辣椒疫霉抗甲霜灵基因的精细定位与克隆

Phytophthora capsici, one of the most destructive oomycetes, can lead to Phytophthora blight to a wide range of hosts, including pepper, eggplant, cucumber, watermelon and pumpkin. Presently, metalaxyl is still a key fungicide for controlling the plant oomycete diseases such as pepper blight that is destructive around world, and now the resistance of the pathogenic oomycete to metalaxyl is becoming more and more serious due to the constantly use of this fungicide. However, the direct researches on the mechanism of metalaxyl resistance development still lack in P. capsici to date. Recently, the metalaxyl resistance mutagenizing, monitoring and inheritance characteristic of P. capsici were performed in our lab and the homozygous strains, resistant or sensitive to metalaxyl, were selected for constructing the F2 progeny population. Based on the studies, the fine mapping of the gene encoding the protein resistant to metalaxyl will be carried out by the combination of SSR and CAPS marker techniques. Then, the cloning of the gene will be performed with the methods of bioinformatics, BAC library selection and Phytophthora transformation techniques together. Our results would give insight of the molecular mechanism in development of metalaxyl-resistance in P. capsici. Meanwhile, they would also provide references to monitoring and controlling of the metalaxyl resistant isolates of P. capsici, as well as studying the metalaxyl resistant mechanism in other oomycetes in Phytophthora.

辣椒疫霉是具有重大危害性的植物病原卵菌，可引致辣椒、茄子、黄瓜、西瓜、南瓜等多种重要作物的疫病。目前甲霜灵是防治由该菌引致的辣椒疫病等植物病害的主要杀菌剂。由于长期大量使用，该菌对甲霜灵抗性日趋严重。但迄今辣椒疫霉对甲霜灵抗性的分子机理尚不清楚，给抗药性治理带来困难。本项目在现有辣椒疫霉对甲霜灵抗性诱变、监测和遗传研究的基础上，以前期研究中获得的纯合的辣椒疫霉甲霜灵抗、感菌株为试验材料，建立F2代群体，采用SSR和CAPS标记技术对辣椒疫霉抗甲霜灵基因进行精细定位，将生物信息学技术、BAC文库筛选及疫霉转化技术相结合，克隆辣椒疫霉抗甲霜灵基因，研究结果可揭示辣椒疫霉抗甲霜灵的分子机制，为辣椒疫霉菌甲霜灵抗药性的监测和治理提供理论依据，同时也为研究其他疫霉菌的抗甲霜灵的分子机理提供重要的参考。

辣椒疫霉引致的辣椒疫病给辣椒生产带来巨大风险。目前，甲霜灵是防治辣椒疫病的主要杀菌剂，由于长期大量使用，抗药性日趋严重。但迄今辣椒疫霉对甲霜灵抗性的分子机理尚不清楚，给抗药性治理带来困难。本研究以SD1亲本敏感菌株为出发菌株，经药剂驯化诱导获得抗性突变菌株SD1-9。通过转录组测序分析和以辣椒疫霉全基因组为参考基因组进行同源比对分析筛选到5个抗甲霜灵候选基因，对其进行克隆和功能验证，主要结果如下：（1）甲霜灵突变菌株SD1-9与亲本菌株SD1的菌落形态基本相似，说明甲霜灵的诱导不会改变其菌落形态。在致病力测定中，亲本菌株SD1在不同植株和叶片、果实上的致病力与突变菌株SD1-9无显著差异。（2）以敏感菌株SD1和抗药性突变菌株SD1-9为试验材料，对其进行转录组测序分析。以已公布的辣椒疫霉全基因组数据库为参考基因组，进行基因表达差异分析，功能预测，GO、KEGG富集分析，筛选到4个表达量显著上调与下调的基因：XLOC_020226（下调）、XLOC_001584（上调）、fgenesh1_pg.C_scaffold_21000144（上调）、fgenesh1_pm.C_scaffold_22000006（上调）。（3）以致病疫霉中RPA190基因的保守功能域（RPOLA-N）在辣椒疫霉中进行同源比对获得基因片段RPA190-pc，通过PEG介导的原生质体转化方法，分别沉默了这5个基因，并对RPA190-pc进行了过表达。结果表明RPA190-pc基因与辣椒疫霉的生长和致病相关，同时也参与调控辣椒疫霉的游动孢子释放量。（4）通过GC-MS代谢组学分析辣椒疫霉在甲霜灵处理下代谢物的变化，结果显示，在甲霜灵处理下，供试4个菌株的氨基酸代谢物的种类及含量都显著增加，而在脂肪酸类代谢物中，4个菌株的硬脂酸和十六酸的含量都随甲霜灵浓度的增加而显著下降。脂肪酸作为真菌膜的主要成分，脂肪酸含量下降会影响细胞膜的流动性，导致细胞膜运输能力受损、碳素运输与能量代谢受阻，从而致使辣椒疫霉生长受到抑制。代谢产物上调的数据表明，与病原菌抗药性有关的膜刚度增加。本研究结果揭示了辣椒疫霉抗甲霜灵的分子机制，为辣椒疫霉对甲霜灵的抗药性监测和治理提供了试验依据。