1,6二氧大花旋覆花内酯对辣椒疫霉菌分子作用机制研究

Previous studies have shown that 1, 6-O, O-Diacetylbritannilactone (ABLOO) isolated from Inula britannica had very strong antifungal activities against Phytophthora capsici in vitro and in vivo conditions. The compound exhibited good potential for being lead compounds and new fungicides. In this proposal, P. capsici was target organism and proteomics technology was used for studying the mechanism of action of ABLOO. Mycelium of P. capsici were treated with ABLOO at different concentration and different time. Total protein from mycelium were extracted and then the protein were pured and quantitatived. Two-dimensional electrophores were employed and gels were scanned. Protein expression profile differences were analysed. Differentially expressed proteins were separated and cut down and identified by MALDI-TOF-MS/MS. According to the position of differentially expressed proteins in biochemical pathways, biochemical pathways of ABLOO against P. capsici were speculated. The effect of ABLOO to biochemical pathways of the target organism were analyzed by some biochemical research methods and mainly biochemical pathways were preliminarily determined. Gene coding differentially expressed proteins that played a key role in the biochemical pathways were deleted and complemented by homologous pair exchange. The sensitivity of deletion and complementary mutation to ABLOO was determined and physiological and biochemical characteristics of the mutations were analyzed. Molecular mechanism of action of ABLOO was revealed. This study will provide new scientific evidence for further understanding the mechanisms of action of pesticides against plant pathogenic fungi. The new target is expected to find and the theory of pesticide science is to rich. Our research findings will fill in the blank in the study of mechanisms of fungicides in China and lay theoretical foundations for reasonably designing new pesticidal compounds based on molecular targets and studying their structure-activity relationship.

从欧亚旋覆花（Inula britannica）花中分离的1,6-二氧大花旋覆花内酯（ABLOO）对辣椒疫霉菌（Phytophthora capsici）离体活性强，防治效果好。本项目拟以辣椒疫霉菌为靶标菌，应用蛋白质组学技术分析ABLOO处理菌体前后蛋白质组的表达差异，分离、鉴定差异蛋白质；Real-time PCR验证差异蛋白质在转录水平上的相应变化；对差异蛋白质进行功能分析，根据其在生化途径中的位置，推定药剂作用的可能生化途径；测定ABLOO对推定生化途径的影响，初步明确药剂作用的主要生化途径；构建编码生化途径中关键差异蛋白的基因敲除/回复突变体，系统分析突变体对药剂的敏感性和生理生化特征，进而阐明ABLOO对辣椒疫霉菌作用的分子机制。研究成果有望发现新的作用靶标，丰富农药学理论，填补我国在杀菌剂作用机理研究的空白，为基于作用靶标的新农药分子合理设计及化合物构效关系研究提供理论依据。

应用蛋白质组学（iTRAQ / TMT）技术对分离自欧亚旋覆花（Inula britannica）的1,6-二氧大花旋覆花内酯（ABLOO）处理的辣椒疫霉菌中识别出3808种蛋白质，检测到794个差异表达蛋白，其中387个蛋白上调，407个蛋白下调； 1.5倍以上差异蛋白有147个，其中26个蛋白上调，121个蛋白下调。采用Real-time PCR对差异蛋白在转录水平上进行了确认。通过GO注释对差异蛋白进行主要生物学功能分析，发现有1697个差异蛋白富集到44个GO条目（二级分类）中，涉及生物过程有代谢、细胞、单一有机体、细胞组织形成、生物调节、刺激反应和信号传导等，其中前三者分别占81.85%、73.68%和55.79%。GO功能富集分析结果表明与生物过程相关的570种差异表达蛋白主要涉及氧化还原过程、氢离子跨膜运输、无机离子跨膜运输和氧化磷酸化；665种差异表达蛋白涉及的分子生物过程包括单价无机阳离子跨膜运输活性、氢离子跨膜转运活性、跨膜转运体活性、氧化还原酶活性、无机阳离子跨膜转运活性和FMN结合；462种差异蛋白主要涉及到细胞色素复合物、膜的固有组分、呼吸链复合体III、膜的有机组分、膜部分和膜。为确定ABLOO对辣椒疫霉菌生物学通路的影响，KEGG富集分析结果表明，差异表达蛋白分布于83个通路中，其中显著富集的通路为碳代谢、核糖体、嘌呤代谢、氨基酸的生物合成、脂肪酸代谢、丙酮酸代谢、内质网蛋白过程、真核生物核糖体生物起源、糖酵解和糖异生和二羧酸代谢。iTRAQ分析发现与β-1,3-葡聚糖相关的1,3-β-葡聚糖酶与胞内1,3-1,4-β葡聚糖酶均为上调，生化测定发现ABLOO对辣椒疫霉碱水溶性和不溶性β-1,3-葡聚糖具明显抑制作用，进而通过透射电镜观察到ABLOO处理后靶标菌细胞壁发生变化。推测ABLOO处理辣椒疫霉菌丝后，菌体细胞内β-1,3-葡聚糖相关酶表达量上调，β-葡聚糖水解酶含量升高，导致大量β-葡聚糖被迅速水解，无法维持正常的细胞内β-葡聚糖含量，而作为细胞壁主要组成成分，β-葡聚糖的减少无疑会对细胞壁形成造成影响，导致细胞壁变薄，完整性遭到破坏，进而影响靶标菌的菌丝生长、孢子囊形成与释放以及孢子萌发等。