Construction Of CDNA Libraries Of Conotoxins And Analysis Of Transcripts

Conotoxins are neurophysiologically active peptides isolated from venomous marine cone snails. Since most conotoxins have a sequence length of 12-46 amino acids with rich disulfide bond and can?target a wide range of voltage- and ligand-gated ion channels and receptors subtypes, which are not only developed directly into drugs, but also used as perfect drug design templates and probes for neurobiological researches. Therefore conotoxins are great potential rools for neuroscience, of several are being developed as diagnostic and novel therapeutic agents. Present studies are trying to illuminate the mechanism of Conus peptide evolution, but few studies are on the transcript differentiation and the evolution characteristic of different Conus species, although these studies are also important for us to expand the understanding of phylogenetic characteristics of different Conus in the same area.This study aims to construct the cDNA libraries of Conus from South China Sea, and analyze the evolution and expression profile of conotoxin and find novel comotoxins and related enzymes. It is basis for the research and related applications. The results were summarized as follow:1. In this work, cDNA libraries derived from venom ducts of C. virgo, C. eburneus, C. imperialis and C. marmoreus, collected from the same area of South China Sea, were constructed using Gateway Technology. The 1312 transcripts from four cDNA libraries were analysized. About 50 percents of total transcripts showed the coding sequences of conotoxins which were grouped into 35 novel conotoxin sequences.2. A common characteristic of transcripts from four Conus species was A-, T- and O-superfamily conotoxins exited in each library. In addition, the other conotoxin superfamilies, such as M-, I-, D-, J-, P- and L-superfamily and non-cysteine-rich conopeptides, such as contryphan and conantoxin, were also identided. Moreover, Analysis of phylogenetic tree and diversifying selective demonstrated that conotoxin evolution of four Conus from the same area exhibited the convergence evolution phenomenon.3. The entire nucleotide sequences of protein disulfide isomerases (PDIs) of C. virgo, C. eburneus, C. imperialis and C. betulinus from the South China Sea were cloned by 3'RACE and 5'RACE and analyzed by Bioinformatics. The entire nucleotide sequences of PDIs of four Conus species were cloned. The homology of the entire nucleotide sequences of PDIs among these Conus species was more than 90%, and higher than that of the Litopenaeus vannamei, Homo sapiens and S.cerevisiao. Two active sites of PDIs were highly conservative between Conus and other organisms, but the substrate binding sites were species specific. Phylogenetic tree analysis showed that the mutants of PDIs from Conus might be affected by their predator diet.