A Role Of Small GTPase Rac1 In Innate Immunity Of Crayfish Procambarus Clarkii

The red swamp crayfish,Procambarus clarkii is one of the most important economically cultured species in China.It distributes in many regions,especially in the rivers,swamps,lakes in the middle and lower reaches of the Yangtze River.However,due to the deterioration of the water environment and the unscientific intensive cultivation and other adverse factors,crayfish suffer from diseases and result in the economic losses.Explorating the immune-related genes and immune mechanism in crayfish is important for theoretical study and useful for improving its disease resistance and disease-resistant breeding.Ras-related C3 botulinum toxin substrate 1(Rac1)is a key regulator,which regulates many biological processes such as transport,membrane transport,cell-cell adhesion and cell migration.In this study,Rac1 gene of Procambarus clarkii was cloned and its immunological function was discussed.The results were listed as follows:1.The open reading frame(ORF)of Rac1 gene of Procambarus clarkii was cloned and its length was 579 bp.It encoded 192 amino acids and the predicted molecular weight of the protein was 21.4 kDa.The protein had a conserved Rho domain.Multiple sequence alignment and phylogenetic tree analysis showed that Rac1 protein was conserved in both invertebrates and vertebrates,and had the closest relationship with Fenneropenaeus chinensis.2.The recombinant expression plasmid pET-32a-Rac1 was constructed and transformed into E.coli BL21.The protein expression was induced by IPTG at different concentrations.SDS-PAGE and western blot results showed that Rac1 recombinant protein was successfully expressed in E.coli.The recombinant protein(1.3 mg/mL)was purified by Ni-NTA affinity chromatography column and the corresponding mouse polyclonal antibodies were prepared.3.The distribution of Rac1 gene in different tissues was detected by real-time PCR.Racl gene was extensively expressed in the hemocytes,hepatopancreas,stomach,heart,gill,intestine and muscle.The highest expression level was found in the hemocytes.4.After LPS and poly I:C challenges,the transcription level of Racl gene in hemocytes,hepatopancreas and intestine was significantly up-regulated,and the transcription level of Rac1 gene in hemocytes was higher than that in hepatopancreas and intestine.5.The expression levels of immune-related genes like lectin,TNF Toll,ALF,crustin and cactus in hemocytes obviously changed after RNA interference of Racl gene.6.Far-western blot analysis confirmed that Racl protein can interact with the PBD domain of PAK1 directly in vitro.