海藻糖合成酶调控椭圆食粉螨低温低氧交互耐受性机制

Aleuroglyphus ovatus is seriously endangering stored grain. We found hypoxia can improve its tolerance to the low temperature, which affect pest control.Trehalose synthases (TPS/TPP) play an important role on anti-stress and have not yet been studied on cross tolerance between hypoxia and low temperature. Based on obtained partial sequence of TPS genes and recombination protein of TPS1 in Aleuroglyphus ovatus, this project intends to adopt biometric, biochemical, molecular biological method to determinate the relationship between the expression pattern of TPS/TPP and the term under low temperature or hypoxia; to explore their mechanism regulating the cross tolerance between low temperature and hypoxia with analysising the effect on cross tolerance by differential expression of TPS/TPP induced by low temperature or hypoxic pretreatment; to verify the function of TPS/TPP in the cross tolerance by comparing the change of the ability to resist low temperature and hypoxia after RNA interference of those genes; to identify the important role of TPS/TPP in the regulation of cross tolerance by analysising the differences in the expression of trehalose metabolism related genes in high and low survival groups with different temperature and oxygen concentration combinations by transcriptome sequencing technology. The project can not only detect molecular mechanism associated with cross tolerance between low temperature and hypoxia by the TPS/TPP in Aleuroglyphus ovatus, but provide the basis for the evolution mechanism of insects adapted to the extreme environmental, and provide a theoretical basis and practical guidance to control mites in stored products.

椭圆食粉螨严重危害储粮，我们发现低氧能提高其耐寒性，影响防治效果。海藻糖合成酶TPS/TPP在抗逆方面具有重要作用，尚未有其调控低温低氧交互耐受性研究。在已获得椭圆食粉螨TPS基因及重组蛋白的基础上，本项目拟采用生测、生化及分子生物学等方法，确定TPS/TPP表达动态与低温低氧胁迫时长的相关关系；分析低温或低氧预处理诱导TPS/TPP差异表达对交互耐受性的影响，探明其调控低温低氧交互耐受性机制；对比RNA干扰TPS/TPP后交互耐受性的变化，验证其在低温低氧交互耐受中的功能；转录组测序技术分析不同温氧交互处理下高存活组和低存活组中海藻糖代谢相关基因表达差异，明确TPS/TPP在交互耐受性调控中的重要地位。项目的实施不仅可探明TPS/TPP在低温低氧交互耐受中的调控机制，为阐明极端环境昆虫的适应进化机理提供依据，同时为储粮害虫防治提供理论基础和实践指导。

椭圆食粉螨严重危害储粮，我们发现低氧能提高其耐寒性，影响防治效果。海藻糖合成酶TPS在抗逆方面具有重要作用，尚未有其调控低温低氧交互耐受性研究。我们测定了椭圆食粉螨不同低温低氧胁迫下存活率，发现12和24h处理的半致死低温为6.636和8.878°C，4°C下半致死时间为10.642h，5%和10%氧气浓度下的半致死时间为9.146和12.284；低温处理下海藻糖含量显著上升，且随温度下降而升高，4°C下达到峰值，葡萄糖和海藻糖的含量存在互补关系；海藻糖酶酶活与海藻糖含量相呼应，也是随温度的降低而显著升高，4°C达到峰值，为308.10±6.51 U/mgprot；低温胁迫下2个TPS基因表达量均上调，在4°C达到最高，10%低氧胁迫显著提升TPS基因表达量，但5%低氧下表达量下调，TPS2甚至低于对照组；利用浸渍法对椭圆食粉螨2个TPS基因进行RNAi，显著降低了该螨对低温低氧的耐受性，表明TPS是防控该螨的有效靶标；获得了椭圆食粉螨2个海藻糖转运蛋白基因Tret-1和Tret1-2全长，2基因在各发育阶段均能表达，低温胁迫下2基因的表达模式类似，均随温度的降低而显著升高，在8°C表达量达到峰值，而Tret酶活在卵期为最高，其他龄期表达量均较低，但各龄期表达量低温处理均显著高于对照组，且在8°C含量最高，这与Tret基因表达量一致；克隆获得了椭圆食粉螨天敌基氏蠊螨和剑毛帕厉螨的线粒体全长，分别为17,314和16,468 bp，均存在基因重排，全长的获得为椭圆食粉螨防治——天敌的应用奠定了理论基础；完成马六甲肉食螨对椭圆食粉螨的捕食功能反应，该肉食螨对不同螨态椭圆食粉螨的功能反应均符合Holling II型，马六甲肉食螨对该螨幼虫和若虫表现出正偏好，而对其成螨和卵表现出负偏好，为其防治提供理论依据和实践指导。海藻糖代谢是椭圆食粉螨应对环境胁迫的重要环节，TPS和Tret在其中扮演了重要角色，抑制其表达量可以有效降低其抵御环境胁迫的能力，是防控储藏物害螨的有效靶标。