Role Of Peptidoglycan Recognition Protein-A Gene In Innate Immunity Of The Cotton Bollworm, Helicoverpa Armigera

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In this study we identified and characterized a PGRP from the cotton bollworm, Helicoverpa armigera and named HaPGRP-A. we used prokaryotic expression, protein function analysis, RNAi and other methods to investigate the specific function of this gene in the innate immunity of H. armigera. The results are as follows.1. cloning and sequence analysis of HaPGRP-A:we identified a PGRP from the transcriptome of H. armigera hemocytes and named HaPGRP-A, the full-length cDNA sequence of HaPGRP-A is 1245 bp long, contained an ORF of 576 bp, which encoded 191 amino acids. The predicted MW of rHaPGRP-A was 21,358 kDa; A multiple sequence alignment indicates that PGRP-A amino acid sequences have significant similarities with PGRP sequences from other lepidopterans, but sequence comparison suggested that HaPGRP-A is not an amidase-type PGRP.2. Tissue specific expression: RT-PCR was used to compare transcript abundance of HaPGRP-A in epidermis, fat bodies, midguts and hemocytes of H. armigera. The result showed that HaPGRP-A was expressed in all of four tissues, which was expressed at a higher level in midguts.3. The expression of HaPGRP-A in H. armigera after the injection of microbes or beads:The result of qRT-PCR indicated, the expression levels of HaPGRP-A in both the fat body and the hemocytes were upregulated after the injection of microbes or beads. This result suggests that HaPGRP-A gene may play a important role in in innate immunity of the H. armigera.4. Prokaryotic expression and purification of recombinant protein: we constructed the expression vector of pET-32a-HaPGRP-A, then expressed the recombinant protein in Escherichia coli and purified by Ni column, finally obtained the recombinant protein HaPGRP-A.5. The function assays of rHaPGRP-A:(1) rHaPGRP-A could bind and agglutinate Gram-negative E. coli and Gram-positive Staphylococcus aureus, the results indicate that agglutinating activities of the rHaPGRP-A towards bacteria are Zn2+-dependent to S. aureus but not to E. coli (2) rHaPGRP-A enhanced prophenoloxidase activation in vitro, (3) rHaPGRP-A promoted the formation of melanotic nodules and enhanced melanization of chromatography beads in vivo.6. RNA interference assays:When the expression of HaPGRP-A in H. armigera larvae was inhibited by dsHaPGRP-A injection in vivo, phenoloxidase activity in bacteria-challenged larval hemolymph and the Clearing ability of hemolymph to E. coli were significantly decreased.These results indicated that rHaPGRP-A acts as a pattern recognition receptor to trigger prophenoloxidase activation system of host upon binding to invader, and the activated phenoloxidase may take part in the melanization process of nodulation and encapsulation responses. Thereby eliminating pathogens and play an important role in insects immune response.