昆虫学报 ›› 2022, Vol. 65 ›› Issue (4): 417-426.doi: 10.16380/j.kcxb.2022.04.002

• 研究论文 • 上一篇    下一篇

甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定

李亚子1, 赵丹1, 郭晓昌1, 吴涵1, 刘兆瑞1, 郭巍1, 2,*   

  1.   (1. 河北农业大学植物保护学院, 河北保定 071001; 2. 中国农业科学院研究生院, 北京 100081)
  • 出版日期:2022-04-20 发布日期:2022-03-24

Cloning and functional characterization of the peptidoglycan recognition protein gene SePGRP-SA in Spodoptera exigua (Lepidoptera: Noctuidae)

LI Ya-Zi1, ZHAO Dan1, GUO Xiao-Chang1, WU Han1, LIU Zhao-Rui1, GUO Wei1,2,*   

  1. (1. College of Plant Protection, Agricultural University of Hebei, Baoding, Hebei 071001, China; 2. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China)
  • Online:2022-04-20 Published:2022-03-24

摘要: 【目的】本研究旨在阐明甜菜夜蛾Spodoptera exigua幼虫体内肽聚糖识别蛋白(peptidoglycan recognition protein, PGRP)在响应苏云金芽胞杆菌Bacillus thuringiensis(Bt)感染过程中的功能。【方法】利用PCR方法扩增甜菜夜蛾幼虫肽聚糖识别蛋白基因SePGRP-SA全长cDNA;采用qRT-PCR分析SePGRP-SA在甜菜夜蛾不同发育阶段(卵、1-5龄幼虫、预蛹和蛹)及4龄幼虫不同组织(中肠、马氏管、围食膜、脂肪体、血淋巴和表皮)中的表达。通过RNAi技术沉默SePGRP-SA基因72 h后,qRT-PCR检测SePGRP-SA沉默效率及甜菜夜蛾4龄幼虫中肠抗菌肽相关基因(Ceropin, AttacinDefensin)和细菌载量的变化。RNAi沉默SePGRP-SA 24 h后,以苏云金芽胞杆菌菌株Bt-GS57饲喂甜菜夜蛾4龄幼虫0, 24, 48, 72, 96和120 h,计算幼虫校正死亡率;饲喂甜菜夜蛾4龄幼虫Bt-GS57后0, 24, 48和72 h,利用qRT-PCR检测中肠SePGRP-SA, Ceropin, AttacinDefensin的相对表达量。【结果】克隆获得甜菜夜蛾SePGRP-SA全长DNA(GenBank登录号:MW265930),开放阅读框长576 bp,编码191个氨基酸,其编码蛋白的预测分子量为21.59 kD。序列分析结果表明,SePGRP-SA具有典型的PGRP和Ami2保守结构域,信号肽为19个氨基酸,为分泌型蛋白;系统进化分析发现,SePGRP-SA与斜纹夜蛾Spodoptera litura的SlPGRP亲缘关系最近,氨基酸序列一致性达91.1%。发育表达谱结果表明SePGRP-SA在甜菜夜蛾4和5龄幼虫、预蛹和蛹中高表达;组织表达谱结果表明,SePGRP-SA在4龄幼虫各组织中均表达,其中以血淋巴中表达量最高。与注射dsEGFP(对照)相比,注射dsSePGRP-SA的甜菜夜蛾4龄幼虫在72 h时中肠SePGRP-SA基因表达量下调了95.26%,Cecropin, AttacinDefensin表达量显著下调,中肠细菌载量显著升高。注射dsEGFP和dsSePGRP-SA的甜菜夜蛾4龄幼虫饲喂Bt-GS57,72 h时幼虫校正死亡率分别为50.00%和73.33%,表明幼虫对Bt-GS57的敏感性明显增加。甜菜夜蛾4龄幼虫取食Bt-GS57后,中肠SePGRP-SA, Cecropin, AttacinDefensin表达量在48 h均显著增加,72 h时降低。【结论】Bt侵染能够引起甜菜夜蛾SePGRPSA基因激活抗菌肽相关基因Cecropin, Attacin和Defensin的表达。

关键词: 甜菜夜蛾, 免疫调控, 肽聚糖识别蛋白, 基因克隆; RNAi

Abstract: 【Aim】 This study aims to elucidate the function of peptidoglycan recognition protein (PGRP) in Spodoptera exigua larvae in response to Bacillus thuringiensis (Bt) infection. 【Methods】 The fulllength cDNA of SePGRP-SA from S. exigua larvae was cloned by PCR method. The relative expression levels of SePGRP-SA in different developmental stages (egg, 1st-5th instar larval, prepupal and pupal stages) and different tissues of the 4th instar larvae (midgut, Malpighian tubules, peritrophic membrane, fat body, hemolymph and epidermis) of S. exigua were analyzed by qRT-PCR. At 72 h after silencing SePGRP-SA by RNAi, the silence efficiency of SePGRP-SA, the changes in the expression levels of antimicrobial peptide-related genes (Ceropin, Attacin and Defensin) and the bacterial load in the midgut of the 4th instar larvae were detected by qRT-PCR. At 0, 24, 48, 72, 96 and 120 h after the 4th instar larvae of S. exigua were fed with B. thuringiensis Bt-GS57 strain following silencing SePGRP-SA by RNAi for 24 h, the corrected mortality rates of larvae were calculated. The relative expression levels of SePGRP-SA, Ceropin, Attacin and Defensin in the migdut of the 4th instar larvae fed with Bt-GS57 for 0, 24, 48 and 72 h were detected by qRT-PCR. 【Results】 The full-length cDNA of SePGRP-SA (GenBank accession no.: MW265930) was successfully cloned. Its ORF is 576 bp in length encoding 191 amino acid residues with the molecular weight of 21.59 kD. Sequence analysis results indicated that SePGRP-SA contains typical conserved PGRP and Ami2 domains and a 19-amino-acid signal peptide, being a secretory protein. Phylogenetic analysis indicated that SePGRP-SA is most closely related to SlPGRP of Spodoptera litura, sharing 91.1% amino acid sequence identity. Developmental expression profiles revealed that SePGRP-SA was highly expressed in the 4th and 5th instar larval, pre-pupal and pupal stages of S. exigua. Tissue expression profiles showed that SePGRP-SA was expressed in various tissues of the 4th instar larvae, with the highest expression level in the hemolymph. After the 4th instar larvae of S. exigua were injected with dsSePGRP-SA for 72 h, the expression level of SePGRP-SA in the midgut was down-regulated by 95.26%, while those of Cecropin, Attacin and Defensin were significantly down-regulated, and the microbial load in the midgut was significantly increased as compared to those in the control (infection with dsEGFP). At 72 h after the 4th instar larvae of S. exigua were fed with Bt-GS57 following injection with dsEGFP and dsSePGRP-SA, the corrected mortality rates of larvae were 50.00% and 73.33%, respectively, indicating the significantly increased sensitivity of larvae to Bt-GS57. After the 4th instar larvae of S. exigua were fed with Bt-GS57, the expression levels of SePGRP-SA, Cecropin, Attacin and Defensin in the midgut were increased significantly at 48 h after feeding, but decreased at 72 h after feeding. 【Conclusion】 SePGRP-SA gene of S. exigua can activate the expression of antimicrobial peptide-related genes Cecropin, Attacin and Defensin after infection of Bt.

Key words: Spodoptera exigua, immune regulation, peptidoglycan recognition protein, gene cloning, RNAi