【作者】 王敏；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2012， 硕士

【摘要】 晚疫病是马铃薯生产中最具毁灭性的病害。晚疫病抗性分为垂直抗性和水平抗性,垂直抗性由主效基因控制,具有小种转化性,抗性常因晚疫病菌生理小种变异而迅速丧失。水平抗性是由微效多基因控制的,对马铃薯晚疫病具有广谱性,稳定性的特点。因此,选育广谱,稳定的水平抗性品种已成为马铃薯晚疫病抗病育种的重要目标。本实验窒前期从马铃薯晚疫病水平抗性相关的ESTS中克隆出一个可以调控马铃薯晚疫病抗性的胞质类受体激酶基因StPK1。植物激酶是一类重要的蛋白激酶,它们在抗病性,抗逆和生长发育等方面都具有重要作用。前期研究表明,StPK1基因可被马铃薯晚疫病诱导表达,但其功能有待进一步试验证实。同时,StPK1蛋白是否具有激酶活性亦为探讨该基因的作用机理所必须。本课题通过基因StPK1转基因株系的抗病性鉴定、蛋白激酶原核表达及激酶活性测定等研究,探讨基因stPK1在抗晚疫病中的作用机理。主要结果如下：1.利用Realtime-PCR筛选己获得的StPK1超量转基因株系和干涉转基因株系,其中StPK1干涉株系干涉效率最高为70%,超量表达株系表达量最高为对照E3的600倍。后续晚疫病抗性鉴定选用了以上5株超量表达和4株干涉表达效果好的转基因株系进行晚疫病接种实验。统计病斑面积结果,超量表达株系病斑扩展速率显著小于对照(E3)病斑扩展速率,干涉株系的病斑扩展速率显著大于对照(E3)病斑扩展速率,表明StPK1基因参与了马铃薯晚疫病抗病性反应。2.构建了StPKl的绿色荧光表达载体GFP-StPK1,转化大肠杆菌,抽提质粒后,用基因枪轰击法转化洋葱表皮细胞,暗培养12小时后,激光共聚焦显微镜观察。发现,GFP-StPK1绿色荧光主要出现在细胞质中,说明蛋白激酶StPK1是在马铃薯细胞质内行使功能。3.构建了StPK1基因的原核表达载体,转化大肠杆菌,用IPTG诱导融合蛋白体外表达。GST柱式透析纯化蛋白质,用同位素标记的γ-32P-ATP测定蛋白激酶的磷酸化活性。结果表明,蛋白激酶StPK1与ATP发生反应,有较强的自磷酸化活性,是一个有活性的蛋白激酶。蛋白激酶stPK1磷酸化活性的证实,为寻找其磷酸化互做底物及深入探讨其抗病性的作用机理奠定基础。更多还原

【Abstract】 Late blight, the most devastating disease of potato production, is divided into vertical resistance and horizontal resistance. The former is controlled by dominant resistance (R) genes and have race specialty, which can be easily overcomed by the rapid evolution of new virulent races of P. infestans, and the later is controlled by many interacting genes and expected to be more durable and reliable. Therefore, selecting the horizontal resistance variety against late blight has become the pivotal aim on potato breeding. StPK1, a cytoplasmic receptor-like kinase, was cloned from the horizontal resistance variety of potato aganist late blight by the previous work in our lab. It may have some important function in the singal transduction of late blight resistance. Plant protein kinase is one of the most important kinases which play a very important role in disease resistance and stress tolerance as well as plant growth. The preliminary experiments show that, the StPKl can be induced by P. infestans, but the function remains to be identified with further experiments. At the same time, to ascertain the activity of StPK1is required to further understand the mechanism of the gene. Late blight resistance of the transgenic plants and the expression of the fusion protein in E.coil as well as the protein activity were further analysised in this study. The main results are as follows:1. Over expression and RNAi transgenic plants were slected by Realtime-PCR.It showed that the highest silencing efficiency of the RNAi transgenic plants reached to70%, and the higest expression level of StPKl of overexpression lines was600times than that of control.5overexpressed and4interference lines which were slected from the transgenic lines were subjected to late blight resistance assessment. The results demonstrated that lesion expansion rate of the overexpression plants were significantly lower than that of control (E3), and it was significantly greater in the RNAi transgenic plants. It showed that StPKl involved in the potato late blight resistance.2. GFP-StPKl vectors was constructed and transferred into E. coli. The fusion vector was transfomed into epidermal cells of onions by the particle bombardment method and cultivated in the dark for12h. Laser confocal microscopy displayed that GFP-StPK1mainly located in the plasma,Which implicated that StPKl exercised its function in the intracellular space.3. Bacterial expression vector of pGEX-6p-1-StPK1was constructed and the fusion protein can be induced in Rosetta by IPTG. The fusion protein was purified by GST dialysis pillar in vitro and the protein activty was identified by γ-32P-ATP which was marked by isotope labeling. The results showed that the purified fusion protein can ineteract with ATP and it can autophorylate. So StPK1is a protein kinase. It has lay the foundation to find the substrate of StPK1for phosphorylation and further analysis the resistance mechanism of the gene.更多还原