Identification And Function Of Yippee-2 And Yippee-5 From Red Crayfish,Procambarus Clarkii

Procambarus clarkii is one of the important economic cultured shrimp species in China.Research on the immune and developmental regulation mechanism of P.clarkii has important theoretical and practical significance for improving its resistance to stress,breeding production and new species breeding.Yippee-like protein is a zinc-finger binding protein that is highly conserved in eukaryotes.It is widely involved in the regulation of a variety of cell physiological processes,but its function in the immune response of crustaceans is still unclear.In this experiment,two genes Yippee-2 and Yippee-5 in the Yippee-like protein family were cloned,and their effects on the immune response of P.clarkii were discussed.The results obtained are listed as follows:1.The open reading frame(ORF)of Yippee-2 and Yippee-5 gene of Procambarus clarkii were cloned and which contains 345 bp and 360 bp,respectively.Yippee-2gene encoded 115 amino acids and the predicted molecular weight of the protein was13 k Da.While Yippee-5 gene encoded 120 amino acids with a predicted molecular weight of 14 k Da.Both Yippee-2 and Yippee-5 proteins have a conserved Yippee-Mis18 domain.Multiple sequence alignment and phylogenetic relationship analyzed shown that the amino acid sequence of Yippee-2 and Yippee-5 were highly conserved in eukaryotes.Among them,the similarity between P.clarkii Yippee-2 and Pomacea canaliculata Yippee-2 is 95%,and the similarity between P.clarkii Yippee-5 and Penaeus vannamei Yippee-5 is 84%.2.The recombinant expression plasmid pGEX4T-1-Yippee-2 and PET-32a-Yippee-5 were constructed and the protein expression were induced by IPTG with different concentrations.SDS-PAGE and western blot results showed that Yippee-2 and Yippee-5 recombinant proteins were successfully expressed in E.coli.The Yippee-2 recombinant protein was purified by GST affinity chromatography column and the Yippee-5 recombinant protein was purified by Ni-NTA affinity chromatography column,also the corresponding mouse polyclonal antibodies were prepared.3.Far-western blot analysis confirmed that Yippee-2 protein can interact with the Yippee-5.4.The distribution of Yippee-2 and Yippee-5 gene in different tissues was detected by real-time PCR.The results shown that Yippee-2 and Yippee-5 genes were extensively expressed in the hemocytes,hepatopancreas,stomach,heart,gill,intestine and muscle.Among them,the highest expression level of Yippee-2 gene was found in the heart,while the highest expression level of Yippee-5 gene was found in the hemocytes.5.After LPS and poly(I:C)challenges,The m RNA expression levels of Yippee-2and Yippee-5 gene have significant changes among intestines,muscle,gills and hepatopancreas.Yippee-2 and Yippee-5 genes are most sensitive to LPS and poly(I:C)stimulation in gills.Comparison of LPS with poly(I:C),it is found that the changes in m RNA levels of Yippee-2 and Yippee-5 are more sensitive to LPS treatment.6.After RNA interference of Yippee-2 and Yippee-5 genes,the expression levels of the apoptosis-related genes and immune genes including Bax,Cyto C,p53,Caspase-3,ATF2,Crustin,ALF and Cactus in gills,muscles and hepatopancreas tissues were significantly affected,indicating that they may be involved in the regulation of p53 and Toll signaling pathways.