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为进一步了解小麦Dof(DNA binding with one figner)转录因子在小麦种子发育过程中的作用和功能,本文利用RTPCR技术,克隆到一个小麦PBF蛋白的等位基因cDNA序列(Gen Bank登录号为KX058135),命名为TaPBFB。该序列包含完整的开放阅读框(ORF)987 bp,编码了328个氨基酸的蛋白质,理论等电点8.75,理论分子量为33.9 kDa,这与原核表达后SDS-PAGE和Werstern blot检测结果一致。蛋白质序列分析表明,该基因编码的氨基酸序列与粗山羊草(Aegilops tauschii,AHZ44510)Ata PBF蛋白相似性最高为92%,而与已知的‘中国春’小麦(Triticum aestivum,AGR34055)WPBF蛋白相比同源性为91.5%。保守结构域分析表明,该蛋白中具有典型的Dof结构域,以及NLS核定位信号,推测该蛋白定位于细胞核并具有调控储藏蛋白基因表达的功能。凝胶阻滞实验结果也表明,融合蛋白能与小麦低分子量麦谷蛋白GluD-LMWGS基因上游的顺式元件Prolamin box(PB)特异结合,证明TaPBFB具有DNA结合活性。这为深入研究多个等位基因的小麦PBF转录因子如何共同调控储藏蛋白的表达机制提供研究材料,为进一步在转录调控水平实现农作物的遗传改良奠定基础。
In order to further understand the function and function of Df (DNA binding with one figner) in wheat seed development, we cloned the allelic cDNA sequence of a wheat PBF protein (GenBank accession number KX058135) using RTPCR technique. , Named TaPBFB. The sequence contained a complete open reading frame (ORF) of 987 bp encoding a 328 amino acid protein with a theoretical isoelectric point of 8.75 and a theoretical molecular mass of 33.9 kDa, consistent with the results of SDS-PAGE and Werstern blot after prokaryotic expression. Protein sequence analysis showed that the amino acid sequence of this gene was 92% identical to that of Aegilops tauschii (AHZ44510) Ata PBF protein, but similar to the known WPBF protein of Triticum aestivum (AGR34055) The homology is 91.5%. Conserved domain analysis showed that the protein has a typical Dof domain, and NLS nuclear localization signal, suggesting that the protein located in the nucleus and has the function of regulating the gene expression of storage proteins. The results of gel retardation assay also showed that the fusion protein could specifically bind to the cis-element Prolamin box (PB) upstream of GluD-LMWGS gene of wheat low-molecular-weight glutenin, indicating that TaPBFB has DNA-binding activity. This provided a research material for further study on how the wheat PBF transcription factors of multiple alleles co-regulate the expression mechanism of storage proteins, and laid the foundation for further genetic improvement of crops at the level of transcriptional regulation.