| Conotoxins come from the venom of gastropod mollusk cone shells which is sarcophagy and distribute in the tropical waters. Conotoxin is a kind of cysteine-rich neuropeptide, which has construction of disulfide bonds. Conotoxins selectively act on various kinds of receptors of neurotransmitters, ion channels and neurokinins, so conotoxins can be used as research tools of ion channels and membrane receptors.Thus, conepeptide have attracted extensive attention with their potentials to be developed as new research tools in neuroscience and as novel medications for treatment of pain.Genomic DNA was isolated from venom glands, hepatopancreas and muscle of Conus vexillum using three methods of CTAB, spin column and magnetic bead respectively. The results showed that the genomic DNA could be extracted by all the three methods. The purity and yield of DNAs isolated by CTAB and centrifugal column methods using venom gland of Conus vexillum were good and high, which could meet the following experimental requirements.While the yield of the magnetic bead method is less and the purity is not good. The length of most DNA fragments isolated by the centrifugal column method was less than 9 kb, while most fragments'sizes isolated by CTAB method were more than 20 kb. Among the three kinds of organs, hepatopancreas had highest yield of DNA, but its fragment was dispersed with degradation happened, the DNA yield of venom glands was high but less than hepatopancreas slightly, its integrity was the best; however the DNA yield of muscle was lowest and its fragments was smaller. The high quality genomic DNA fragments with different sizes were separated and prepared successfully by optimized pulsed-field gel electrophoresis. In conclusion, using CTAB method to isolateConus vexillum DNAis better than the other methods for fosmid genomic DNA library construction.The prepared DNA was end-repaired and then the size selected fragments which lager than 36kb were recovered from low melting point gel using different agarose enzyme. The ones using GelaseTM appeared to have better result.The collected fragments were ligated into the fosmid vectors and then packaged to construct the library. The final fosmid library with an average insert size of 36 kb, and the frequency of recombinant clones was calculated to be 97.5%. The fosmid stability assays indicated that inserted DNA was stable during propagation. The fosmid library will serve as a useful resource for end sequences, physical mapping, and positional cloning of Conus vexillum. It provides a base for further genetic analysis in this species and will be important for a better understanding of the genome.Thus, aeeording to the known sequences of the conserved gene sequence which encod sigal peptide and the conserved sequence of intron, and the 3'-UTR, pairs of primers were used to designed to screen novel a-CTX from the genomic DNAs with PCR amplification respectively in this study, which were both abstraeted from Conus vexillum and recombinant plasmids in the fosmid library. After sequencing and identify them in to peptide sequences,comparations were made out with the other known conopeptides. Basedonthesejob,64 different ?-CTx precursor genes were identified. There are two a 4/4 CTx, 5 ? 4/6 and 55 ? 4/7 CTx, all of which have a typical CC-CC cysteine skeleton pattern, and the cysteine skeleton is CC-CC-CC of two O-CTx. The number of amino acids in these toxin gene maturation peptide sequences ranged from 16 to 26, and the two O-superfamily toxin genes is 32. |
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