绿僵菌干扰飞蝗CaN调控途径解除飞蝗免疫研究

As an important second messenger, Ca2+ may regulate the immune of insect by calcineurin (CaN). In the previous study, we found that the toxicity of Metarhizium anisopliae was related with its capability in inhibition the increasing of the activity of CaN, superoxide dismutase (SOD) and phenol oxidase (PO) in Locusta migratoria manilensis(Meyen), and the activators of the ryanodine (Ry) receptor (RyR)-Ca2+ channel can enhance this inhibition to synergist the toxicity of M. anisopliae. So, we deduced that the interference of destruxin of M. anisopliae to the balance of Ca2+ in host can disturb the regulation of CaN to SOD and PO and overcome the immune of host to M. anisopliae. In order to clarify this mechanism, we have been cloned the cDNA sequences of CaN, SOD, PPO(Prophenoloxidase), actin and elongation factors of L. migratoria manilensis and obtained the antibodies of these protein that was obtained by expressing in Escherichia coli. On the basis of these results, we will reveal the mechanism of regulate in mRNA level and the mode of action in protein level of CaN regulate SOD and PPO in the immune of L. migratoria manilensis to M. anisopliae by detect spatial-temporal dynamics in mRNA level of CaN, SOD and PPO in the process of L. migratoria manilensis that was infected by various concentrations of IMI330189 (one strain of M. anisopliae has high toxicity to L. migratoria manilensis )and IBC200614 (one strain of M. anisopliae has low toxicity to L. migratoria manilensis ) with Real-Time Quantitative PCR, and detect the spatial-temporal dynamics in protein level of CaN, SOD and PPO in the process of L. migratoria manilensis that was infected by various concentrations of IMI330189 and IBC200614 with fluorescent western blotting system. And the same times, we will verify the immune function of CaN, SOD and PPO of L. migratoria manilensis to M. anisopliae, and confirm the mechanism of M. anisopliae overcome the immune of SOD and PO in L. migratoria manilensis by interfere CaN of L. migratoria manilensis by RNA interferences of CaN, SOD and PPO and adding enzymes inhibitors of CaN, SOD and PO. These results can help us to understand the toxicity mechanism of M. anisopliae, and provide guidance in toxicity evaluation and toxicity improve by transformation of themselves genes of M. anisopliae.

钙离子作为细胞第二信使，可通过钙调磷酸酶(CaN)启动昆虫细胞免疫。申请者研究发现绿僵菌毒力与其对飞蝗CaN、超氧化物歧化酶(SOD)、酚氧化酶(PO)活力上升的抑制程度相关，鱼尼丁受体激动剂可使抑制增强，推测绿僵菌分泌的绿僵菌素导致寄主细胞钙离子失衡后，可干扰CaN对SOD和PO的调控，从而解除寄主对绿僵菌的免疫。为此，我们克隆了飞蝗CaN、SOD、PPO基因及内参基因并制备了抗体，拟通过RT-PCR明确3个基因的mRNA在高毒株与弱毒株处理的3龄蝗蝻中的时空动态，并经荧光蛋白印迹法明确3种酶蛋白水平的时空变化，揭示飞蝗CaN调控SOD和PPO对绿僵菌免疫在基因水平的调控机制及蛋白水平的作用方式。通过RNAi和酶抑制剂验证它们对绿僵菌的免疫及绿僵菌解除该免疫的机制，从而阐明绿僵菌干扰飞蝗CaN主动解除SOD和PO对其免疫的调控途径，为明确绿僵菌的毒力机制、毒力评价和自身基因改造提供参考。

经过4年的系统研究，本项目在如下方面取得了进展。1）明确了飞蝗卵的最佳储存条件，结合飞蝗人工饲料的改进，在室内建立了遗传背景单一的飞蝗室内种群；2）通过比较不同致病力菌株对飞蝗的侵染，发现了绿僵菌主动破坏宿主血细胞的现象，揭示了绿僵菌致病力与宿主酶活变化的相互关系，阐明了绿僵菌侵染的关键时期；3）分子水平和蛋白水平比较发现，GNBP1和Toll可能是高毒力绿僵菌的靶标，高毒力绿僵菌可在转录和蛋白水平上主动攻击GNBP1以抑制PPO2的免疫应答，在转录水平抑制Toll降低抗菌肽的转录和表达；4）阐明了氯虫苯甲酰胺对飞蝗解毒代谢酶的影响，共毒实验发现氯虫苯甲酰胺与绿僵菌IMI330189存在明显共毒现象，并阐明两者共毒的生化机理是通过与宿主中钙离子破坏机制的协同实现的；5）两者共毒的分子和蛋白方面机制是，绿僵菌和氯虫苯甲酰胺的协同作用在转录水平和蛋白水平上抑制了寄主GNBP1对绿僵菌识别，在转录水平抑制了寄主Toll和dMyD88参与抗菌肽合成信号传导，以及抑制了寄主PPO1和PPO2参与的酚氧化酶级联反应，说明GNBP1和Toll可能是绿僵菌抑制寄主免疫的分子靶标；6）发现曲酸对绿僵菌增效的原因是曲酸不但可以明显抑制寄主的酚氧化酶，而且可以明显干扰其他生化酶活性；7）通过抑制剂活体实验，阐明了肌醇三磷酸受体、钙调素和钙调磷酸酶在调控飞蝗免疫相关酶中的作用。以上结果对绿僵菌高效剂型的开发、增效剂的筛选、高度菌株的构建提供了理论指导。