C-型凝集素RfCTL-S1在红棕象甲幼虫免疫防御中的作用

doi:10.16380/j.kcxb.2021.12.004

摘要/Abstract

摘要：

摘要: 【目的】明确红棕象甲Rhynchophorus ferrugineus C-型凝集素RfCTL-S1在其幼虫免疫防御中的作用。【方法】对红棕象甲RfCTL-S1序列特征进行生物信息学分析；利用RT-qPCR检测RfCTL-S1在红棕象甲4龄幼虫不同组织(头、体壁、前肠、中-后肠、血淋巴和脂肪体)以及大肠杆菌Escherichia coli DH5α和金黄色葡萄球菌Staphylococcus aureus注射感染(注射1 μL OD₆₀₀=1.6的菌液)后在红棕象甲4龄幼虫脂肪体和肠道中的表达量，以及RfCTL-S1基因RNAi后红棕象甲4龄幼虫脂肪体和肠道中抗菌肽基因RfAttacin, RfCecropin, RfColeopericin和RfDefensin表达量；分析RfCTL-S1基因通过RNAi被沉默后对红棕象甲4龄幼虫血淋巴EGFP标记的大肠杆菌的清除能力以及肠道中可培养细菌菌落数量的影响。【结果】生物信息学分析发现红棕象甲RfCTL-S1基因编码的蛋白无跨膜结构域，但包含一个信号肽和糖类识别结构域，该结构域中含有EPN基序，表明RfCTL-S1是一种能结合甘露糖的分泌型蛋白。RT-qPCR检测发现，RfCTL-S1在红棕象甲4龄幼虫血淋巴中高表达。在红棕象甲幼虫脂肪体中，与注射PBS的对照组相比，注射感染大肠杆菌24 h后RfCTL-S1的表达量显著下调；注射感染金黄色葡萄球菌6 h后红棕象甲幼虫脂肪体中RfCTL-S1的表达量显著高于注射后24 h。在红棕象甲幼虫肠道中，注射感染金黄色葡萄球菌12 h后，该基因的表达量显著高于注射后6和24 h。RNAi 干扰RfCTL-S1后，红棕象甲幼虫脂肪体中抗菌肽基因RfCecropin, RfColeoptericin和RfDefensin的表达量都显著下降；红棕象甲幼虫血淋巴对感染EGFP标记的大肠杆菌的清除能力显著弱于对照组，然而RNAi组和对照组间幼虫肠道中的可培养细菌菌落数没有显著差异。【结论】分泌型RfCTL-S1蛋白能够通过识别细菌并激活红棕象甲幼虫脂肪体中相应的免疫信号通路促进抗菌肽基因表达，从而实现对入侵病原体的清除。

关键词: 红棕象甲, C-型凝集素, 天然免疫, 抗菌肽, 病原, RNA干扰

Abstract: 【Aim】 This study aims to determine the role of a C-type lectin, RfCTL-S1, in immune defense in larvae of the red palm weevil, Rhynchophorus ferrugineus. 【Methods】 The sequence characteristics of RfCTL-S1 of R. ferrugineus were analyzed by bioinformatics methods. The expression levels of RfCTL-S1 across the different tissues (head, integument, foregut, mid-/hindgut, hemolymph and fat body) of the 4th instar larvae of R. ferrugineus were assayed by RT-qPCR. The abundance of RfCTL-S1 transcripts in the fat body and gut of the 4th instar larvae of R. ferrugineus challenged with Escherichia coli DH5α and Staphylococcus aureus by injection with 1 μL bacterial suspension with the OD₆₀₀ value of 1.6 was also detected by RT-qPCR. Moreover, the expression levels of the antimicrobial peptide genes, including RfAttacin, RfCecropin, RfColeopericin and RfDefensin, in the fat body and gut of the 4th instar larvae of R. ferrugineus after RNAi of RfCTL-S1 were also quantified by RT-qPCR. After RfCTL-S1 was silenced by RNAi, the ability of R. ferrugineus individuals to clear the invaded EGFP-tagged E. coli in the hemolymph and the number of cultivable bacterial colonies in the gut of the 4th instar larvae of R. ferrugineus were evaluated. 【Results】 The bioinformatics analysis revealed that RfCTL-S1 contains a signal peptide and the carbohydrate recognition domain (CRD) with EPN motif, but without transmembrane domain, suggesting that RfCTL-S1 is a secretory protein with mannose-binding activity. RT-qPCR results showed that RfCTL-S1 was highly expressed in the hemolymph of the 4th instar larvae of R. ferrugineus. In the fat body of R. ferrugineus larvae at 24 h after injection of E. coli, the expression level of RfCTL-S1 was significantly down-regulated as compared with that of the control injected with PBS. The expression level of RfCTL-S1 in the fat body of R. ferrugineus larvae at 6 h after injection of S. aureus was significantly higher than that at 24 h after injection of S. aureus. In the gut of R. ferrugineus larvae, the expression level of RfCTL-S1 at 12 h after challenge with S. aureus was significantly higher than those at 6 and 24 h after challenge with S. aureus. After RfCTL-S1 was silenced by RNAi, the expression levels of three antimicrobial peptide genes RfCecropin, RfColeoptericin and RfDefensin in the fat body of R. ferrugineus larvae were significantly reduced, and the ability of R. ferrugineus larvae to clear the invaded EGFP-tagged E. coli in the hemolymph was significantly impaired as compared to the control. However, no significant difference was determined in the number of cultivable bacterial colonies in the larval gut between the RNAi group and the control group. 【Conclusion】 Secretory RfCTL-S1 protein can mount the expression of antimicrobial peptide genes by recognizing bacteria and activating the corresponding immune signaling pathways in the fat body of R. ferrugineus larvae, thus to eliminate invaded pathogens.

Key words: Rhynchophorus ferrugineus, C-type lectins, innate immunity, antimicrobial peptide, pathogen, RNA interference

鲁盛平, 刘惠惠, 苏治平, 刘倩霞, 侯有明, 石章红. C-型凝集素RfCTL-S1在红棕象甲幼虫免疫防御中的作用[J]. 昆虫学报, 2021, 64(12): 1388-1397.

LU Sheng-Ping, LIU Hui-Hui, SU Zhi-Ping, LIU Qian-Xia, HOU You-Ming, SHI Zhang-Hong. Role of a C-type lectin, RfCTL-S1, in immune defense in larvae of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae)[J]. Acta Entomologica Sinica, 2021, 64(12): 1388-1397.

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C-型凝集素RfCTL-S1在红棕象甲幼虫免疫防御中的作用

Role of a C-type lectin, RfCTL-S1, in immune defense in larvae of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae)

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