Cloning, Expression And Evolution Of AmphiSDHD And AmphiS27 Genes From Amphioxus Branchiostoma Belcheri Tsingtauense

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 cloning, expression and phylogenetic analysis of AmphiSDHD and AmphiS27 from the gut cDNA library of amphioxus Branchiostoma becheri tisingtauense are carried out.AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It consists of 1429 bp, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids containing rich basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.AmphiS27, encoding ribosomal protein S27, was identified from the gut cDNA library of amphioxus B. belcheri tsingtauense. It consists of 607 bp, which contains a255 bp open reading frame (ORF) corresponding to a deduced protein of 84 amino acids with a calculated molecular mass of 9,488 Da and an isoelectric point (pI) of 9.500. Alignment of the deduced AmphiS27 amino acid sequence with other 12 known S27 protein sequences unraveled that AmphiS27 shares 94% to 99%, 84% to 94% and 69% to 72% homology, respectively, to its counterparts from vertebrates, invertebrates and other eukaryotes, suggesting AmphiS27 appears more closely related to vertebrate S27 than invertebrate one. Southern blot analysis exhibits a single copy of S27 gene present in the genome of amphioxus B. belcheri tsingtauense. This favors that amphioxus retains a genome uncomplicated by extensive gene duplication.