新疆荒漠甲虫光滑鳖甲免疫防御机制的初步研究

Desert insects evolved an unique antimicrobial immune adaptation mechanism in the extreme environments. So far, the immune defense mechanism of desert Tenebrionidae insects has not been reported. In this study, the widespread beetle Anatolica polita in the Junggar desert was used as the research object. The artificial breeding system of Anatolica polita has been established. The subtractive cDNA library of Anatolica polita against bacteria has been constructed. Based on this, the transcriptional level of immune-associated genes including peptidoglycan recognition protein gene, serine protease gene, serine protease inhibitors gene, antibacterial peptide genes will be assayed by using real-time fluorescence quantitative PCR and the immune response to bacteria of Anatolica polita will be studied. The full-length cDNA of immune-associated genes will be cloned based on the conservative sequence screening, and will be expressed by prokaryotic or eukaryotic expression system. The recombinant proteins encoded by the immune-associated genes will be purified and functionally assayed. The effects of RNA interference on the expression of immune-associated genes will be studied by dsRNA injection, and the immune recognition and signal transmission in the desert beetle Anatolica polita immune defense response will be further illuminated. This study will provide some evidences for the clarification of immune defense mechanism of Anatolica polita and has the significance for the development of immune-regulation theory of desert insects.

荒漠昆虫在极端环境中进化形成了独特的免疫抗菌适应机制。目前有关荒漠拟步甲科昆虫免疫防御机制的研究尚未见报道。基于此，本研究以准噶尔荒漠广布的光滑鳖甲为研究对象，建立其人工养殖体系，在已构建细菌诱导的抑制差减cDNA文库基础上，通过实时荧光定量PCR检测幼虫被细菌感染后免疫相关基因（肽聚糖识别蛋白基因、丝氨酸蛋白酶基因、丝氨酸蛋白酶抑制剂基因、抗菌肽基因）的转录水平，研究光滑鳖甲对细菌免疫应答的变化规律。基于保守序列筛选、克隆免疫相关的重要基因全长cDNA，并通过原核或真核表达、纯化免疫相关基因编码的重组蛋白，进行其功能鉴定；采用注射dsRNA的方法，研究RNA干扰免疫相关基因的表达，从而探讨光滑鳖甲免疫防御系统的免疫识别及信号传递模式，初步揭示光滑鳖甲的免疫防御机制。这将对发展荒漠昆虫的免疫调控理论具有重要的科学意义。

本项目建立了光滑鳖甲的人工养殖体系，通过qRT-PCR检测了幼虫被细菌感染后免疫相关基因（肽聚糖识别蛋白基因ApPGRP-FD1、β-1,3-葡聚糖识别蛋白基因ApβGRP、丝氨酸蛋白酶基因ApSP-RE32、丝氨酸蛋白酶抑制剂基因ApSerpin-FA72、抗菌肽基因Attacin2、Attacin1、Coleoptericin）的转录水平；克隆了免疫相关重要基因的全长cDNA，并进行了其重组蛋白的功能鉴定；使用RNA干扰技术干扰了ApPGRP基因的表达，进而研究了免疫相关基因的表达情况。结果表明重组His-ApPGRP具有与E.coli和S.aureus结合的功能，并且能够与细菌细胞壁的肽聚糖Lys-type PGN和Dap-type PGN相结合；重组TrxA-ApSerpin-FA72能抑制胰蛋白酶及胰凝乳蛋白酶的活性；重组TrxA-ApβGRP具有与酵母菌结合的活性；重组TrxA-ApAMP1015能够有效抑制大肠杆菌的生长。光滑鳖甲幼虫受到细菌刺激时，ApβGRP倾向于识别E.coli，而ApPGRP倾向于识别S.aureus。ApβGRP与细菌结合后激活信号通路，转录产生抗菌肽Attacin2、Attacin1、Coleoptericin、Defensin；而ApPGRP与细菌结合后，可能激活信号通路，转录产生抗菌肽Attacin2、Attacin1、Coleoptericin，同时参与酚氧化酶原激活系统产生有活性的酚氧化酶，引起黑化反应。这些结果初步揭示了光滑鳖甲对细菌免疫应答的变化规律，为进一步阐明荒漠昆虫的抗菌防御机制，发展荒漠昆虫的免疫调控理论奠定了基础。