Cloning and functional characterization of the peptidoglycan recognition protein gene SePGRP-SA in Spodoptera exigua (Lepidoptera: Noctuidae) LI Ya-Zi, ZHAO Dan, GUO Xiao-Chang, WU Han, LIU Zhao-Rui, GUO Wei
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【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 fulllength 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 S ePGRP-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 o f 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 ds SePGRP-SA for 72 h, the expression level of SePGRP-SA in the midgut was down-regulated by 95.26%, while those o f 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 dsE GFP). At 72 h after the 4th instar larvae of S. exigua were fed with Bt-GS57 following injection with dsE GFP and ds SePGRP-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.