节节麦pbf基因的克隆、序列分析及原核表达

醇溶蛋白盒结合因子(prolamin-box binding factor,PBF)通过调控籽粒蛋白的表达效率进而影响面粉的加工品质。为给深入研究小麦籽粒PBF对籽粒蛋白质表达调控的分子基础提供参考依据,进而为小麦面粉加工品质的改良提供候选基因资源,利用特异引物组合pbfF1/pbf R1分别从两份节节麦(AS90、AS2386)中克隆出pbf基因后进行序列分析,并进一步构建pbf基因的原核表达体系。结果表明,从两份节节麦中克隆得到了2个不同类型的pbf基因(GenBank登录号分别为KJ544771和KJ544772),其中来源于AS2386的KJ544772与来源于普通六倍体小麦的1个pbf基因序列完全相同。推导的氨基酸序列分析表明,KJ544771和KJ544772所编码的蛋白质均为弱碱性的亲水蛋白,具有典型的DOF蛋白结构域。与其他远缘物种来源的PBF比对结果表明,该类蛋白在NLS核心、DOF结构域及Ser铰链区相对保守,而在C-端调控区变异较大,说明PBF蛋白具有种属特异性。同时,系统演化树显示,来源于节节麦AS2386的pbf基因与迄今已知的全部普通小麦的pbf基因具有高度的相似性,因此推测该种类型的节节麦很可能参与了最初的普通六倍体小麦的形成。此外,本研究成功构建了pbf基因的原核表达体系,可为后续功能验证的开展奠定基础。

Cloning, Sequence Analysis and Prokaryotic Expression of pbf genes from Aegilops tauschii

PBF (prolamin box binding factor), as a trans acting factor, is involved in seed storage protein expression and thus determined the processing quality of wheat flour. To provide a reference for studying the regulation characteristics of wheat PBF and further enhanced the gene resources for wheat quality improvement, two pbf genes were isolated from Ae.tauschii lines of AS90 and AS2386 with the primer pbf F1/pbf R1 and then a prokaryotic expression system was constructed. Nucleic acid sequence analysis showed that two pbf genes (GenBank accession numbers: KJ544771 and KJ544772) belonged to two different kinds and KJ544772 was identical to the pbf gene from Chinese Spring. Deduced amino acid sequence analysis revealed that PBFs encoded by pbf genes in this study were slightly alkaline and hydrophilic. And the PBFs had typical domains of DOF protein. While regulatory region of PBF C terminal showed significant differences, with NLS core, DOF domain and Ser hinge were relatively conserved, through alignment among PBFs from different species. This result indicated PBFs had the species specificity as was showed in phylogenetic analysis. Further, phylogenetic analysis showed pbf gene from AS2386 had high similarity with all known pbf genes from common hexaploid wheat so far, which indicated Ae.tauschii like AS2386 was probably involved in the original formation of common hexaploid wheat. In addition, to lay foundation on further studies including functional validation, prokaryotic expression system of pbf gene was constructed successfully.