Characterization And Functional Analysis Of A₂M And Fndc3a Gene In Amphioxus Branchiostoma Japonicum

Amphioxus or lancelet, a protochordate, located at the base of vertebrates, haslong been regarded as a model animal for gaining understanding of the origin ofvertebrates. Its genetic information on gene sequence and expression pattern has beenwidely used for interspecies comparative genome studies and developmentalhomology analysis. here we report the cloning of alpha-2 Macroglobulin (α₂M) geneand part of fibronectin typeⅢdomain containing 3a (fndc3a) gene from amphioxusBranchiostoma japonicum and part sequence of fndc3a was expressed withProkaryotic expression in the paper.In this study, a cDNA encodingα₂M was cloned from Branchiostoma japonicumby RT-PCR, 5'RACE and 3'RACE. The gene spans 5545 bp with a 5′untranslatedregion (UTR) of 78 bp, a 3′-UTR of 874 bp and an open reading frame (ORF) of 4593bp encoded a polypeptide of 1530 amino acids. The first 16 amino acid residues in theN-terminus were recognized as signal peptide. The deduced molecular mass of Bjα₂Mmature protein (1514 amino acids) is 164.2 kDa with estimated pI of 4.6. There werefour potential N-linked glycosylation sites in Bjα₂M located at the residual positions728, 978, 1298 and 1412. The Bjα₂M sequence is the same with putative functionaldomains including a bait region, an internal thiol ester site and a receptor-bindingdomain as other animalα₂Ms. The phylogenetic tree found that Bjα₂M formed anindependent cluster with ascidianα₂M and located at the base of vertabrates,conforming the important position of amphioxus in evolution.Northern blot revealed the presence of an approximate 5500 bp trancriptcorresponding in size to the full-length Bjα₂M cDNA we cloned in Branchiostomajaponicum. In situ hybridization histoehemistry demonstrated that Bjα₂M transcriptwas most abundant expressed in hepatic caecum and hind-gut, and present in theovary and gill. Real-time PCR demonstrated that hepatic caecum and hind-gut havehigher expression than gill, muscle, neutral tube, testis, ovary and testis. These indicated that Bjα₂M was mainly expressed in the hepatic caecum and hind-gut in atissue-specific manner. Immunohistochemistry revealed that Bjα₂M was abundantexpressed in hepatic caecum consistent with Real-time PCR. Challenge withlipopolysaccharide (LPS) soon (8h) resulted in an up-regulation of Bjα₂M expression,suggesting that Bjα₂M may be a key acute phase protein in amphioxus and involve inthe host immune defense.In this study, we also isolated part of Bjfndc3a gene which encoded 873 aminoacids from Branchiostoma japonicum. The fndc3a cDNA fragment contained eightfibronectin typeⅢdomain, and a transmembrane segment. Northern blot analysisdemonstrated that the presence of two trancripts corresponding to 4.4 kb and 4.7 kbrespectively which is similiar to human HUGO (fndc3a) gene. Challenge withV.anguillarum soon (12h) resulted in an up-regulation of Bjfndc3a expression,suggesting that Bjfndc3a may be an immune defense-relevant molecule in amphioxusand involve in the host immune defense.The expression primers were designed according to part of fndc3a cDNAsegment. The constructed recombinant expression plasimid was induced by IPTG,The expression product was analyzed by SDS-PAGE. The result indicated thatrecombination protein was successfully expressed in Escherichia Coli. The expressionproduct was a 14 kDa protein.