Multiple alignment analysis of the α-gliadin amino acid sequences of other published species revealed that these genes have the typical structural characteristics of α-gliadin genes, as well as the absence of bases, insertion and SNPs . Based on the cloned target gene, we obtained the target protein of high purity through the E. coli expression in vitro and gel-purified, and expressed the gene. Seven α-gliadin gene sequences were cloned, and the gene with accession number KC421089 was successfully expressed in prokaryotic system. The target protein was obtained by gel excision, which laid the foundation for the further utilization of Seeds goat grass to improve wheat processing quality basis.