热胁迫下抗性和敏感小菜蛾线粒体凋亡路径信号传导与上游调控机制

Because of global warming, it would be important to study the physiological response and its molecular basis under heat stress in S and R insect trains. Our previous results, published in Pestic. Biochem. Physiol. (2008), Mol. Biol. Rep. (2011) and J. Econ. Entomol.(2004), respectively indicated that under heat stress, insecticide-susceptible (S) Pltuella xylostella (DBM) showed higher advantages in life-history parameters, higher heat shock protein 70 expression, lower apoptosis of sperm and ovum cell and lower caspases-3 expression (which was a effector caspases in the downstream), as compared to insecticide-resistant (R) DBM.Significantly seasonal change of insecticide resistance had been observed. Because the individuals with high insecticide-resistant levels in the field population of DBM had significantly lower biological fitness under heat stress, the levels of insecticide resistance in the field DBM population declined sharply in July, but maintained a high level in other seasons. However, the comparison on the effects of HT on the physical and biochemical responses between S and R insect strains was sparse. In particular, the comparison on the signal conduction and regulation of mitochondrial apoptotic pathway in R and S insect trains under heat stress could not be found in the literature. This study was designed to investigate the cell morphology and the signal conduction and regulation of mitochondrial apoptotic pathway in S and R DBM under heat stress: 1) full-length cDNA identification of the key genes (including Bax, Bcl-2, Cytc and caspase-9) in the upstream of the mitochondrial apoptotic pathway; 2) the temporal and spatial dynamics of signal conduction among the four genes of the upstream pathway; 3) the upstream regulations on the pathway by Bax/Bcl-2 expression; and 4) the expression dynamics of caspase-3 in the downstream pathway and apoptotic morphology analysis. Based on the results, we hope to know: 1) why higher extent of apoptosis happaned in R DBM than in S DBM under heat stress by understanding the molecular basis in the signal conduction and regulation of the mitochondrial apoptotic pathway; 2) how the related detection technology system was established to monitor the dynamics of the mitochondrial apoptotic pathway; 3) why the resistant level of field R DBM declined sharply in summer and why R DBM showed significantly lower biological fitness under heat stress. The research in this study would be beneficial to know how HT affected the evolution of insecticide resistance in insects. In addition, the study on the evolution trends of insecticide resistance under heat stress will be important issue in IPM stratagem to modify insecticide application in the field with the consideration of seasonal effect as an additional IPM measure.

全球变暖将深刻影响田间昆虫抗药性进化。本人前期工作已证实高温下抗药性（R）小菜蛾精子和卵子细胞凋亡及线粒体凋亡路径下游基因caspase-3表达量显著高于敏感（S）小菜蛾，但国内外相关的分子机制研究尚很薄弱。本申请拟从细胞凋亡形态学和分子水平上研究R和S小菜蛾：1)线粒体凋亡路径上游关键基因（Bax、Bcl-2、Cytc、caspase-9）克隆、信号激发与传导的时空动态与亚细胞定位；2) Bax/Bcl-2对上游基因信号传导动态的调控；3)路径下游基因caspase-3对上游信号的应答动态及细胞凋亡形态学观察；从而：1)揭示热胁迫下R和S小菜蛾线粒体凋亡路径信号传导、凋亡发生及Bax/Bcl-2调控机制，构建相应的检测技术体系；2)阐明夏季田间R小菜蛾因适合度较低而导致抗药性急剧下降的分子机制。成果对揭示全球变暖对田间昆虫进化的影响、提出害虫抗性治理新理论和新方法有重要意义。

成功克隆了小菜蛾线粒体调亡路径的6个关健基因，在此基础上，建立了相应的基因表达qPCR检测、蛋白质表达EllASA检测、酶活性试剂盒检测方法。完成了热胁迫对抗性（R）和敏感（S）小菜蛾细胞调亡、生命表参数的影响以及热胁迫下Bcl-2和 Bax基因对抗性和敏感小菜蛾线粒体调亡路径的上游调控机制的研究。结果表明，热胁迫对抗性小菜蛾的生长发育与繁殖的抑制显著高于对敏感小菜蛾的抑制，在热胁迫条件下，抗性小菜蛾精子及卵子DNA损伤及翅脉发育损伤程度均显著高于敏感小菜蛾。成功克隆了线粒体凋亡路径6个最关建的基因，包括上游调控者：Bcl-2和Bax、信号传导调亡体：cytochrome c、Apaf-1、 caspase-9和下游调亡执行者：caspase-7。热胁迫下，抗性小菜蛾细胞调亡基因caspase－9、－7表达上调幅度显著高于敏感小菜蛾，调控基因bax表达水平上调，而bcl-2表达水平下调，但热胁迫下抗性小菜蛾Bax基因表达上调幅度显著高于敏感小菜蛾。与敏感小菜蛾相比，热胁迫下，抗性小菜蛾表现出显著更低的hsp基因家族(hsp69s, hsp70s and hsp20)上调表达、更低的保护酶活力（SOD,、CAT、POD酶)、更高的自由基含量、根严重的精子和卵子细胞凋亡。此外，为研究热胁迫对抗性和敏感小菜蛾翅脉损伤影响分子机制，先后克隆了翅脉发育路径相关的9个关健基因，并进行了热激表达的初步研究。上述结果表明，热胁迫下，抗性小菜蛾表现了显著较低的生物学适合度及更大的伤害与其较低的热休克蛋白及体内保护酶系统保护及较高的细胞调亡及相关基因表达有关，而抗性小菜蛾更高细胞调亡及相关基因上调表达与热胁迫下抗性小菜蛾Bax基因更大幅度的上调有关，bax和bcl-2在抗性和敏感小菜蛾中的差异表达在调控抗性和敏感小菜蛾种群细胞调亡基因表达差异中可能起重要作用。研究结果对揭示热胁迫下抗性和敏感小菜蛾线粒体路径细胞调亡的差异机制，进而揭示热胁迫影响小菜蛾抗药性进化机制及全球变暖背景下田间小菜蛾的抗药性进化机制有重要意义。