Amphioxus Phgpx Gene Identification, Expression And Evolutionary Analysis

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.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,the cloning,verification,expression and phylogenefic analysis of PHGPx gene from the gut cDNA library of amphioxus Branchiostoma becheri tisingtauense are carried out.Phospholipid-hydroperoxide glutathione peroxidases(PHGPx)were functionally characterized as a peculiar type of glutathione peroxidases(GPX)reducing hydroperoxides of phospholipids and cholesterol.PHGPx fast attracted interest by its ability to directly interact with peroxidized lipids even if these complex lipids are integrated in biomembranes,and it has therefore been considered the main line of enzymatic defense against oxidative biomembrane destruction.Studies of PHGPx are mostly from mammalian,however in other vertebrate and invertebrate there is only rare reports.Two full-length cDNAs(GenBank accession number:EU599642)encoding PHGPx, were isolated from the cDNA library of amphioxus Branchiostoma belcheri tsingtauense.The clone is 943bp long and encodes a full-length sequence protein.Initial BLASTP search at NCBI reveales that the protein has a unique feature of PHGPx proteins,and shares 55-71%identity to other known PHGPx proteins.It is thus further compared with other members of PHGPx family.To verify that thePHGPx gene is from amphioxus,we performed Northern blotting analyses.Northern blotting reveales the presence of a single band of 1000bp transcript, corresponding to the size of F1721.In addition,in situ hybrdizafion histochemistry demonstrated a tissue-specific expression pattern of the clone F1721 in amphioxus. All these exhibit that the clone F1721 represents an amphioxus PHGPx gene.The un-rooted phylogenetic tree constructed using the amino acid sequence of amphioxus PHGPx protein and that of other known PHGPx proteins from various species showes that the envolvement location of amphioxus PHGPx is between PHGPx proteins from vertebrates and invertebrates.This suggests that the divergence of PHGPx genes probably occurs the same time to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago.In situ hybridization histochemistry reveales a conspicuous expression of amphioxus PHGPx gene in the gonad including ovary and testis,gill and hepatic caecum,and hind-gut and in the muscle.No signals are seen in endostyle,neural tube and notochord.It is clear that PHGPx gene is expressed in amphioxus in a tissue-specific manner.Taken the high degree of sequence conservation into consideration,it is highly likely that PHGPx protein may perform some critical but specialized physiological function in different tissues of amphioxus.However,this needs studying at the protein level.