活性氧在豌豆蚜免疫系统中的作用机理研究

Aphids are devastating agricultural pests around the world. Currently chemical insecticides are the most used means for controlling aphids. However, the efficiency is not as good as expected. Utilization of entomopathogenic microorganisms is a promising strategy for aphid controlling. However, the mechanism of the immune interaction between pathogenic microorganisms and aphids is yet unclear, limiting the application and efficiency of entomopathogenic microorganisms in aphid controlling. Genome analysis and some studies showed that the pea aphid Acyrthosiphon pisum does not have antimicrobial peptide pathway, implying that the pea aphid might have unique mechanisms to defend themselves against invading pathogens. Recent studies indicated that reactive oxygen species (ROS) is involved in pea aphid defense responses. In this project we will investigate the correlation between ROS levels, bacterial viability and aphid survival. By using quantitative PCR, we will profile the expressional changes of the genes participating in ROS generation and clearance, followed by knockdown of the key genes through RNA interference and gene cloning, recombinant protein activity assays to verify the function of ROS in the pea aphid immune system. We will also investigate the effect of change of ROS level on densities of aphid’s symbiotic bacteria. Through this project, we will reveal the roles of ROS in aphid immune system, and will have a better understanding of the interaction between aphid, ROS and invading pathogens, setting up the basis for employing microorganisms to control aphids.

蚜虫是一类重要的农业害虫，目前主要依靠化学农药进行防治，但效果很不理想，而利用昆虫病原微生物防治蚜虫是最好的选择之一。了解病原微生物与蚜虫免疫相互作用的机理是增强微生物防蚜治蚜效果的前提。基因组学分析表明，豌豆蚜不具有抗菌肽通路，其防御病原侵染的机制可能比较独特。申请者前期研究表明活性氧（ROS）在豌豆蚜的免疫防御反应中起着重要作用，值得进一步深入研究。本项目拟系统地分析ROS水平与入侵细菌增殖及豌豆蚜之间的相关性，利用定量PCR分析感染后蚜虫ROS代谢相关基因表达变化，对关键基因通过RNA干扰和体外活性测定验证它们在豌豆蚜防御感染过程中的作用，并分析ROS水平的变化对豌豆蚜共生菌群落密度的影响。本项目的实施，有望揭示ROS在豌豆蚜免疫防御系统中的作用，加深对蚜虫-ROS-入侵细菌之间关系的认识，为利用微生物防治蚜虫奠定理论基础，也为其他蚜虫的生物防治提供借鉴。

与其它昆虫相比，豌豆蚜的免疫防御系统比较独特，不具有抗菌肽通路。本项目通过RNA干扰、重组蛋白体外活性测定等手段，发现细菌感染引发豌豆蚜产生ROS，ROS可以阻止细菌在蚜虫体内的增殖；但另一方面，ROS也对蚜虫造成氧化胁迫，且这是主要的作用，因此对蚜虫具有不利的影响。Peroxiredoxin-1、-2和Catalase对感染后蚜虫体内的H2O2水平、细菌CFU和蚜虫的成活率有显著的影响。更重要的是，抗氧化基因Peroxiredoxin-1、Catalase、OXR1和GPX的表达受到JNK通路的调控。兼性共生菌S. symbiotica可能在对抗细菌感染引发的氧化胁迫中起重要作用。另外，我们证实了PPO在豌豆蚜的防御反应中起关键作用，发现两个丝氨酸蛋白酶Ap_SPLP和Ap-VP参与PPO激活通路，两个非典型的GNBP蛋白（GNBP1和GNBP2）可能作为识别受体参与PPO激活。我们还发现在蚕豆上饲养的豌豆蚜感染后，其后代数量增加；而在苜蓿上饲养的豌豆蚜在感染后，其后代中有翅蚜的比例显著升高，这是一种非免疫性防御策略，值得深入研究其分子机制。本项目揭示了ROS和PPO在豌豆蚜免疫防御反应中的作用，为利用微生物防治蚜虫奠定了理论基础，也为其他蚜虫的生物防治提供了借鉴。