钙离子通道蛋白在辣椒疫霉菌生长发育及致病过程中的作用机制

The calcium channel genes are involved in calcium absorption, and calcium-mediated signal transduction leads to transient increases in cytosolic free calcium and the simultaneous occurrence of calcium influx along with the calcium efflux during infection. The calcium channel genes also have roles in the mycelium growth, sexual reproduction, turgor generation, cell wall synthesis and toxicity of filamentous fungi, but the role of calcium channel genes remains unclear in the development and pathogenicity of Phytophthora capsici. In our previous works, the Cch1 and Mid1 coding calcium channel had been identified from Phytophthora capsici through genome sequence analysis. Both genes contain calcium binding domain and show high similarity with their homologies from filamentous fungi. In order to elucidate the function of calcium channel, Cch1 and Mid1 are silenced and over-expressed in wild type P. capsici to exam the biochemical metabolism, morphogenesis and pathogenesis in host plants. The Real-time PCR is performed to monitor the expression patterns of Cch1 and Mid1 during infection. The localization of Cch1 and Mid1 in cell is additionally studied by expressing the proteins fused with GFP. To confirm the forming of calcium channel, interaction between Cch1 and Mid1 are studied by Yeast two-hybridization, pull-down and BiFC experiments. These results will contribute to the findings of novel targets of fungicide, providing new disease control strategies for vegetable production.

钙离子通道蛋白是信号刺激时病原菌胞质游离钙离子浓度发生短暂而复杂的变化所必须的，广泛参与丝状真菌的菌丝生长、有性生殖、膨压产生、细胞壁合成及毒性改变等多种生命过程，但钙离子通道蛋白在辣椒疫霉菌等卵菌生长发育和致病过程中的作用机制尚未见报道。前期研究发现，辣椒疫霉菌中存在编码钙离子通道蛋白Cch1和Mid1的同源基因，本项目拟对其在生长发育和致病过程中的作用机制进行研究。利用疫霉稳定转化获得基因沉默及过表达转化子，检测其在生化代谢、细胞结构、形态发育及致病性等方面的表型变化；运用Real-time PCR和GFP融合蛋白表达，研究基因表达的时序性和蛋白定位；借助酵母双杂交、pull-down和双分子荧光互补技术验证Cch1和Mid1蛋白之间的互作，从而明确钙离子通道蛋白在辣椒疫霉菌生长发育和致病过程中的作用机制。研究结果将为设计新型抗疫病杀菌剂，提出新的植物病害控制策略提供理论基础。

辣椒疫霉菌（Phytophthora capsici Leonian），属茸鞭生物界(Stramenopilia)卵菌门(Oomycota)疫霉属(Phytophthora)，寄主范围很广，能引起22个科500多种作物病害，每年在生产上也造成重大经济损失。已有研究表明，在模式生物酿酒酵母（Saccharomyces cerevisiae），粗糙脉孢菌（Neurospora crassa）中钙离子通道蛋白Mid1和Cch1在细胞的形态建成、营养生长、细胞壁合成以及致病性方面有着重要的作用，但是，在辣椒疫霉菌中Mid1和Cch1的功能还不清楚。为了分析辣椒疫霉菌钙离子通道蛋白在生长发育和致病过程中的功能，我们采用基因沉默的方法， 将Mid1和Cch1编码框序列正向和反向插入的转化单元转化辣椒疫霉菌（LT1534），利用辣椒疫霉稳定转化系统，获得了该基因的沉默突变体。研究结果表明，Mid1和Cch1沉默转化子的生长速率与野生型相比，均下降了约60%；沉默Mid1和Cch1后严重影响了游动孢子的产生，并且在菌丝和孢子囊形态等方面与野生型菌株有着显著差异。接种结果表明Mid1和Cch1沉默突变体降低了对辣椒和烟草叶片的致病性。Mid1和Cch1沉默突变体在菌丝生长，孢子囊形态等方面的表型十分相似，然而在游动孢子的产生以及对辣椒和烟草叶片的致病性方面，Mid1沉默突变体表现出更少的游动孢子产生和更弱的致病能力。部分回补发现钙离子能回补辣椒疫霉菌Mid1和Cch1突变体的生长表型，进一步的功能回补明确了辣椒疫霉菌Mid1和Cch1能回补酵母突变体的功能。除此之外，Mid1和Cch1可能参与外源H2O2胁迫的应答。以上结果说明辣椒疫霉菌钙离子通道蛋白Mid1和Cch1调控辣椒疫霉菌的生长发育和致病性。