小麦Wrab基因家族分离于一种抗寒性小麦品种,该家族基因受冷胁迫或脱落酸诱导,在冷胁迫或脱落酸诱导后有高表达.通过构建转录组数据库,发现Wrab 18基因在小麦抵抗叶锈菌侵染的过程中有明显上调.本试验以小麦品种‘洛夫林10’(简称‘L10’)与叶锈菌生理小种260组成不亲和组合,采用RT-PCR技术克隆Wrab18全长序列;应用生物信息学方法分析该基因的序列特征;通过与GFP蛋白融合进行亚细胞定位;采用实时荧光定量PCR (RT-qPCR)方法检测Wrab 18基因的表达;利用VIGS技术沉默Wrab18基因后,探究Wrab18基因在小麦与叶锈菌互作过程中的作用.结果克隆得到510 bp的Wrab18基因开放阅读框全长,生物信息学分析表明,Wrab18可能不含有跨膜结构,为亲水性蛋白;亚细胞定位显示Wrab18定位于细胞质及细胞核中;Wrab18基因在接菌后8h表达量有明显增高;通过VIGS技术沉默‘L10’中的Wrab18基因,在接种叶锈菌后96h,发现叶锈菌的吸器母细胞数量增多,引起的小麦HR细胞面积增大,表明Wrab18基因参与了小麦抵抗叶锈菌侵染的过程,并在该过程中发挥正调控作用.%The wheat Wrab gene family was isolated from a cold resistant wheat,which was induced by cold stress or abscisic acid,and was expressed in cold stress or abscisic acid.By constructing a transcriptome database,we found that the Wrab18 gene was up-regulated in wheat resistance to Puccinia triticina.The experiment was conducted with an incompatible combination of the wheat variety'Lovrin10'(abbreviated as L10) and the physiological race 260 of P.triticina.The Wrab18 gene was isolated and obtained by RT-PCR method.The sequence characteristics of Wrab18 was analyzed by bioinformatics tools.The subcellular localization of Wrab18 was analyzed through fusing with GFP protein.the expression level of Wrab18 gene was detected by real-time quantitative PCR (RT-qPCR).Using VIGS technology to silence Wrab18 gene,we wanted to explore the role of Wrab18 gene in the interaction between wheat and P.triticina.A full-length Wrab18 of 510 bp was cloned from'L10'.Bioinformatics analysis revealed that Wrab18 probably did not contain transmembrane structures and was hydrophilic proteins.Subcellular localization showed that Wrab18 was localized in cytoplasm and nucleus.The expression of Wrab18 gene was increased significantly at 8h after inoculation.The Wrab18 gene was silenced by VIGS technology.The silencing plants of 'L10'was inoculated with P.triticina race 260,found the number of haustorial mother cells was increased and the HR area of wheat cell was increased also 96 h after inoculation.The result indicated that the Wrab18 gene was involved in the process of wheat resistance to P.triticina,and play a positive regulatory role in this process.
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