Identification And Characterization Of Immunological Function Of BmPTP In Silkworm, Bombyx Mori

Protein tyrosine phosphatases remove phosphate group from Tyr residues on protein in eukaryotes. The importance of these enzymes in cellular physiology was underscored before, now much more research has focused on them Protein tyrosine phosphatases are important regulatory molecules in cell signal transduction and cell cycle. They take part in cell growth, differentiation, transformation, reproduction, immunity and many other physiological processes. Research on protein tyrosine phosphatase was mainly concentrated in mammals. In insects there was little report, especially in immune function research. In this study, we identified and classified protein tyrosine phosphatase in Bombyx mori by bioinformatics, and determined BmPTP’s immune function. Firstly we evaluated the expression changes of BmPTP in a spatio-temporal manner and after microbial infection. Then we overexpressed or knocked down BmPTP at cellular level, and examined the replicative production of BmNPV in those cells. RNA interference at silkworm individual level has also been carried out to test and verify the result of cellular level. The main results are as follows:1. Identification and classification of protein tyrosine phosphatase in silkwormBased on the Japanese KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/) and the Chinese Silkworm Genome Database (http://www.silkdb.org/silkdb/) we identified 41 gene sequences that contain protein tyrosine phosphatase catalytic domain.Then we performed a phylogenetic tree alignment of these 41 genes, and classified them by catalytic mechanism. We also determined the target of this study, BmPrP(BMgn016959).2. The functional research on BmPTPFirstly we examined the expression profile of BmPTP after silkworm were challenged with Bacillus bombyseptieus, Serratia marcescens or Bombyx mori nucleopolyhedrovirus. We found that expression of BmPTP was much higher at 48 h after BmNPV infection. And it remarkly changed after injection of Bacillus bombyseptieus and Serratia marcescens to silkworm larvae. In order to make a further study on the immunological function of BmPTP, We constructed its eukaryotic and prokaryotic expression vector. We confirmed its protein tyrosine phosphatase activityusing cell lysate. We also synthesized a double stranded RNA against BmPTP to knock it down. Overexpression of BmPTP in BmE cells resulted in higher proliferation of BmNPV, and partially silence of BmPTP resulted in lower production of BmNPV dsRNA mediated-RNAi on silkworm larvae confirmed the result in cell.