Molecular Cloning And Expression Analysis Of Immune-relevant Genes P38, PIAS And HSP90of The Mud Crab, Scylla Paramamosain

In recent years, diseases caused by kinds of bacteria and virus have been known to as an obstacle for the development of crustacean farming. Currently, initiating the study of crustacean innate immunity and exploring the defensive mechanism against invading pathogens have become a necessary research area. Performing these studies will facilitate exploitation and application of immunoenhancement and novel antimicrobial agents, in order to control crustacean diseases. The mud crab, Scylla Paramamosain is one kind of marine crab with crucial economical and nourishing value. The taxonomy for mud crab is Arthropoda, Crustacea, Malacostraca, Decapoda, Pleocyemata, Portunidae, Syclla De Harm, Scylla Paramamosain. Now diseases has become the biggest threat and led to a huge economic loss to this industry of mud crab farming. Therefore, in order to prevent the mud crab from diseases, this study focused on three innate immune related genes P38, PIAS and HSP90. In this study, we cloned their full-length cDNA sequences and described their characterization of the the deduced amino acid sequences, the relative expression profiles against different immune stimulation, All these results suggested that these three genes involved in the defense system of mud crabs against pathogenic bacteria and virus. Specific studies are as follows:1. Molecular cloning and expression pattern analysis of P38and PIAS of the mud crab, Scylla paramamosainP38mitogen-activated protein kinases (MAPKs) are one of the key members in P38MAPK pathway expressed from invertebrate to vertebrate, and protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of various signaling pathways as negative regulator, also play a role in P38MAPK pathway. However, the functional research of these two genes in invertebrates is limited. To further understand the role and mechanism of them in immune and stress response, In present study, two sequences fragment of P38and PIAS homologue from mud crab Scylla paramamosain was isolated through transcriptome sequencing with a mixture of hepatopancreas and hemocytes. The full-length cDNA of them were obtained through rapid amplification of cDNA ends (RACE) approaches, P38gene from Scylla paramamosain contained an open reading frame of1095bp which encoding a365-amino-acid protein. Tissue expression profiling of SpP38mRNA in hemocytes, heart, hepatopancreas, gills, stomach, intestines, muscle, connective tissue and gonad of S. paramamosain calculated by Real-time PCR showed that SpP38expressed the most in hemocytes. After been challenged by the Staphylococcus aureus, Vibrio harveyi and WSSV, rising expression levels were all detected. SpPIAS gene contained an open reading frame of2364bp encoding a polypeptide of788amino acids with all the four PIAS family signatures. the expression levels of SpPIAS were up-regulated after challenge with Staphylococcus aureus, Vibrio harveyi and WSSV for2-24h. These results suggested that SpP38and SpPIAS may play an important role in the innate immune system of mud crabs.2. Molecular cloning, expression pattern analysis and characterization of Heat Shock Protein90(HSP90) of Scylla paramamosain.Heat shock protein90(HSP90), functions as a molecular chaperone in protein biosynthesis playing an important role in signal transduction, immune responses and embryogenesis. We have isolated the the fragment of HSP90homologue from mud crab Scylla paramamosain through transcriptome sequencing of mixture of hepatopancreas and hemocytes, The full-length cDNA of HSP90is harvested through RACE technology and it contains73bp5’terminal UTR,577bp3’terminal UTR and2373bp open reading frame for a protein of790amino acid residues which was named SpHSP90. BLASTP results demonstrate that SpHSP90share higher identity with Tribolium castaneum and Locusta migratoria than other reported crustacean. Phylogenetic tree based on comparison of HSP90proteins show that SpHSP90as well as Daphnia pulex forms a unique cluster while other reported crustacean HSP90s are grouped into another branch. The tissue distribution was tested by quantitative real-time PCR and the results showed that the expected PCR products were amplified from each examined tissue suggesting that HSP90was constitutively expressed in Scylla paramamosain. The highest expression of HSP90was found in hepatopancreas, the following was in heart, and the lowest expression of it was found in blood. The expression pattern of SpHSP90was also analyzed by quantitative real-time PCR and the result showed that SpHSP90was elevated after challenge with the Gram-positive bacterium(Staphylococcus aureus) Gram-negative bacterium (Vibro harveyi) and virus (white spot syndrome virus, WSSV). All these results suggested that SpHSP90was a unique gene and involved in the defense against invading pathogens. Furthermore, this encoding gene was expressed in prokaryotic expression system, and the recombinant protein with His-tags was obtained from His-Tag protein purification system. The result of microorganism binding was displayed by Western blot assay, revealed that SpHSP90could bind to E.Coli and V. harveyi. Those provide an important reference material for unraveling the function and the mechanism of action of this protein in the future.