Cloning Of JAK/STAT Pathway Related Major Genes Of Silkworm, Bombyx Mori And The Response Of JAK/STAT Pathway On Virus And Microsporidian Infection

The JAK/STAT signaling is a multiple cytokines shared pathway. Despite thenumber and diversity of cytokines, the related proteins and fundamental functionsappear to be highly conserved. To identify the elements of JAK/STAT pathway ofsilkworm, Bombyx mori.JAK/STAT pathway related major genes in silkworm wereobtained through electronic cloning and conventional RT-PCR, Domain analysis on therelated protein were conducted by using bio-softs online, and analysis the function ofJAK/STAT pathway related major genes in silkworm. The expression profile of thesegenes in diverse tissues were analysed through microarray database or qPCR. Thesilkworm JAK/STAT pathway related major genes including BmSTAT, BmHOP,BmSOCS2, BmSOCS5A, BmSOCS5B, BmSOCS6, BmDRK,Bmken, BmPIAS1,BmPIAS2and BmPI3K were cloned respectively, but the homologues of DrosophilaUpd1, Upd2and Upd3. Sequence analysis showed that BmHOP contained CRD_FZ,Kringle domains, transmembrane region and Pkc superfamily protein kinase catalysisdomain TyrKc, BmSTAT had two isoforms, which were consisted of SH2, STAT_bind,STAT_int and STAT_alpha domains; Both BmSOCS2A and BmSOCS6A had aconserved SH2domain and a SOCS box in cloned three genes of BmSOCS family;BmDRK was consisted of SH3and SH2domains that connected in series to formSH3-SH2-SH3; BmKen contained BTB and zinc finger domains; BmPIAS1andBmPIAS2contained LCR domains; BmPI3K had two conserved SH2domains. TheJAK/STAT pathway related major genes were cloned successfully in silkworm. Theanalysis of conserved structural domains suggesting that BmHOP is one of JAK kinases,which phosphorylates the BmSTAT transcription factor and lead to activation of targetgenes; BmSOCS members, Bmken, BmPIAS1and BmPIAS2are negative feedback regulators in the JAK/STAT pathway.The Real-Time PCR results displayed that the aboundence of BmSOCS6mRNA was highest insilkworm testis and lowest in haemocyte.Mouse polyclonal antibody against BmSOCS6wasprepared with recombinant protein BmSOCS6expressed in E. coli.Western Blotting result displayedthat polyclonal antibody could specifically bind to BmSOCS6. Localization revealed that BmSOCS6mainly localized in cytoplasm of BmN cells. And the Immunohistochemistry revealed thatBmSOCS6is expressed both in testis and ovary and also in spermatophore and ovarioleIn order to investigate the immune response pathway on anti-virus and anti-microsporidian insilkworms, the expression levels of BmSTAT were determined by real-time PCR, the results showedthat the expression levels of BmSTAT in haemlymph were increased by14and12times at48and72hours post infection with BmNPV, increased by5times in midgut at5days post-infection withBmDNV, but obvious change was not found in midgut at4days post infection with BmCPV andmicrosporidium. So the JAK/STAT signaling pathway may be contributed to the antiviralresponse. These results will help us to further understand JAK/STAT signaling pathway insilkworm, the role of JAK/STAT pathway on immune response to virus infection...