Identification And Characterization Of Immune Regulatory Molecule BmFAF2in Silkworm,Bombyx Mori

Antimicrobial peptides are produced after critical transcription factors Dorsal/Dif or Relish in Toll or Imd signaling pathways are activated when silkworm immune system makes response to immunal signals. After removal of immune stimulus, the activities of some critical transcriptors in Toll or Imd signaling pathways will be restrained, as a result the expression of antimicrobial peptides will be ceased and the immune homeostasis will be maintained. In fruit flies, it has been reported that a variety of immune regulatory molecules can reduce or prevent the overactivation of immune system by repressing the nuclear transcription factor. Mutation or knocking out of these molecules leads to excessive activation of the immune system. To obtain a more comprehensive understanding of the immune system, studies on immune homeostasis research is required. However, the mechanism of immune homeostasis in silkworm is unclear. In this study, multiple molecules involved in the immune homeostasis in silkworm have been identified using bioinformatics analysis. Expression patterns of these molecules in a wide variety of immune organs have been evaluated after infection of pathogenic microorganisms. Overexpression or interference of BmFAF2at a cellular level and then induced by immune stimulation results in a change of the expression level of antibacterial peptides. We also studied survival rate of silkworm against pathogenic microorganisms infection after knocking down BmFAF2using RNAi.. The main results are as follows:1. Identification of immune homeostasis moleculars in silkwormBased on the Silkworm Genome Database, immune homeostasis molecular such as BmWnt, BmUbc9, BmFAF2and BmPOSHgenes have been identified. There are9genes containing wnt domain which corresponding Numbers are BGIBMGA006145, BGIBMGA006146,BGIBMGA006147,BGIBMGA006148,BGIBMGA007580,BGIBM GA006004,BGIBMGA013783, BGIBMGA013787and BGIBMGA013981; The corresponding Numbers for Ubc9are BGIBMGA005425and BGIBMGA010222; The corresponding Numbers for FAF2is BGIBMGA008874; The corresponding Numbers for POSH is BGIBMGA012467.2. Expression pattern analysis of Immune homeostasis molecules in silkwormBmWnt, BmUbc9, BmFAF2and BmPOSH are expressed throughout the life span of silkworm. After inducing by several pathogenic microorganisms, we studied the expression pattern of BmWnt, BmUbc9, BmFAF2and BmPOSH. Generally, for the first6to12h after infection, the expression level of these genes is low, but they rapidly increased in the mid-late period of infection, followed by a rapid decrease to the starting level or even below.3. The functional research on BmFAF2Overexpression and RNAi by dsRNA of BmFAF2were used in BmE cells. We found, after cells were induced by LPS, overexpression of BmFAF2can inhibit the expression of antimicrobial peptides, BmAttacin A, BmMoricin A and Bmcecropin A, but RNAi of BmFAF2led to a increase in theexpression of BmAttacin A and BmMoricin A. In survival test, the survival rate and the expression of antimicrobial peptides in silkworm are significantly increased after RNAi of BmFAF2gene compared to the controls, suggesting that FAF2acts as one of immune homeostasis molecules in silkworm and controls the activation of immune system. We further constructed BmRelish expression vector for study the mechanism of immune regulatory function of BmFAF2in IMD signaling pathway.