小麦新型Avenin-like type b基因克隆、功能预测及品质相关分析

Cloning,Functional Prediction and Quality-Related Analysis of New Avenin-like Type b Genes in Wheat

为进一步明确Avenin-like type b基因的功能特性,以小麦品种西农188为材料,通过设计简并引物克隆小麦Avenin-like type b基因,对克隆得到的序列进行同源性比对,分析编码蛋白的理化性质、结构特点,结合系统发育树、亚细胞定位预测及结构域分析探讨其可能具有的功能特性,同时对该基因进行体外原核表达及品质功能相关分析。结果表明,从西农188克隆得到4条序列,Genbank登录号为KY794919~KY794922,其中KY794922在信号肽区段碱基缺失导致移码突变。KY794919~KY794921序列与Aveninlike type b家族中大麦、柱穗山羊草、二穗短柄草及节节麦的同源性较高。蛋白序列分析表明,Avenin-like type b编码氨基酸序列具有较高的保守性,其Cys的数目及位置也较为保守,具有两个典型的AAI结构域,推测其可能为一类具有α-淀粉酶抑制剂活性的蛋白。亚细胞定位显示,该类蛋白可能在合成加工后通过运输小泡转运至细胞外行使功能。SDS-PAGE分析表明,KY794919~KY794921体外原核表达成功。通过纯化表达产物及掺粉试验表明,KY794919~KY794921编码蛋白对小麦面粉品质均具有显著正向效应,但仅有KY794919可显著提高面粉吸水量进而延长面筋扩展时间,说明其可能在品质功能研究资源中更具有优越性。

To further clarify the functional properties of Avenin-like type b gene, this experiment took the wheat cultivar Xinong 188 as the material. The Avenin-like type b genes were cloned from the material with degenerate primers. The possible functional properties of the genes were discussed by making a homologously comparison, analyzing physicochemical properties and structural characteristics of their encoded protein, predicting subcellular localization, constructing the phylogenetic tree and analyzing the particular domain. In addition, they were expressed in prokaryocyte and their quality related function was analyzed. The result showed that there were four sequences cloned from Xinong 188,with the Genbank accession number KY794919-KY794922,of which, the base deletion in signal peptide region leads to a frameshift mutation of KY794922. The sequences of KY794919-KY794921 have high homology with Avenin-like type b families in Hordeum vulgare, Aegilops cylindrica, Brachypodium distachyon and Aegilops tauschii.Protein sequence analysis showed that the amino acid sequences encoded by Avenin-like type b genes,as well as the number and position of the cysteine,are more conserative.The Avenin-like type b protein has two typical AAI structural domains, and it may be for a class of protein with alpha amylase inhibitor activity. Subcellular localization showed that the protein may be transported to the extracellular by transport vesicles after processing for functional implementation. SDS-PAGE analysis showed that the prokaryotic expression in vitro of KY794919-KY794921 is successful. The results of the expression products purification and supple mentation experiment showed that the proteins encoded by KY794919-KY794921 have significant positive effects on wheat flour quality, however, only the protein encoded by KY794919 can significantly increase water absorption of flour and prolong extension time of gluten, which indicating that KY794919 might be more advantageous in quality and function research resources.