胞质型肽聚糖识别蛋白RfPGRP-L2在维持红棕象甲肠道菌群稳态中的作用  

Function of the cytoplasmic peptidoglycan recognition protein RfPGRP-L2 in maintaining the homeostasis of gut microbiota in Rhynchophorus ferrugineus(Coleoptera:Dryophthoridae)

作　　者：肖蓉 王兴红 李雄伟 刘惠惠 鲁盛平 侯有明[1,2] 石章红 XIAO Rong;WANG Xing-Hong;LI Xiong-Wei;LIU Hui-Hui;LU Sheng-Ping;HOU You-Ming;SHI Zhang-Hong(State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Provincial Key Laboratory of Insect Ecology,College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学,闽台作物有害生物防控国家重点实验室,福州350002 [2]福建农林大学植物保护学院,福建省昆虫生态学重点实验室,福州350002

出　　处：《昆虫学报》2021年第3期348-362,共15页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31470656);国家重点研发计划(2017YFC1200605);福建省自然科学基金项目(2018J01705);福建省高校新世纪人才计划项目(KLa16058A)。

摘　　要：【目的】明确入侵害虫红棕象甲Rhynchophorus ferrugineus胞质型肽聚糖识别蛋白RfPGRP-L2在肠道菌群稳态的维持和调控过程中的作用,将为靶向破坏肠道菌群稳态的害虫控制新策略研发提供新的科学依据和作用靶标。【方法】利用生物信息学方法分析RfPGRP-L2的序列特征。利用RT-qPCR分析RfPGRP-L2在健康红棕象甲4龄幼虫不同组织(头、脂肪体、表皮、前肠、中-后肠、血淋巴)以及大肠杆菌Escherichia coli DH5α和金黄色葡萄球菌Staphylococcus aureus经注射(注射1μL OD 600=1.6的菌液)和喂食(取食涂抹1 mL OD 600=1.6的菌液的甘蔗薄片)两种不同方式分别感染后红棕象甲4龄幼虫肠道和脂肪体中的表达量;进行RfPGRP-L2原核表达,利用体外孵育方法检测重组蛋白RfPGRP-L2对大肠杆菌DH5α和金黄色葡萄球菌的凝集和抑菌活性;RNAi干扰RfPGRP-L2后,检测红棕象甲4龄幼虫血淋巴和肠道中大肠杆菌菌落数的变化;利用RT-qPCR分析RNAi干扰RfPGRP-L2后红棕象甲4龄幼虫脂肪体和肠道中抗菌肽基因表达量的变化;利用基于细菌16S rRNA的高通量测序分析RNAi干扰RfPGRP-L2对健康红棕象甲4龄幼虫肠道菌群结构组成的影响。【结果】SMART预测发现红棕象甲RfPGRP-L2基因编码的蛋白中无跨膜结构域也无信号肽,这表明RfPGRP-L2是一种胞质型肽聚糖识别蛋白。RT-qPCR检测发现,RfPGRP-L2主要在健康红棕象甲4龄幼虫血淋巴、肠道和脂肪体等免疫组织中表达;被注射感染大肠杆菌和金黄色葡萄球菌6 h和12 h后,红棕象甲4龄幼虫脂肪体中RfPGRP-L2的表达量分别显著上调;被喂食感染大肠杆菌6 h后,红棕象甲4龄幼虫肠道中RfPGRP-L2的表达量显著增加。重组表达蛋白RfPGRP-L2能引起大肠杆菌和金黄色葡萄球菌发生凝集反应,这说明RfPGRP-L2能够识别这两种细菌。当RfPGRP-L2被干扰后,红棕象甲4龄幼虫对肠道和血淋巴中感染EGFP标记的大肠杆菌的清除能力显著【Aim】To determine the function of a cytoplasmic peptidoglycan recognition protein,RfPGRP-L2,in the maintenance and regulation of homeostasis of gut microbiota in the invasive insect pest,Rhynchophorus ferrugineus,so as to provide scientific basis and action targets for the development of new insect pest control strategies targeting to destroy the homeostasis of gut microbiota.【Methods】The sequence characteristics of RfPGRP-L2 were analyzed by bioinformatics methods.RT-qPCR was used to analyze the expression levels of RfPGRP-L2 in the different tissues(head,fat body,epidermis,foregut,mid-/hindgut and hemolymph)of the healthy 4th instar larvae of R.ferrugineus and in the gut and fat body of the 4th instar larvae of R.ferrugineus challenged with Escherichia coli DH5αand Staphylococcus aureus by injection with 1μL bacterial suspension with the OD 600 value of 1.6 and oral feeding with sugarcane slices smeared with 1 mL bacterial suspension with the OD 600 value of 1.6,respectively.Prokaryotic expression of RfPGRP-L2 was carried out,and in vitro assays were applied to determine the agglutination and antibacterial activity of the recombinant RfPGRP-L2 to E.coli DH5αand S.aureus.After RNAi of RfPGRP-L2,the number of gut bacterial colonies of E.coli in the hemolymph and gut of the 4th instar larvae of R.ferrugineus was determined.The expression levels of antimicrobial peptide genes in the fat body and gut of the 4th instar larvae of R.ferrugineus after RNAi of RfPGRP-L2 were detected by RT-qPCR.By bacterial 16S rRNA-based high-throughput sequencing,the effect of RNAi of RfPGRP-L 2 on the gut microbiota composition of the healthy 4th instar larvae of R.ferrugineus was analyzed.【Results】The SMART analysis revealed that RfPGRP-L2 has no transmembrane domain and signal peptide,suggesting that RfPGRP-L2 is a cyoplasmic peptidoglycan recognition protein.The RT-qPCR results showed that RfPGRP-L2 was highly expressed in the immunity-related tissues,such as the hemolymph,gut and fat body of the healthy 4th instar l

关 键 词：红棕象甲 肽聚糖识别蛋白 表达谱 肠道菌群 免疫反应 免疫稳态 

分 类 号：S433.5[农业科学—农业昆虫与害虫防治]