Molecular Cloning, Expression And Function Analysis Of Key Molecules In Toll-like Receptor Signaling Pathway And ALF Of Mud Crab, Scylla Paramamosain

In the past decades, diseases caused by kinds of bacteria and virus have been refrred to as an obstacle for the development of crustacean farming. Currently, initiating the study of crustacean immunity and exploring the defensive mechanism against invading pathogens have become a necessary research area. Performing these studies will facilitate exploitation and application of immunopotentiator and novel antimicrobial agents, and thus help 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 Hann, Scylla Paramamosain. Now diseases has become the biggest theat and led to a huge economic loss to this industry of mud crab farming. Thus, in order to prevent the mud crab diseases, this study focused on two key gene of Toll singnaling pathway and an antimicrobial peptide (SpALF) and further investigated their functions. The details are as follows.1. Transcriptome sequencing and Toll signal genes screeningIn order to complete transcriptome sequencing, total RNA was extracted from the tissues mixture of hemocytes and hepatopanceas of WSSV-infected mud crab. After solexa high-throughput sequencing, clustering and splicing, and annotation, we harvested plenty of cDNA sequences of mud crab. When screened by KEGG database, two key gene, spaetzle and Toll in Toll signaling pathway and one antimicrobial peptide (SpALF) were found.2. Toll-like receptors and Spaetzle Cloning and characterizationTo obtain SpToll and Spspaetzle mRNA full long sequences form known intermediate fragment, RACE was emploied, SpToll is aprotein encoded1006amino acids with molecular weight about115.2Kda. domain prediction suggest that the conserved TIR domain and IRR domain exist. Spspaetzle is an encoding288amino acid protein, Spaetzle only been found in arthropods, the domains and tertiary structure has not yet been reported. through hydrophilic amino acid sequence prediction analysis we could see its may be a conservative (3-sheet structure at275aa, the number of amino acids396-400, and445-447has the (3-pleated sheet structure are also conservative, besides, a turn structure at410aa is also conservative. Scylla to be adopted in the WSSV stimulation, we found that two genes transcript raise both and peaked at around12hours, while SpToll has a dramatic decline after it reach the peak however Spspaetzle has not.3. SpALF cloning and antibacterial propertiesProduct of complete sequence cloned based from sequences in the solexa database, we have successfully expressed SpALF in prokaryotic system (E. coli BL21-Pgex4Tl), although majority of expressed products are inclusion bodies, we completely dissolved with urea renaturation and by chromatographic column purified and obtain relatively pured SpALF. By functional analysis we found that at a very low concentration (of Vibrio Parahaemolyticus56-70, Escherichia coli,26-52) will be able to effectively inhibit the growth of Gram-negative bacteria. antimicrobial peptides on recombinant SpALF research for prevent future biological disease can reduce the use of antibiotics as well as provide a very good way for biological immunity modified.In short, we cloned tow key genes in the Toll signaling pathway and one antimicrobial peptide gene, and further analysis their function, the results indicated that Spspaetzle and SpToll play an important role in the immune responsive of mud crab, and SpALF was an important immune effector molecules with potent bacteria-bindding and bacterostatic activities.