Characterization, Evolution And Expression Of Amphioxus Calcium-binding Proteins Genes

Amphioxus, a cephalochordate, is the closest living relative to vertebrate, and has been widely known as the most important animal to study the origin and evolution of vertebrates. Studies on gene structure, function and expression in amphioxus will contribute to the understanding of the origin and evolution of the vertebrates. In this paper, the characterization, expression and phylogenetic analysis of AmphiCaM1a, AmphiCaM1b, AmphiCalbin, AmphiCaRP1 and AmphiCaRP2 are carried out.Two full-length cDNAs encoding the highly conserved calmodulin1 (CaM1) proteins, named AmphiCaM1a and AmphiCaM1b, were isolated from the cDNA library of amphioxus Branchiostoma belcheri tsingtauense. There are only two nucleotide substitutions within their open reading frames and one amino acid difference between AmphiCaM1a and AmphiCaM1b, which have been verified by genomic hemi-nested PCR. The phylogenetic analysis indicates that the CaM1 sequence in all three amphioxus species appears to be the conventional CaM and CaM2 might be the gene duplication product of CaM1. Southern blotting suggests that there are two copies CaM1 genes in the genome of B. belcheri tsingtauense. Northern blotting analysis shows the presence of two AmphiCaM1a mRNA transcripts. In addition, whole mount in situ hybridization and tissue-section in situ hybridization as well as Northern blotting prove that AmphiCaM1a shows substantial expression in the digestive system starting from late neurulae stage and continuously lasting to its adulthood stage. These observations denote that it is possible that the AmphiCaM mainly involved in the differentiation of the digestive system after the neurulae stage is completed, and plays a role in both gut and gonads when it sexually matures. The presence of two CaM1 proteins and the expression pattern regarding CaM are the first reported in cephalochordate amphioxus,which has long been regarded as the extant invertebrate most closely related to the proximate ancestor of vertebrates.AmphiCalbin, encoding a novel EF-hand calcium-binding protein (EFCaBP), was another gene cloned from the gut cDNA library of amphioxus. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Secondly, AmphiCalbin falls at the base of vertebrate unnamed EFCaBPs clade, probably representing their prototype. This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin with the high levels in the digestive system and gonad. It is proposed that AmphiCalbin might play a role in the digestive system and gonad, which lays foundation for further understanding the function of the unnamed EFCaBPs.The last two genes reported here obtained from the gut cDNA library of amphioxus are AmphiCaRP1 and AmphiCaRP2, which encoding calcium-related proteins. Both sequence and phylogenetic analysis show that AmphiCaRP1 and AmphiCaRP2 are new members of calium-binding proteins without EF-hand calcium-binding domain, and are clubbed together. RT-PCR analysis indicates that both AmphiCaRP1 and AmphiCaRP2 expressed in all tissues examined, with the relative abundance in the hepatic caecum, suggesting AmphiCaRP1 and AmphiCaRP2 might implicate the role of food digestion. Whole mount in situ hybridization reveals that AmphiCaRP1 is firstly detected in the primitive gut of 48 hr larvae, whereas AmphiCaRP2 is firstly detected in the primitive gut of 24 hr larvae, suggesting that AmphiCaRP2 plays its physiological role in advance compared with AmphiCaRP1.