Cloning, Characterization And Expression Of SRA Genes From Amphioxus Branchiostoma Belcheri Tsingtauense

Amphioxus belongs to the cephalochordate, and is exposed to the position of the invertebrates to vertebrates. As a result, amphioxus has long been regarded as the most important animal to study the origin and evolution of vertebrates. SRA, a steroid receptor coactivator, is the first found molecule that has the unusual property to function both at the RNA and the protein levels. sites. Numerous investigations have been carried out in invertebrates, but little is known to date about it in the amphioxus Branchiostoma belcheri.In this paper, we used5'-rapid amplification of cDNA ends (RACE) to obtain the full-length sequence of SRA from Amphioxus Branchiostoma belcheri tsingtauense, named AmphiSRA. The full length of AmphiSRA cDNA (GenBank accession number: JN384108) obtained was1542bp long with a5'-untranslated region (UTR) of165bp and a3'-UTR of852bp with the polyadenylation signal and a polyadenylation tail. The ORF of AmphiSRA encoded a polypeptide of174amino acids with a calculated molecular weight of about19.5kDa. The BLASTp searching at NCBI showed that the deduced protein had a full SRA family domain.Northern blot showed three bonds in amphioxus B. belcheri. They were more than4kb,1.6kb and less than500bp in length, respectively. This result indicated that the isolated cDNA we have got was likely to be full length. And other transcripts may be existed in the amphioxus. To study the expression pattern of AmphiSRA in adults and embryos/larvaes, whole mount in situ hybridization was carried out. AmphiSRA was expressed in almost all of the organzation of amphioxus, especially in gill, intestine, testis, neural tube, notochord and ovary. What is in accordance with the expression in embryos/larvaes.To investigate whether AmphiSRA is a translation subtype, we constructed recombinant vector pET-28a-SRA and transferred into E.coli BL21. Induced by IPTG for expression and western blotting, the result indicated that AmphiSRA was expressed and its molecular weight was approximately19.5kDa. That identified our conlusion, AmphiSRA is a translation subtype.