The Cloning, Expression And Molecular Phylogenetic Analysis Of Amphioxus GDF8/11, ACTIVIN And NM23-Bbt2 Gene

Amphioxus, a cephalochordate, is believed to be the closest living relative of the vertebrate. It is often regarded as an important model animal for phylogenetic studies on the evolution of animals from invertebrate to vertebrate. In this study, GDF8/11, ACTIVIN and NM23-Bbt2 were identified from amphioxus (B. belcheri tsingtausenese), and the expression of these genes in embryo and adult tissues was analyzed by RT-PCR. The evolution of these genes was also analyzed.The full-length genome of the amphioxus GDF8/11 is 9.9 kb. The amphioxus GDF8/11 gene consists of five exons flanked by four introns, which have two more exons and introns than that of other species. In intronIII, a possible transposable element was identified. This suggested this intron might derive from transposon. The amphioxus GDF8/11 cDNA encodes a polypeptide of 419 amino acid residues that showed high homology with MSTNs of molluscan, teleostean, avian, and mammalian species and GDF11 of mammalian and zebrafish species. Phologenetic analysis shows that the GDF8/11 is at the base of vertebrate MSTNs and GDF11s. This result might prove that the GDF8/11 derived from one ancestral gene, and the amphioxus GDF8/11 may be the common ancestral, and also the gene duplication event generating MSTN and GDF11 occurred before the divergence of vertebrates and after or at the divergence of amphioxus from vertebrates. RT-PCR results showed that GDF8/11 gene was expressed in new fertilized cell, early gastrulation and knife-shaped embryo, which was different from that in mammals. It suggested that GDF8/11 gene might possess additional functions other than regulating muscle growth in amphioxus.The genomic sequence of amphioxus ACTIVIN is 6.1 kb long. The promoter was about 447 bp long. Compared with cDNA sequence revealed that there were two exons and one introns and the boundary of exon and intron abided by the role of GT…AG strictly. ACTIVIN gene encoded a 410-amino-acid proprotein. The proprotein contained signal domain, N-terminal domain and C-terminal domain. Comparison of deduced amino acid sequences of amphioxus ACTIVIIN with vertebrate showed that ACTIVIN was conserved. Phylogenetic analysis shows that vertebrate and invertebrate ACTIVIN are each grouped together, with amphioxus falling at the base of vertebrate ACTIVIN clade, and suggests that the amphioxus ACTIVIN may represents the ancestral type of ACTIVIN homologue gene.The NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species. All of them contained high conserved motifs that play important roles in the function of NM23. RT-PCR reveals a ubiquitous expression pattern of amphioxus NM23-Bbt2 in all adult tissues and it was also expressed in all stages during the embryogenesis. Phylogenetic analysis shows that vertebrate and invertebrate NM23-H2 are each grouped together, with amphioxus falling at the base of vertebrate NM23-H2 clade, and suggests that the amphioxus NM23-Bbt2 may represents the ancestral type of NM23-H2 homologue gene. Comparison of the gene structures (four introns and five exons) of NM23-H2 homologue from invertebrate to vertebrate suggested that the gene structure (four introns and five exons) may be generated before the divergence of amphioxus from vertebrates.