稻瘟病菌乙酰乳酸合成酶催化亚基MoIlv2互作蛋白的鉴定及其功能分析

Acetolactate synthase is a key enzyme in the branched-chain amino acid biosynthetic pathway, catalyze synthesis of branched-chain amino acids leucine, isoleucine and valine. Our previous study revealed that acetolactate synthase catalytic subunit MoIlv2 was key pathogenic factor, involved in the development, conidiation and pathogenesis of M. oryzae. In this study, MoIlv2 and 8 candidate binding proteins will be further confirmed respectively by co-IP and yeast two hybrid system. The confirmed candidates will be selected to knock out, complement and functionally characterized. The gene expression characteristics and subcellular localization of gene expression products will be studied via Real-time PCR and GFP fusion expression methods. These results will not only reveal the branched-chain amio acid biosynthetic pathway involving in growth, development and virulence of M. oryzae, but also provide insight into the underlying molecular mechanisms and designing novel disease management strategies targeted with MoIlv2 binding proteins.

乙酰乳酸合成酶是支链氨基酸合成途径中的第一个关键酶，催化生成亮氨酸、异亮氨酸和缬氨酸。申请者前期研究发现稻瘟病菌乙酰乳酸合成酶催化亚基MoIlv2是支链氨基酸合成途径中的关键致病因子，参与该病菌的生长发育、产孢及对水稻的致病性过程。本项目拟采用co-IP和酵母双杂交技术分别对前期筛选的8个MoIlv2候选互作蛋白进行鉴定；通过构建关键互作蛋白编码基因的敲除突变体和互补突变体，分析该基因对稻瘟病菌生物学特性和致病性的影响；进一步运用Real-time PCR和GFP融合表达技术，研究关键基因的实时表达特征和基因表达产物的亚细胞定位。研究结果不仅有助于深入揭示稻瘟病菌中支链氨基酸生物合成途径的分子机制，而且可为开发以MoIlv2互作蛋白为靶标的新型杀菌剂提供参考。

氨基酸的生物合成及代谢机制参与调控稻瘟病菌的生长发育与致病过程，特别是生物合成途径中的关键酶大多数是稻瘟病菌的致病因子。我们前期从稻瘟病菌中鉴定到一个ALS催化亚基MoIlv2，其通过参与支链氨基酸生物合成过程从而调控该病原菌的生长、产孢及对水稻的致病力。本项目通过co-IP技术捕获MoIlv2的互作蛋白，测序及质谱分析筛选到1个候选蛋白MoLeu2A。进一步对互作蛋白的编码基因MoLEU2A进行敲除，并分析其生物学表型，从而阐明MoLEU2A在稻瘟病菌生长发育及致病过程中的作用机制。研究结果不仅有助于深入揭示亮氨酸合成途径介导的分子机制，而且可为开发以亮氨酸合成相关蛋白为靶标的新型杀菌剂提供参考。