Cloning And Sequence Analysis Of The Conotoxin And Kunitz-type Toxin Intron In Four Kinds Of Conus Native To Hainan

Cone snails use the venoms secreted in their venom ducts for paralyzing prey or defending against predators,which are mainly composed of small peptides called conotoxins(conopeptides).They are distinctive by their structure,strong biological activity,and high selectivity on ion channels and receptors.Thus,they have gradually attracted extensive attention with their potentials to be developed as new research tools in neuroscience,as well as medicine precursors.At present,more than 830 species of cone snails have been reported in the world.As the venom of each cone snail species consists of about 3 000 conopeptides or conotoxins,there are more than 1.5 million different bioactive conotoxins to exist,less than 1% of which have been identified.Moreover,cone snails also can secrete a variety of protease inhibitors to inhibit the activity of proteases in the prey,which help maximize the effects of conotoxins.However,few of them have been reported so far.So,there are many conotoxin resources to be developed and utilized.To solve the problems above,the following research was carried out focusing on the conotoxin resources native to Hainan.1.We obtained 108 DNA sequences encoding conotoxin from Conus litteratus and Conus marmoreus by three methods,including 89 genes of A-superfamily,15 genes of O-superfamily,1 gene of T-superfamily and 3 genes of J-superfamily toxin sequences.A total of 72 conotoxin genes were identified in C.litteratus as follow.(1)46 A-superfamily conotoxins were cloned based on the conserved sequences of intron and 3’-UTR(untranslated region)of A-superfamily genes and O-superfamily,including 45 genes of A-superfamily conotoxins and 2 gene with 3-Cys framework.Besides,11 toxin sequences of O-superfamily were also obtained using the method above,including 9 genes with VI/VII framework and 2 gene with 5-Cys framework.(2)4 toxin sequences of A-superfamily with signal region were cloned based on the conserved DNA sequences of signal peptide and 3’-UTR of A-superfamily genes.(3)The c DNA of C.litteratus was obtained using 3’-RACE(Rapid Amplification of c DNA Ends).Then 11 conotoxin genes were cloned,including 3 genes of A-superfamily,4 genes of O-superfamily,1 gene of T-superfamily and 3 genes of J-superfamily.The remaining 36 conotoxin genes were found in the C.marmoreus based on the conserved sequences of intron and 3’-UTR.2.A total of 13 kunitz-type toxin genes were obtained from three species of cone snails by two methods as follow.(1)Using the primers designed based on the novel 098 and 069 x kunitz-type toxin sequences discovered from C.litteratus transcriptome in our laboratory,11 kunitz-type toxin genes containing introns were cloned,including 2 genes for 098kunitz-type toxin and 2 genes for 069 x kunitz-type toxin from C.litteratus,4 genes for 098kunitz-type toxin from C.marmoreus and 3 genes for 098 kunitz-type toxin from C imperialis,respectively.(2)The c DNA of C.literatus was obtained using 3’-RACE,and 2genes of 069 x kunitz-type toxin without introns were successfully cloned.The discovery of new genes encoding conotoxin provides a basis for the artificial synthesis,activity evaluation and medicinal development of conotoxin.The evolutionary tree analysis of the kunitz-type toxin of cone snails shows that the novel sequences found in this study are not in the same branch as kunitz-type toxins from other cone snails,which means they are kinds of relatively new type of Kunitz-type toxin.In addition,we obtained and analyzed the intron sequences of Kunitz-type toxin from the genome of cone snails for the first time.The results showed that the repetition of microsatellite DNA in introns may affect the expression of kunitz-type toxin,which enriched the study of Kunitz-type toxin of cone snails and provide the foundation for the subsequent research.