印楝素诱导柑橘木虱中肠细胞凋亡及其影响黄龙病菌定殖的分子机理

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the only natural vector of Candidatus Liberibacter asiaticus that causes citrus huanglongbing (HLB), a most destructive disease of citrus. Currently, no remedial therapy exists for the disease, and so effective control of ACP is very important in curbing the transmission of the disease. Azadirachtin is an effective systemic insecticide for control of ACP. However, its effects on the interaction between ACP and HLB are still unclear. We had proved that azadirachtin induced midgut cell apoptosis, and first found that it could suppress ACP infecting the HLB disease. Therefore, we propose that azadirachtin influences huanglongbing colonization in the ACP midgut by inducing the midgut cell apoptosis and destroying its analogous biofilm structure. To reveal the molecular mechanism underlying azadirachtin influences huanglongbing colonization, we first detect the expressions of the apopotosis related protein caspase-3 and the mitochondrial pathway protein Bcl-2 by western blot, and then observe the structures of midgut cells and HLB analogous biofilm using the fluorescence microscope and transmission electron microscope. Also, the HLB colonization in ACP midgut cells under azadirachtin stress will be determined by qRT-PCR. This study will provide a theoretical basis for the scientific use of azadirachtin in citrus and the development of new technologies for ACP and HLB integrated control.

柑橘黄龙病是柑橘毁灭性病害，柑橘木虱是黄龙病唯一传播昆虫媒介，有效防治柑橘木虱是防控黄龙病的关键。印楝素具有内吸性，可到达柑橘韧皮部，有效防治柑橘木虱。但印楝素被取食后会不会影响木虱和黄龙病菌互作尚不明确。申请人已证明了印楝素可诱导昆虫中肠细胞凋亡，并首次发现印楝素可降低柑橘木虱获得黄龙病菌的水平。据此，申请人提出印楝素通过诱导木虱中肠凋亡，破坏黄龙病菌形成伪生物膜结构，抑制黄龙病菌在中肠定殖。本项目拟采用荧光显微镜、透射电镜、qRT-PCR、western blot等技术研究印楝素作用下木虱中肠细胞凋亡蛋白caspase-3和Bcl-2的表达，中肠细胞结构、黄龙病菌伪生物膜结构的变化，以及黄龙病菌定殖情况，明确印楝素破坏柑橘木虱与黄龙病菌互作的方式，揭示印楝素抑制黄龙病菌在中肠定殖的分子机理。本研究将为印楝素在柑橘上科学的使用，以及研发新型柑橘木虱和黄龙病综合防治措施提供重要的理论依据。

柑橘木虱是黄龙病的唯一传播媒介昆虫，而黄龙病菌可在木虱中肠定殖。尽管印楝素可有效防治柑橘木虱，但是印楝素被取食后是否会影响木虱和黄龙病菌互作尚不明确。本项目首先研究测定印楝素对柑橘木虱若虫和成虫的生物活性，明确了印楝素可以抑制若虫的生长发育，并与作用剂量呈正相关，但对成虫无生物活性；在此基础上，研究发现印楝素作用下木虱中肠细胞cleavage-caspase 3蛋白表达量显著增加，表明可诱导中肠细胞发生凋亡；检测发现印楝素作用前后柑橘植株内的黄龙病菌含量没有显著变化，说明没有直接抑制活性，但是印楝素作用前后柑橘木虱体内黄龙病菌的含量发生了变化，说明印楝素影响了黄龙病菌在中肠细胞定殖；最后检测发现印楝素处理后中肠细胞的Bcl-2蛋白表达升高，而细胞色素c从线粒体内释放到胞质，揭示了印楝素通过线粒体途径诱导诱导木虱中肠细胞发生凋亡，进而抑制黄龙病菌定殖的分子机理。以上研究成果可为印楝素在柑橘木虱和黄龙病防控上应用提供理论依据，为研发作用于木虱肠道的新型药剂提供了新的思路。受本项目资助，在国内外期刊发表论文3篇，其中SCI论文1篇。