簇毛麦中一个E3泛素连接酶基因Hv-CMPG的克隆及功能鉴定
首发时间:2013-01-31
摘要:小麦近缘物种簇毛麦高抗小麦白粉病,携带广谱高抗白粉病基因Pm21, 为了克隆簇毛麦中的抗白粉病关键基因,阐明其白粉病抗性的分子机制,本实验室在前期利用大麦基因芯片分析簇毛麦受白粉菌诱导表达谱,发现多个基因受白粉菌诱导后上调或下调表达。本研究发现其中一个探针受白粉菌诱导后快速上调表达,半定量 RT-PCR 进一步证明该表达特征。选择该基因根据其探针序列设计引物筛选簇毛麦受白粉病诱导的叶片cDNA文库,克隆到其全长序列。序列分析结果表明,该基因全长1677bp, ORF1362bp,编码454个氨基酸、具有保守的U-Box结构域的蛋白质,定名为Hv-CMPG。染色体定位分析表明,该基因定位于簇毛麦6V染色体长臂上,其同源基因定位于大麦6HL、小麦6AL、6BL、6DL、短柄草Bd3L和水稻R2染色体的共线区域;体外泛素化试验结果证明,Hv-CMPG具有E3泛素连接酶活性,推测其通过泛素化介导的蛋白质降解途径在植物抗病信号传导中发挥作用;亚细胞定位结果表明Hv-CMPG定位在细胞核和细胞质内;构建了Hv-CMPG基因的超量表达载体,利用基因枪方法进行转基因功能互补试验,结果发现该基因的超量表达显著提高了转基因受体品种扬麦158的白粉病抗性。表明Hv-CMPG在簇毛麦抗白粉病中发挥重要作用,其作用的分子机制有待进一步研究。
关键词: 簇毛麦 白粉病 Hv-CMPG E3泛素连接酶 转基因小麦
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Cloning and functional analysis of a E3 ubiquitin ligase gene Hv-CMPG in Haynaldia villosa
Abstract:Haynaldia villosa, a wheat related species, shows high and broad-spectrum resistance to wheat powdery mildew. The powdery mildew resistance gene Pm21 from H. villosa has been introduced into common wheat by the production of wheat-alien chromosome lines, and the T6VS/6AL translocation are now widely used in wheat breeding. For better understanding of the molecular mechanism of powdery mildew resistance mediated by Pm21, and cloning of the critical genes related to powdery mildew resistance, barley GeneChip was used for microarray analysis. Among the differentially expressed genes upon the infection of Blumeria graminis f.s.p. tritici (Bgt) in H. villosa, a rapidly up-regulated gene induced by Bgt was selected for further analysis. Semi RT-PCR showed that this gene was rapidly up-regulated by Bgt specifically in the leaves of H. villosa. The full cDNA sequence of this gene was obtained by screening the cDNA library constructed using cDNA from leaves of H. villosa inoculated with Bgt. Its full length was 1,677bp, with an ORF of 1362bp encoding a protein of 454 aa. The protein has a conserved U-Box domain, showed high similarity with the CMPG gene from Parsley and other species, and hence was designated as Hv-CMPG. Hv-CMPG was mapped to the long arm of chromosome 6V, and the expressed Hv-CMPG was localized both in the nucleus and cytoplasmic. In vitro ubiquitination activity assay proved that the Hv-CMPG had the E3 ligase activity in vitro. The Hv-CMPG gene was transformed into a wheat variety Yangmai158, which is moderate susceptible to powdery mildew, and it was found that the over-expression of Hv-CMPG led to significantly improvement of the powdery mildew resistance, indicating that Hv-CMPG plays an important role in powdery mildew resistance of H. villosa.
Keywords: Haynaldia villosa Powdery mildew resistance Hv-CMPG E3 ligase transgenic wheat
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