【作者】 谌江华；

【导师】 郑重； 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2006， 硕士

【摘要】 蛋白激酶在植物生长、激素调节和生物与非生物逆境胁迫反应中起着重要的作用。实验室的前期研究中,我们已克隆到两个水稻抗病相关的MAPK蛋白激酶。为进一步研究抗病反应中的蛋白激酶,我们克隆鉴定了水稻中一个新的丝/苏氨酸蛋白激酶基因,OsBIWNK1。OsBIWNK1基因的cDNA全长2576bp,开放阅读框(ORF)2115bp,编码一个含704个氨基酸的蛋白,预期分子量79.8kD,等电点5.50。OsBIWNK1位于水稻7号染色体上,含有7外显子和6个内含子。OsBIWNK1蛋白N-端含有一个由11个丝/苏氨酸蛋白激酶亚结构域组成的蛋白激酶催化结构域,后面跟着一个大约60个氨基酸的PWNK模序。其中Ⅱ亚结构域中保守的赖氨酸(K)与拟南芥AtWNK1一样被天冬酰氨(N)所代替。OsBIWNK1蛋白氨基酸序列与拟南芥中的WNK(with no lysine(K)kinase)激酶组成员具有很高的相似性,因此,我们将该基因命名为OsBIWNK1(Oryza sativa L.BTH induced WNK1)。 为了解OsBIWNK1基因在抗病反应中的功能,我们将水稻OsBIWNK1基因的编码区插入到花椰菜花叶病毒CaMV-35S启动子下游,与载体系统CHF3构建水稻OsBIWNK1基因的植物转化双元表达载体。利用根癌农杆菌(Agrobactrium tumifaciens)介导的叶盘法转化烟草无毒苗,经卡那霉素(Kan~+)抗性筛选获得10株转OsBIWNK1基因T_o代烟草植株。通过提取转基因烟草基因组DNA,然后进行PCR检测,结果表明OsBIWNK1基因都整合到了烟草基因组中。同时我们也发现,转基因烟草植株的顶芽、腋芽、叶表现异常,开花期推迟。因此,我们认为OsBIWNK1蛋白还可能植物生长发育过程中起一定作用。更多还原

【Abstract】 Protein kinases play important roles in plant growth and development, hormone response as well as in response to abiotic and biotic stress. Previously, we have found that two rice mitogen-activated protein kinases are involved in rice disease resistance responses, to further study putative protein kinses in disease resistance response, we cloned and characterized a noval rice serine/threonine protein kinase gene, OsBIWNK1. The full-length cDNA of OsBIWNK1 is 2576bp with a predicted 2115bp open reading frame. OsBIWNK1 encodes a protein of 704 amino acids with a calculated molecular weight of 79.8KD and the isolectic point of 5.50. OsBIWNK1 gene is located on chromosome 7 of rice genome and consists of 7 exons and 6 introns. The deduced OsBIWNK1 protein contains a ser/thr protein kinase catalytic domain consisting of 11 conserved ser/thr protein kinase subdomains in the N-terminal, followed by a PWNK motif of about 60 amino acid, which was found in plant WNKs only. The conserved lysine (K) of subdomain II was replaced by the asparagines (N) as well as in Arabidopsis AtWNK1 protein. The amino acid sequence of OsBIWNK1 showed high levels of similarly to a number of protein kinases that belong to the WNK (with no lysine (K)) kinase group, and thus was named as OsBIWNK1 (Oryza sativa L. BTH indeced WNK1).To understand the function of OsBIWNK1 in disease resistance response, we performed a functional analysis of OsBIWNK1 in transgenic tobacco plants. The OsBIWNK1 coding region was cloned into a plant transformation binary vector pCHF3 under the control of the CaMV35s promoter and transformed tobacco leaf discs via agrobacterium-mediated feaf disc transformation. Ten kanamycin-resistant OsBIWNK1 transgenic tobacco lines were obtained and confirmed by PCR detection. Some transgenic lines overexpressing OsBIWNK1 showed morphological abnormalities in top-buds, axillary-shoot, leaf and delayed flowering, suggesting arole for OsBIWNKl in development process.更多还原