Characterization, Expression And Evolution Of AmphiANT And AmphiTRAPβ Genes From Amphioxus

Amphioxus or lancelet, a cephalochordate, has been regarded as the closest living invertebrate relative of the vertebrates. Studies on the gene structure, function and expression in amphioxus will greatly contribute to understanding of the origin and evolution of the vertebrates. Its genetic information on gene sequence and expression pattern has been widely used for interspecies comparative genome studies and developmental homology analysis. In this paper, It is reported the cloning, characterization, phylogenetic analysis and expression analysis of AmphiANT and AmphiTRAPβ from the gut cDNA library of amphioxus Branchiostoma becheri tisingtauense.AmphiANT, was identified from the gut cDNA library of amphioxus B. becheri tisingtauense, consists of 1289 bps and contains an 897 bps open reading frame (ORF) corresponding to a deduced protein of 298 amino acids. AmphiANT possesses 74%, 76 % and 74% identity to human , zebrafish and fruit fly ANT proteins, respectively and includes the ADP/ATP carrier signature, RRRMMM, and three homologous sequence repeats of about 100 amino acid residues each and each repeat has a signature motif P-X-[D/E]-X-X-[R/K]. The unrooted phylogenetic tree constructed identified two ANT clades, one consisting of invertebrate ANT and the other comprising vertebrate ANT, and AmphiANT appears more closely related to invertebrate ANT clade. Northern blotting analysis revealed a ubiquitous expression pattern of AmphiANT in the different tissues of adult amphioxus. However, AmphiANT transcripts were more abundant in eggs, neurula, hatched larvae and 1-day larvae, and to a lesser extent in zygotes, blastulae and gastrulae. It is proposed that the difference in AmphiANT transcript levels in the different stage embryos is not only parallel to the requirements for mitochondrial energy at different developmental processes but also dependent on the balance of transcription of AmphiANT gene and its mRNA translation.AmphiTRAPβ, identified from the gut cDNA library of amphioxus B. becheri tisingtauense. consists of 687 bp and contains a 564-bp open reading frame (ORF)corresponding to a deduced protein of 187 amino acids with a calculated molecular mass of 20.4 kDa. The protein AmphiTRAPP encoded by the cDNA possesses 60%, 59% and 56% identity to zebrafish, canine and human TRAPp molecules, respectively. It also contains two glycosylation Asn sites and a transmembrane domain. In all four cDNAs, the human, the canine, the zebrafish and the amphioxus, transmembrane regions are identical, and their positions in the polypeptide as well as the positions of the two Asn residues that are conserved. These indicate that the cloned gene in amphioxus codes for TRAPp protein, and is thus designated AmphiTRAPβ. The two phylogenetic trees indicated that AmphiTRAPp is intermediately positioned between invertebrate TRAPβ and vertebrate TRAPp, well reflecting the established phylogeny of the chosen organisms. Southern blotting revealed that there is only a single copy gene encoding TRAPp in the genome of B. belcheri tsingtauense. Northern blotting showed that AmphiTRAPβ transcript was detected in all tissues tested. This is obviously in accord with the previous observation by Mangos et al. (2000) that TRAPβ mRNA is maternally supplied to the zebrafish egg during oogenesis. However, the role of TRAPP in cell adhesion and migration during development awaits further study.