小菜蛾PxMRP和PxIDGF对蝉拟青霉的免疫识别作用研究

Fungal infections occur in arthropods as well as insect. Entomopathogenic fungi had taken some effective measures to defend themselves against insect innate immunity. As for insects, many researches focused on cellular immunity (like phagocytosis, nodulation and encapsulation) against entomopathogenic fungi infection. And the molecular mechanism of insect humoral immune against entomopathogenic fungi infection is not clear. The diamondback moth, Plutella xylostella, was a notorious insect pest of brassicaceous crops because it evolves resistance to many classes of insecticides. The elucidation of the mechanism of the immunity of P. xylostella to pathogens can provide a theoretical basis for new approaches to biologically control this pest. In this project, the innate immune response of P. xylostella against an entomopathogenic fungus Paecilomyces cicadae challenges was surveyed. Among the immunity-related genes potentially involved in innate immunity, two recognition molecules PxMRP and PxIDGF gene will be cloned by 3'RACE and 5'RACE. Expression profiles of them in different tissues (such as fat body, hemocytes, and midgut) and to different pathogen infections will be performed using qPCR and Western blotting. We will express PxMRP and PxIDGF in a baculovirus-insect cell system and then purify them. The binding patterns of these two proteins to different microorganisms (bacteria and fungi) and pathogen associated molecular patterns (such as LPS, PG and β-1,3-glucan) will also be assayed. In order to identify the function of these two recognition molecules, dsRNA or siRNA specific to PxMRP and PxIDGF gene are prepared and injected or feeding into larval. Injection of antibody of PxMRP and PxIDGF protein are also conducted. Knockdown of the target genes or proteins are evaluated by qPCR, antimicrobial activity, PO activity, survival analysis and electron microscope at selected times. The elucidation of the recognition mechanism of P. xylostella against entomopathogenic fungi will provide new insights into the molecular mechanisms during the innate immunity of insect against entomopathogenic fungi infection. In application, this research can provide a theoretical basis for new approaches (such as RNAi) to biologically control pests. This also will give us new target for biological pesticide and improve the efficiency of the insecticidal fungi pesticides.

昆虫病原真菌采取了一些有效对策来抵御昆虫的天然免疫保护反应。昆虫抵御病原真菌侵染在细胞免疫上研究的较多。而昆虫是如何体液免疫病原真菌的分子机理尚不清楚。本项目拟以小菜蛾为研究对象，在前期已筛选出的免疫相关基因中，对体液免疫通路中的2个识别分子PxMRP和PxIDGF基因进行克隆，利用qPCR和Western blotting分析它们免疫特征，通过表达纯化的PxMRP和PxIDGF重组蛋白与病原物细胞、病原物细胞壁成分结合实验鉴定出它们的识别对象，并利用RNAi（或抗体封闭）技术明确它们在小菜蛾体液抵御蝉拟青霉侵染中的作用。深入研究和阐明小菜蛾对病原真菌的免疫识别机制，理论上可以丰富我们对昆虫体液免疫病原真菌的分子机理的认知；在应用上，一方面可为新的害虫防治方法提供思路和靶标，探索利用昆虫天然免疫进行害虫防治的潜在可能性，另一方面可以为真菌生物杀虫剂找到新的靶标，提高真菌杀虫剂的杀虫效率。

昆虫病原真菌采取了一些有效对策来抵御昆虫的天然免疫保护反应。昆虫是如何体液免疫病原真菌的分子机理尚不清楚。本研究以小菜蛾为研究对象，在前期已筛选出的免疫相关基因中，对PxMRP和PxIDGF基因进行克隆。PxMRP基因编码473个氨基酸，与烟草天蛾GNBP-like蛋白相似性很高。PxIDGF基因编码433个氨基酸。利用半定量RT-PCR、qRT-PCR、western blotting、基因芯片和iTRAQ的方法分析了它们的免疫特征。半定量RT-PCR检测显示PxMRP、PxIDGF两个基因在幼虫、蛹和成虫均有分布。qPCR结果表明小菜蛾经不同微生物诱导后，2个基因表达水平均有所提高。Western blotting分析结果表明：在未诱导的幼虫血淋巴中可以检测到MRP和IDGF两种蛋白的表达。不同微生物处理后，与对照相比两种蛋白的表达量均有不同程度的提高。芯片分析同样证实两个基因在不同微生物诱导后均有不同程度的诱导表达。将PxMRP和PxIDGF基因重组到pCzn1载体上，转化到宿主菌中表达。SDS-PAGE和western blotting检测证明PxMRP和PxIDGF重组蛋白均成功表达。通过Ni柱亲和纯化，将纯化的PxMRP和PxIDGF重组蛋白与与不同的病原物细胞共孵育。结果表明PxMRP重组蛋白可以结合革兰氏阴性细菌E. coli、阳性细菌S. aureus，也可以结合病原真菌P. cicadae。而PxIDGF重组蛋白仅可以微弱的与S. aureus、E. coli和P. cicadae产生结合反应。将编码PxMRP和PxIDGF蛋白基因的 dsRNA分别微量注射到小菜蛾幼虫。与对照相比，注射小菜蛾PxMRP和PxIDGF蛋白基因的dsRNA后，两个基因的转录水平均有不同程度降低。注射dsMRP的小菜蛾对病原真菌蝉拟青霉的抵抗力减弱，注射PxIDGF蛋白基因dsIDGF和注射GFP基因的 dsGFP的干涉效果类似，它们之间无显著差异。深入研究和阐明小菜蛾对病原真菌的体液防卫机制，在理论上丰富了我们对于昆虫天然免疫反应的认知；在应用上，一方面可为新的害虫防治方法提供思路和靶标，探索利用昆虫天然免疫进行害虫防治的潜在可能性，另一方面可以为真菌生物杀虫剂找到新的靶标或途径，提高真菌杀虫剂的杀虫效率。