簇毛麦金属转运蛋白基因Hv-FP3克隆及其功能初步分析
首发时间:2013-01-31
摘要:白粉病是小麦重要的病害之一,小麦近缘物种簇毛麦表现高抗白粉病,携带抗白粉病基因Pm21。本实验室在前期研究中利用基因芯片杂交结合cDNA文库筛选在簇毛麦中克隆了一个编码E3连接酶的基因Hv-CMPG,证明该基因在抗白粉病中发挥重要的正调控作用。为了进一步阐明簇毛麦抗白粉病的分子机制,本研究在构建簇毛麦酵母双杂交文库的基础上,通过筛选发现一个与Hv-CMPG互作的金属转运蛋白基因,并在簇毛麦中克隆获得该基因全长,命名为Hv-FP3(已申请专利,专利申请号:201210574738X)。实时定量RT-PCR结果表明,该基因在白粉菌诱导的簇毛麦叶片中上调表达,并且其表达受ET诱导上调;亚细胞定位结果显示,该基因定位于细胞核和细胞膜上;瞬间表达结果表明,在扬麦158叶片中超量表达Hv-FP3后,白粉菌的吸器指数明显下降;转基因结果表明,在中感小麦白粉病的小麦品种扬麦158中,过量表达Hv-FP3的转基因植株高抗小麦白粉病。综合上述结果,Hv-FP3基因参与簇毛麦的白粉病反应,并发挥重要作用。
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Cloning and preliminary functional analysis of a metal transporter gene Hv-FP3 from Haynaldia villosa L.
Abstract:Powdery mildew is one of the most serious diseases threaten wheat production. Haynaldia villosa L., a wheat relative species, shows high powdery mildew resistance and confers the resistance gene Pm21. In our previous study, by using the Barley GeneChip microarray and cDNA library screening, a Hv CMPG gene encoding an E3 ligase has been cloned and was found to play an important role in the defense response to the powdery mildew disease. For furthee elucidating the molecular mechanism, a yeast two-hybrid library of H. villosa was constructed and used to screen the interact genes of Hv CMPG. A metal transporter gene was found to be interact with the Hv CMPG, and the full length was cloned from H. villosa and was designated as Hv-FP3 (Patent application number: 201210574738X). Analysis by quantitative RT-PCR showed that Hv-FP3 was rapidly up-regulated in the leaf of H. villosa after Blumeria graminis f.sp.ritici (Bgt) inoculation. The subcellular localization of the Hv-FP3 was in the nuclei and plasma membrane of the epidermal cell of onion. In the transient overexpression assay, it was found that the transcient over-expression of Hv-FP3 led to significantly decreased of the haustoria index (HI) compared with the vector conctrol in the epidermal cell of wheat variety Yangmai158, which is moderate susceptible to powdery mildew disease. The transgenic plants with the Hv-FP3 over-expressed in Yangmai158 was obtained and the positive transgenic plants showed improved powdery mildew resistance than the receptor. These results indicated that the Hv-FP3 participated in the powdery mildew defense responses.
Keywords: wheat powdery mildew yeast two hybrid metal transporter
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