Cloning, Evolution And Functional Analysis Of STATa And STATb Genes Of Amphora Platyphylla

Signal transducer and activator of transcription is an important transcription factors,which play a variety of biological functions in organism.However,the STAT gene family has yet not been reported in amphioxus to date.In this study,using molecular cloning,real time PCR,Co-IP and other molecular biological techniques and bioinformatics methods,we analyzed the evolution,function and mechanism of the STAT family in Branchiostoma belcheri.The experimental results are as follows:1.The full length of STATs gene sequences were obtained by RT-PCR and RACE-PCR techniques(designed as AmphiSTATa and AmphiSTATb).The total length of AmphiSTATa cDNA was 3901bp containing the 5'-UTR region of 140 bp,ORF of 2739 bp,and the 3'-UTR of 1022 bp.The total length of AmphiSTATb cDNA was 3878 bp containing the 5'-UTR region of 173 bp,ORF of 2436 bp,and the 3'-UTR of 1269 bp.2.Sequence conservation and structural analysis showed that AmphiSTATa and AmphiSTATb proteins are closely related to the STAT5 subfamily.AmphiSTATb has a higher similarity with the STAT5 subfamily.The phylogenetic analysis showed that AmphiSTATa and STAT1-STAT4 share a common ancestor gene;AmphiSTATb was clustered together with the STAT5 and STAT6 of other species.These evidences indicated that AmphiSTATa and AmphiSTATb are new members of STAT family.3.The analysis of the tissue expression pattern of amphioxus AmphiSTATa and AmphiSTATb genes showed that the AmphiSTATa and AmphiSTATb genes were expressed in all the examined tissues(gill,hepatic caecum,intestine,muscle and notochord)and the expression level in gills is relatively high.The expression of AmphiSTATa AmphiSTATb genes at different time points(2h,4h,6h,12h,24h and 48h)after LPS stimulation showed that the AmphiSTATa and AmphiSTATb genes was upregulated at 4h and 6h after LPS stimulation.The result indicated that AmphiSTATa AmphiSTATb genes are involved in innate immune responses.4.Using Native-PAGE and Co-IP technology,it has been proved that homodimers or heterodimers can be formed between AmphiSTATa and AmphiSTATb proteins;Subcellular localization technology shows that AmphiSTATa and AmphiSTATb proteins can be localized on the nucleus.These evidences indicate that AmphiSTATa and AmphiSTATb have similar functions and mechanisms to the STAT in vertebrate.In summary,our research provided a new insight into evolution and molecular mechanism of AmphiSTATa and AmphiSTATb genes in Branchiostoma belcheri.