一个调控抗菌肽表达的小菜蛾肽聚糖识别蛋白(PxPGRP-SA)

A peptidoglycan recognition protein(PxPGRP-SA) regulating the expression of antimicrobial peptides in Plutella xylostella(Lepidoptera:Plutellidae)

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是昆虫免疫系统中一类重要的模式识别蛋白。本研究旨在阐明经苏云金芽孢杆菌Bacillus thuringiensis侵染后,小菜蛾Plutella xylostella PGRP-SA基因(命名为Px PGRP-SA)在体内的表达模式和对抗菌肽基因的表达调控。【方法】本研究利用实时荧光定量PCR(qRT-PCR)技术分析B.thuringiensis侵染小菜蛾幼虫后Px PGRP-SA的转录模式,通过RNAi技术结合抗血清封闭实验检测Px PGRP-SA对小菜蛾抗菌肽基因的表达调控作用。【结果】qRT-PCR检测表明,小菜蛾4龄幼虫在注射具有活性的B.thuringiensis 6 h后,Px PGRP-SA在脂肪体和血细胞中表达量迅速上升,其中脂肪体中的表达量在注射24 h后达到高峰,而在血细胞中的表达量在18 h后达到高峰。RNAi沉默小菜蛾4龄幼虫Px PGRP-SA的转录后,可显著降低小菜蛾脂肪体中cecropin,moricin-2,lysozyme和defensin 4个抗菌肽基因及Dorsal和Sptzle基因的mRNA转录水平;注射anti-Px PGRP-SA封闭小菜蛾体内Px PGRP-SA的活性后,也可降低小菜蛾脂肪体中4个抗菌肽基因的mRNA转录水平;Px PGRP-SA转录沉默后,同时导致添食B.thuringiensis的小菜蛾幼虫的存活率明显降低。【结论】Px PGRP-SA参与了小菜蛾体内抗菌肽cecropin,moricin-2,lysozyme和defensin基因的表达调控,并在免疫防御B.thuringiensis的侵染过程中起了重要的作用。

【Aim 】 Peptidoglycan recognition proteins（ PGRPs） are one kind of important pattern recognition proteins in insect immune system. In this study,we aim to investigate the expression patterns of PGRP-SA from Plutella xylostella（ designated as Px PGRP-SA） and its involvement in the regulation of the expression of antimicrobial peptide genes in P. xylostella after infection by Bacillus thuringiensis.【Methods】The transcription pattern of Px PGRP-SA in P. xylostella larvae infected by B. thuringiensis was analyzed by quantitative real-time reverse transcription PCR（ qRT-PCR）,and the regulation of the expression of antimicrobial peptide genes by Px PGRP-SA in P. xylostella was investigated by RNAi technology and antiserum blocking assay. 【Results】qRT-PCR analysis revealed that the transcripts of Px PGRP-SA increased significantly at 6 h after injection of live B. thuringiensis into the fat body and hemocytes of the 4th instar larvae of P. xylostella,with the highest expression level at 24 h in fat body and 18 h in hemocytes,respectively. The transcript of Px PGRP-SA in the 4th instar larvae of P.xylostella decreased due to RNAi. Blocking down the transcript of Px PGRP-SA by RNAi also reduced the transcriptional levels of four antibacterial peptide genes including cecropin,moricin-2,lysozyme and defensin in the 4th instar larvae and the mRNA levels of Dorsal and Sptzle genes significantly. Blocking the activity of Px PGRP-SA by injection of anti-Px PGRP-SA serum into the 4th instar larvae of P.xylostella also decreased the transcription levels of antibacterial peptide genes in the fat body rapidly.Silence of the expression of Px PGRP-SA decreased the survival rate of P. xylostella larvae feeding live B.thuringiensis significantly. 【Conclusion】Px PGRP-SA is involved in regulating the expression of downstream antibacterial peptide genes including cecropin,moricin-2,lysozyme and defensin in P. xylostella,and plays an important role in the immune defense of P. xylostella against B. thuringiensis infection.