Molecular Cloning And Expression Patterns Of Peptidoglycan Recognition Protein C In The Cotton Bollworm,Helicoverpa Armigera

Invertebrates lack the adaptive immune system, but they can resist rapidly and effectively to infection of various pathogens through innate immune system.The innate immune system recognizes pathogen associated molecular patterns through a series of highly conservative pattern-recognition receptors that can activate signal transduction pathways to induce immune response.Peptidoglycan recognition protein(PGRP)can recognize peptidoglycan of microbial surface and plays an important role in immune regulatory pathways.Studies found that PGRP may be involved in the innate immune response such as phagocytosis,melanization and antimicrobial peptides.Peptidoglycan recognition protein have been found in a variety of insects,the study showed that although these PGRPs is conservative in the structure,their function and expression patterns is different. Therefore,in order to comprehensively and thoroughly understand the function of PGRP in the innate immune, we need to carry out research on PGRP in more types of insects. In this study,we cloned a peptidoglycan recognition protein in cotton bollworm and named it HaPGRP-C,at the some time we done a preliminary study on the function and pxpression pattern of the gene in the innate immune.The findings of this experiment can be divided into the following aspects:1,gene cloning and sequence analysis of the peptidoglycan recognition protein HaPGRP-C in the cotton bollworm:Using the technology of Smart PCR,we get the full sequence of a PGRP gene in cotton bollworm. This gene has a complete open reading frame (ORF) and it can encode212amino acids.The protein that the gene encoded has a Ami structure domain and a PGRP structure domain which is located N-terminal from31amino acids to173amino acids.The protein also contains a signal peptide and transmembrane domains;Sequence alignment shows that the similarity between the sequence of the cotton bollworm PGRP and PGRP-C in other Lepidoptera is very high,so we named it HaPGRP-C.2,Tissue specific pxpression analysis of HaPGRP-C: Extract total RNA of four organizations of the cotton bollworm and use RT-PCR to analysis its expression in this four different tissues:epidermis, fat body, midgut and hemolyph,the result shows HaPGRP-C are expressed in the three other tissues in addition to the epidermis and the expression in fat boby is the highest.3,The effects on expression of HaPGRP-C after microbial immune stimulating:Inject Escherichia coli and Staphylococcus auras into the cotton bollworm and then use real time PCR to detect the expression changes of HaPGRP-C in fat boby and blood cell.The result shows that the expression of HaPGRP-C in fat boby has no significant changes in2h and6h after inject Gram-positive bacteria and Gram-negative bacteria, but the expression is up-regulate after12h.The expression level of HaPGRP-C in hemolymph is up-regulate at2h after inject Gram-positive bacteria and the level has no change at6h and12h, but the expression also increase at12h after inject Gram-negative bacteria.These results suggest that HaPGRP-C mat be involved in the regulation of the innate immune.4,The effects on expression of HaPGRP-C after injection of hormone:Inject DMSO,JHA(juvenile hormone analogues) and20E(ecdysone) into the cotton bollworm and then use real time PCR to detect the expression changes of HaPGRP-C in fat boby and blood cell.The result shows that after injection of two hormones, the expression of HaPGRP-C didn't change siginificantly in fat boby or hemolymph.5,Prokaryotic expression of HaPGRP-C:Amplified the ORF area of HaPGRP-C and then clone it to prokaryotic expression vector pET32a(+) in order to construct recombinant expression plasmid which was then transformed into E. coli BL21strains, after induce expression of the transformed bacteria,we use Ni-NTA affinity column chromatography to available the recombinant protein。6,Binding activity of recombinant protein and bacteria:Mixed recombinant protein and a certain amount of gram-negative bacteria or Gram-positive bacteria, then observe the binding activity of recombinant protein and bacteria under the microscope.we found that there is no binding phenomenon between Escherichia coli and recombinant protein,but binding phenomenon is evident between Staphylococcus auras and recombinant protein.In summary, through this experiment we get a peptidoglycan recognition protein and conducted some preliminary research on function of HaPGRP-C.The results showed that HaPGRP-C may be used as a pattern recognition protein and involved in the regulation of the innate immune of cotton bollworm.