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为了获得优良品种陕253有关ω-醇溶蛋白基因的相关信息,根据已报道的ω-醇溶蛋白基因序列设计特异引物,采用PCR方法对小麦(Triticum aestivumL.)品种陕253总DNA进行扩增,回收1000bp左右的片段,经纯化、克隆并测序,获得5个不同的ω-醇溶蛋白新基因,命名为Gli-S253-1、Gli-S253-2、Gli-S253-3、Gli-S253-4和Gli-S253-5,GenBank登录号分别为:GQ423428、GQ423429、GQ423430、GQ423431和GQ423432。序列分析表明:(1)ω-醇溶蛋白基因沉默主要有4种类型(Q→*、I→*、R→*和W→*),并以第一种类型为主(占66.6%);(2)从三联体密码角度看,谷氨酰胺(Q)的两种编码方式最容易发生沉默突变(CAA→TAA和CAG→TAG);(3)从突变位点来看,沉默主要发生于三联体密码的第一位碱基(占77.8%),第二位碱基发生突变的频率占33.3%,但未见第三位沉默突变;GQ423428出现了罕见的ATA→TAA类沉默,同时涉及两个位点的变异;(4)就基因沉默突变的数目而言,GQ423428和GQ423429各高达6次,其中4个位点(349、373、478和592)为上述5条记录所共有;(5)从编码成熟蛋白的结构上看,沉默突变主要集中于ω-醇溶蛋白重复区,信号肽区及N-端区未发现此类变异;(6)进化分析表明,小麦族的ω-gliadin/secalin分成3支,其中小麦来源的ω-gliadin聚成一支。上述结果表明,本研究从陕253成功分离到5条存在丰富多样性的ω-醇溶蛋白编码基因,为深入研究优质的来源及分子辅助育种提供参考。
In order to obtain the related information about the ω-gliadin gene of Shan 253, a specific primer was designed according to the reported ω-gliadin gene sequence, and PCR was used to amplify the total DNA of Triticum aestivum L. strain Shan 253 , And a fragment of about 1000bp was recovered. After purification, cloning and sequencing, five new ω-gliadin genes were obtained and named Gli-S253-1, Gli-S253-2, Gli-S253-3 and Gli-S253 -4 and Gli-S253-5, GenBank accession numbers are: GQ423428, GQ423429, GQ423430, GQ423431 and GQ423432, respectively. Sequence analysis showed that: (1) there are four main types of ω-gliadin gene silencing (Q → *, I → *, R → * and W → *), with the first type being dominant (66.6% ; (2) Silencing mutations (CAA → TAA and CAG → TAG) are most likely to occur in the two coding forms of glutamine (Q) from the perspective of triplet codon usage; (3) Silence mainly occurs from the point of mutation The first base of the triplet codon (77.8%), the frequency of the second base mutation accounted for 33.3%, but no third silent mutation; GQ423428 rare ATA → TAA class silence, at the same time (4) For the number of gene silencing mutations, GQ423428 and GQ423429 are each up to 6 times, of which 4 sites (349, 373, 478 and 592) are common to the above 5 records; (5) From the point of view of the structure of the encoded mature protein, the silent mutation mainly focused on the ω-gliadin repeat region, and no such mutation was observed in the signal peptide region and the N-terminal region. (6) The evolutionary analysis showed that ω -gliadin / secalin is divided into three branches, of which omega-gliadin comes from wheat. The above results show that in this study, five rich and diverse ω-gliadin genes were successfully isolated from Shaanxi 253, providing a reference for further research on high-quality sources and molecular-assisted breeding.