光授时因子介导的二斑叶螨抗药性季节节律分子机制

As the new requirements of environmental protection and food safety, it is very important to find a new economic tactics to the resistance management of phytophagous mite according to the intrinsic biological organism function. Our previous research found that the sensitivity of Tecranychus urticae to acaricides has obvious seasonal differences, but the underlying mechanism is not clear. In this study, through the combination of indoor experiment and field investigation, the research would make it clear that the relationship between sensitivity to acaricides response and seasonal rhythm, whether resistance has the intrinsic rhythm and induced by photoperiod in Tecranychus urticae. Firstly, genome wide gene expression analysis of intrinsic rhythm in different strains of two-spotted spider mite, the differently expressed genes would be cloned with RNA-seq and RT-PCR method, changes of the mRNA expression of these genes in different strains,and with different photoperiodic induction treated would be analyzed using real-time PCR method. Secondly, seasonal monitoring dynamic of resistance, the main physiological index, GSTs enzyme activity and circadian rhythm gene/differential expression genes of two-spotted spider mite in different strains would be analyzed indoors. Thirdly, seasonal dynamic of resistance of Tecranychus urticae would be monitored in field. Finally, the information about the main physiological index, dynamic changes of detoxification enzymatic activities for GSTs, expression changes of circadian rhythm gene and the resistance would be integrated and their correlativities would be analyzed. The mechanisms about dynamic changes of resistance in Tecranychus urticae would be clarified from physiological, biochemical and molecular levels. The result would elucidate a novel mechanism about acaricide-resistance and rhythmicvariation in Tecranychus urticae theoretically,also provide basis of climate-concerned treatment in management of spider mite resistance.

随着环境保护和食品安全新要求，利用螨类内禀特性寻找新的经济、安全的抗药性治理技术具有重要价值。我们前期研究中发现螨类对药剂敏感性具有明显季节性差异，但相关机制尚不清楚。本项目拟通过室内研究与田间调查相结合，从生理生化和分子水平揭示二斑叶螨药剂敏感性变化具有内禀年度节律性的内在原因，阐明其分子机制。研究首先通过转录组分析二斑叶螨敏感和抗性品系节律差异表达基因，筛选克隆相关差异表达基因。其次在不同光周期处理下，分析二斑叶螨敏感和抗性品系的生理响应、抗药性动态、主要解毒代谢酶系的生化效应、关键昼夜节律基因或相关差异表达节律基因的分子响应机制。最后再结合田间二斑叶螨种群抗药性季节动态调查分析室内研究结果，解析二斑叶螨抗药性与四季节律的相关性，从生理生化和分子水平明确其抗性是否具有內禀性节律，光授时因子是否对其年度节律具有诱导性。研究结果可为生产实践中螨类抗药性“因时制宜”治理的新思路提供理论支持。

二斑叶螨是抗药性最严重的世界性农业害螨，目前化学防治仍是主要防控手段，随着环境保护和食品安全新要求，利用螨类内禀特性寻找新的经济、安全的抗药性治理技术具有重要意义。阐明二斑叶螨对药剂敏感性是否为光授时因子诱导的内禀季节节律，将有助于其防控措施（因季施药、光周期利用等）设计，从而减少药剂使用，延缓害螨抗药性发生发展。本项目以24节气为时间点，通过三个光周期（10L:14D、12L:12D和18L:6D）长期诱导处理，研究了二斑叶螨敏感和抗丁醚脲品系对杀螨剂敏感性季节动态变化，从生理指标（肥满度指数，K）、谷胱甘肽S-转移酶酶活性（GSTs）和生物节律基因（tim、per、cyc和clk）分析了其与药剂毒力季节动态的相关性。主要结果为：（1）二斑叶螨对丁醚脲和炔螨特敏感性具有季节节律性，每天10-18 h光照时长对其无诱导，秋冬季明显高于春夏季。（2）光周期和时间季节皆显著影响二斑叶螨种群发育、形态生理状况和谷胱甘肽S-转移酶活性。在未引起螨滞育的光长下（10-18 h/day），长光照不利于种群增长。二斑叶螨品系间形态生理状况随季节变化差异显著，敏感品系冬春季高于夏秋季，抗性品系春夏季高于秋冬季。螨类药剂敏感性与肥满度和时间季节显著相关，即形态生理状况是影响二斑叶螨药剂耐受性季节变化的因素之一。相同光周期下，二斑叶螨GSTs酶活性峰值出现在春夏季，而秋冬季活性值较低。（3）时间季节显著影响二斑叶螨生物节律相关基因（tim、per、cyc和clk）的mRNA水平表达量，tim和per表达量高于cyc和clk，品系间无显著差异；光周期影响tim 和clk基因的表达，对per和cyc无显著影响，二斑叶螨对丁醚脲的敏感性、tim与per表达量和季节变化具有相关性。研究结果理论上拓展了“环境-害螨-作物”关系研究，在实践中可在“植物工厂”等设施农业中，结合植物光周期调控，将生物节律应用于害螨防控。