Identification And Protein Functional Analysis Of The Lysozyme Anddefensin From Musca Domestica

The housefly lives in the poor environment with much bacteria and spread a lot of disease, but itself is infected seldom. The reason is that in the long-term evolution process, the housefly forming a strong and effective immune and defense mechanisms to deal with all kinds of pathogen infection. The antimicrobial peptides containing defensin and lysozyme in the body are very important factor. Research the role of antimicrobial peptides and antibacterial mechanism has important theoretical value of practical significance to antibacterial peptides for the further research and to bring the new dawn for finding out new antibacterial drugs.This thesis study with housefly, contains2primary parts.Part1:lysozyme from housefly1. The lysozymes from housefly were searched from transcriptome database and were named. With RACE (rapid-amplificationof cDNA ends) technique, full-lengthcDNA (516bp) of lysozyme (named lysozyme3)(GenBank:HQ897688). Lysozyme3contains a429bp open reading frame (ORF) encoding142amino acid residues and containing a putative signal peptide of20amino acids and the mature protein of122amino acids which include8structural cysteines residues which form4pairs of disulfide bonds, with a molecular mass of about13.89kD and pI value of6.45. The amino acid sequence that was named lysozyme3showed the higher homology with Lysozyme2in the light of homologous assay.2. Northern blot analysis show that lysozyme3was highly expressed in gut, and expressed at relatively low levels in hemocyte, but did not expressed nearly in epidermis and fat body.3. RNA interference was designed ably and statisticed the weights of interference group and control group larvaes, which showed that the weights of larvaes reduced obviously because the ability of digestion became lower after lysozyme3of M. domestica were knocked.4. The result of RT-qPCR indicated that lysozyme3gene expression level was highest in intestine, higher in whole larveas and hemocyte, as well as regulated down in starvation24h. The expression level declined from3to12h challenged with bacteria, and can be restored a modicum from12to24h challenged with bacteria, then started to drop.5. The recombinant lysozyme3was accomplished by prokaryotic expression system, and SDS-PAGE result showed purpose strip appeared at expected position.Part2:defensin from housefly1. The defensins from housefly were searched from transcriptome database and were named. A full-lengthcDNA (392bp)(named defensin2) were cloned from housefly by RACE (rapid-amplification of cDNA ends) based on dbEST (the expressed sequence tags database). Defensin2gene contains a276bp ORF encoding142amino acid residues and containing a putative signal peptide of23amino acids, propeptide of28amino acids and the mature protein of40amino acids which include6structural cysteines residues which form3pairs of disulfide bonds, with a molecular mass of about4.0kD and pI value of8.69. The amino acid sequence analysed by Blast showed the higheer homology with insect defensins, and sequence homology with defensin1reported by Wang lai cheng is93%.2. The result of RT-qPCR indicated defensin2gene expression level was highest in fat body and hemolymph. Defensin2gene expressed down-regulated after the larvae were challenged with E. coli and up-regulated after the larvae were challenged with S. aureus. Defensin2gene expression level regulated up rapidly, in process of recovering after the larvae were hot at42℃.3. In the defensin2RNA interference to larvae, the survival rate of RNA interference group is lower obviously than the control group larvae at24h the larvaes challenged by gram-positive bacterium. The survival rate of RNA is same between interference group and the control group larvae at24h the larvaes challenged by gram-positive bacterium.4.678bp promoter sequence was cloned by genome walker. Analysis discovery that+1scores calculated is0.97and that"TATA"box was discovered in proper position. The sequence included The typical sites related with immune and defense, NF-KB, C/EBP (CAAT enhancer-binding protein) and AP family (AP-1, AP-2, AP-3). The promoter was transfected into SF9cells from insect by pEGFP-1with GFP but without promoter. The result shows fluorescent signal. 5. The recombinant defensin2was accomplished by prokaryotic expression system, and SDS-PAGE result showed purpose strip appeared at expected position.