Effect Of Bacteria On The Induction Of Anti-bacteria Peptide And Heat Shock Protein Gene Expression Of Chrysomyia Megacephala

Chrysomyia megacephala，living in abominable environment，is a mechanicalvector for spreading infectious disease, which suggests that its immune system isstrong enough to defense germina. When insects are invaded by pathogens,anti-bacteria peptides (ABP), as the vanward defense factors of humoral immune, aresynthesized promptly by adipocyte and released into hemolymph. Heat shock protein(HSP), not only played a role as molecular chaperone in recovering structure andactivity of damaged protein to maintain environment stability, but also involved inantigen presentation, immune regulation and etc. However, the relationship betweenthese two peptides in response to pathogen microorganism has not been reported. Inthis work, the relationship and roles of these peptides in immune system wereinvestigated preliminarily, based on the relationship between their expressions atdifferent time in3rdinstar larvae of Chrysomyia megacephala after injected bydifferent concentrations of Escherichia coli(E. coli), Staphyloccocus aureus(S. aureus)and PBS.Full-length cDNAs of Cecropin A and HSP70, named CM-CecA and CM-Hsp70,respectively, were cloned here. The full-length cDNA of CM-CecA gene was421bp, including5’ non-coding region (5’-UTR)87bp,3’-UTR142bp and open readingframe of CM-CecA191bp, encoding63amino acids. Relative molecular weight ofORF was6.7KD, with a signal peptide (amino acids1-23), one hydrophilic region,two hydrophobic domains and two α-helix. CM-CecA expressed in all six stages, withthe highest expression in the adult stage and relatively stable expression in the otherstages. The full-length cDNA of CM-Hsp70gene was2281bp, including5’-UTR186bp,3’-UTR136bp and open reading frame of CM-CecA1959bp which encoded652amino acids with the relative molecular weight of70KD. CM-HSP70protein wascomprised of three main domains, that is, ATPase domain highly conserved inN-terminal with the relative molecular weight of44KD, approximately18KDconserved peptides-binding domain and almost10KD variable domain in C-terminal.The end of C-terminal was characterized by the specific motif EEVD, suggesting thatthe protein was located in the cytoplasm. CM-Hsp70reached its highest expressionlevel in the egg stage and expressed relatively stable in the other stages.The induction functions of different concentration of pathogen at different timeto the expression of CM-CecA were different. The times of intensive response toE. coli and S. aureus were both at the twelfth hour,40times and41times, respectively.The highest response intensity to E. coli concentration were OD6000.3and2.0, whileto S. aureus were OD6000.5and1.0. However, there was no significant rise in theexpression of CM-Hsp70, even declined when larvae were stimulated by E. coli or S.aureus, suggesting that CM-CecA gene, no CM-Hsp70protein, could be induced byE. coli and S. aureus. The immune system was inclined to attack the invaders whenthe larvae faced the threat. When infected by pathogen, the expression of CM-CecA inlarvae increased promptly to strengthen the immune defense. Considering the greatvariety of heat shock protein and our limited investigation (only CM-Hsp70), theinduction of pathogen to the expression of heat shock protein needs furtherinvestigation.