Molecular Characterization And Function Analysis Of The Large Yellow Croaker(Larimichthys Crocea)Rac,Rab7 And Rab5A Genes After Immune Challenges

Small GTPase are crucial molecular switches which are believed to be important mediators for cell division,formation of cytoskeleton,vesicle transportation and Nucleo-cytoplasmic transportation.They play an important role in immune challenges.The sequence of Rac1,Rac2,Rac3,Rab7 and Rab5 A were obtained from a comprehensive transcriptome of Larimichthys crocea in our laboratory during screening and were named as lycRac1,lycRac2,lycRac3,lycRab7 and lycRab5 A respecctively.The sequences of these genes were obtained after aligned with the genomic sequences of the L.crocea.We analysed the molecular characterization,tissue expression and the variation in liver,spleen,head-kidney after challenged with LPS,polyI:C,V.Parahemolyticus of these genes.The immune colloidal gold technique was used to locate the lycRAC2 in spleen.We also used the GST pull down assay and mass spectrum identification to determine the proteins that interacted with lyc RAB5 A protein.The obtained results are as follows:1.There are three subtypes in RAC protein which named RAC1,RAC2 and RAC3.The full length of lycRac1 was 2329 bp with an open reading frame(ORF)of 579 bp,encoding putative proteins of 192 amino acids.The full length of lycRac2 was 1221 bp with an open reading frame(ORF)of 579 bp,encoding putative proteins of 192 amino acids.The full length of lycRac3 was2182 bp with an open reading frame(ORF)of 579 bp,encoding putative proteins of 192 amino acids.The multiple amino acids alignment showed that lycRac1 shared hith similarity with Rac1 from Chelonia mydas,Maylandia zebra and Takifugu rubripes when lycRac2 shared highly similar sequence with Rac2 from Lates calcarifer,Poecilia formosa and Xiphophorus maculates,lycRac3 was similar with the Rac3 from Bos Taurus,Xenopus tropicalis and Ictalurus furcatus.The genomic analysis indicated that lycRac1 had 6 exons and 5 introns while lycRac2 had 7 exons and 6 introns and lycRac3 had 7 exons and 6 introns.The phylogenetic tree showed that fish Rac1,Rac2 and Rac3,including lycRac1,lycRac2 and lycRac3,formed one cluster.The quantitative real-time PCR analysis revealed that lycRac1,lycRac2 and lycRac3 transcripts were present in all tested tissues,among which the highest expression of lycRac1 m RNA was in the heart and the weakest expression in the head-kidney.Relatively lower levels of lycRac1 were observed in the blood,brain and gill.The highest expression of lycRac2 mRNA was in the head-kidney and the lest level in the muscle,followed by kidney,spleen,gill andblood when the best expression of lycRac3 mRNA in the blood and the lest level in the intestine.Relatively lower levels of lycRac3 were observed in the spleen and brain.lycRac1,lycRac2 and lycRac3 expression increased in the liver,spleen and head-kidney after lipopolysaccharide,Polyinosinic Polycytidylic acid and Vibrio Parahemolyticus stimulation.Antibody was obained after purified the lycRAC2 protein.Western-blot analysis indicated that the antibody of lycRAC2 had a highly specificity.The subcellular localization revealed that lycRAC2 expressed in the fiber between cells.The results suggested that lycRac1,lycRac2 and lycRac3 might be important immune-related genes,which played indispensible roles in the immune defenses against bacterial infection.The localization of lycRac2 provided a powerful evidence about the signaling function of lycRac2.2.Rab7 GTPase is one of the members of Ras family which belongs to the small-GTPase.The complete cDNA sequence of lycRab7 was 2218 bp with an open reading frame(ORF)of624bp encoding putative proteins of 207 amino acids.Multiple amino acids alignment indicated that lycRab7 shared highly similar homology with Rab7 from Danio rerio,Homo sapiens and Oryctolagus cuniculus.The genomic analysis showd that lycRab7 had 5 exons and 4 introns.The phylogenetic tree showed that fish Rab7,including lycRab7,formed one cluster.The quantitative real-time PCR analysis revealed that lycRab7 transcripts were present in all examined tissues,among which the highest expression of lycRab7 mRNA was in the brain,moderatly expressed in blood and heart,with the lest expression in stomach.The transcripts of lycRab7 was up-regulated in liver,spleen and head-kidney in different level after stimulated by LPS,polyI:C and vibrio.Antibody was obained after purified the lycRab7 protein.Western-blot analysis indicated that the antibody of lycRab7 was specificity.The results demonstrated that lycRab7 played a crucial role in deffence of the bacterial infection.3.Rab5 A is one of the members of the Rab family.In this study,Rab5A(named as lycRab5A)purified fusion protein was acquired.The GST pull-down assay indicated that lycRAB5 A was interacted with Calcium-transporting ATPase,Creatine kinase M-type and Actin,alpha skeletal muscle.The results indicated that lycRAB5 A and the interacted proteins played an important role in immune challenge.