井冈霉素刺激褐飞虱生殖的分子机制研究

Jinggangmycin is a conventional biofungicide with low toxicity which control rice sheath blight in China. The pesticide is commonly overused due to its low toxicity and high efficiency. Our investigation demonstrated that jinggangmycin stimulated reproduction of the brown planthopper(BPH) Nilaparvata lugens and that stimulatory effect of foliar spray with the jinggangmycin on reproduction of BPH was greater than that of topical application. However, molecular mechanisms of jinggangmycin-induced stimulation of reproduction is not understood. The present project will conduct proteome and transcription analysis on BPH treated with jinggangmycin by two-dimensional gel electrophoresis(2-D)and liquid chromatography tandem mass spectrometry (LC-MS/MS) and de-novo sequencing.Key genes responsible for stimulation of reproduction were selected and RNAi experiment to the genes will be conducted, finally, the efficiency of RNAi will be verified by qRT-PCR. Gene interactions are investigated by co-immunoprecipitation after analysis of Kegg and Pathway. The objective of the present study is to understand molecular mechanisms of jinggangmycin-induced stimulation of reproduction of BPH. The findings of the present study have important significance for development of pest resurgent theory for use of positive effect of jinggangmycin against rice sheath blight and avoidance of negative effect of jinggangmycin-induced stimulation of reproduction of BPH. The characteristic and innovative aspacts of this project are as follows: jinggangmycin-induced stimulation of reproduction of BPH is a gap study in pest resurgence in worldwide. In addition, protoem and transciption analysis from fat bodies and ovaries of adult females treated with jinggangmycin and selections of key genes associted with stimulation of reproduction based on the analysis and the determination of their finctions are a original innovative study in worldwide.

井冈霉素是一种低毒生物杀菌剂，是我国防治水稻纹枯病的主打药剂、生产上往往频繁多次使用。我们在国内外首次发现井冈霉素刺激褐飞虱生殖现象，而且叶面喷雾处理的刺激效应大于点滴处理，但刺激生殖的机制至今不清楚。本项目采用双向电泳、LC-MS/MS及de-novo测序技术进行蛋白质组、转录组分析，筛选出与刺激生殖相关的关键基因；然后进行RNAi试验，通过qRT-PCR对RNAi效果验证；通过Kegg 和Pathway方法分析后，对相关基因用免疫共沉淀技术研究基因互作。目标是要阐明井冈霉素这个特殊药剂刺激褐飞虱生殖的分子机制。本项目将对发展害虫再猖獗理论、科学利用井冈霉在防治病害中的积极作用，避免其可能导致褐飞虱再猖獗发生的消极作用等方面具有重要意义。项目的特色和创新点为：研究材料（井冈霉素）特色鲜明，在国内外属空白研究；同时从脂肪体和卵巢的蛋白质组及转录组分析关键基因及其刺激生殖功能在国内外也属首次

背景：褐飞虱是典型的再猖獗型水稻重大害虫。过去有关杀虫剂刺激褐飞虱生殖有许多研究。杀菌剂井冈霉素刺激褐飞虱生殖研究几乎是空白。而井冈霉素是防治水稻纹枯病的主要药剂。如果使用不当，有可能使稻飞虱发生再猖獗。因此有必要系统研究井冈霉素刺激褐飞虱生殖的机制。.研究内容：（1）井冈霉素不同使用方式、不同剂量对褐飞虱生殖的影响；（2）井冈霉素处理的褐飞虱耐高温及其分子机制；（3）井冈霉素处理的褐飞虱蛋白组、转录组分析；（4）刺激褐飞虱生殖的关键基因及功能研究；（5）井冈霉素对自然种群增长的影响。.重要结果：井冈霉素叶面喷雾处理的褐飞虱产卵量显著高于点滴和茎秆喷雾，茎秆喷雾对产卵没有影响；叶面喷雾褐飞虱卵巢和脂肪体蛋白含量也显著高于点滴和茎秆喷雾；不同浓度处理以200ppm产卵量最大，分别比点滴组和茎秆处理组产卵量增加13.3%, 43.5%。100-200ppm明显刺激褐飞虱生殖，大于400ppm影响不明显。井冈霉素处理增强了褐飞虱抗高温能力，高温（40℃）处理后48h有对照组死亡80%以上下降到55%，致死中时间（LT50）也显著延长。RNAi试验表明Hsp70和Acc基因是井冈霉素诱导褐飞虱耐热性增强的关键基因，其中Hsp70与生殖无关，而Acc基因调控生殖和耐高温。蛋白组学分析和RNAi试验证实脂肪合成酶（FAS）和甘油三脂脂肪酶（Atgl）是调控井冈霉素刺激生殖的关键基因。沉默这两个基因消除了刺激生殖效应。进行的透射电镜观察显示井冈霉素处理的羽化1-2天（1DAE， 2DAE）的雌虫卵母细胞中脂滴大小显著下降，1DAE和 2DAE分别下降32.6%和29.8%；蜕皮激素下降36.0%和30.0%；相反保幼激素水平增加49.7%和45.7%。.科学意义：生产上为避免使用井冈霉素而导致褐飞虱再猖獗可以用高浓度茎秆喷雾，或在田间褐飞虱没有发生时用井冈霉素防治纹枯病；对井冈霉素刺激褐飞虱生殖的分子机制研究成果发展了害虫再猖獗理论。