Molecular Diversification Of Peptide Toxins From The Nephila Pilipes Venom

Nephila pilipes is a species of golden orb-web spider.It can be found in China,Singapore,Indonesia,Thailand etc.The spider venom gland,which is a very specialized secretory tissue,can secrete abundant and complex toxin components.To extensively examine the transcripts expressed in the venom glands of Nephila pilipes,we generated 1380 expressed sequence tags(ESTs)from a directional cDNA library.All ESTs were assembled into 601 non-redundancy sequences including 85 clusters and 516 singlets,of which toxin-like sequences accounts for 76.53%,18.80%are similar to the cellular transcripts and 4.67%have no significant similarity to any known sequences.All the cDNA sequences are available for public view in the GenBank database of NCBI(accession numbers:KF433089-KF433818].Here,we identified 431 novel toxin-like sequences which can be classified into thirteen different superfamilies(superfamily A,B,C,D,E,F,Q H,I,J,K,L and M),that means a novel potential mine of ligands for varied ion channels was discovered.With the aid of Gene Ontology terms and homology to eukaryotic orthologous groups,the annotation of the cellular transcripts revealed some cellular processes important for the toxin secretion of venom gland including high protein synthesis,protein folding,tuned posttranslational processing and trafficking,and so on.Spider venoms,as is an increasingly valuable library of natural resources,play an extremely important role for spiders to capture terrestrial insects and protect themselves from natural enemies' attack,which are complex mixtures of biologically active compounds of different chemical composition,from salts to large multidomain proteins.Here we investigated how the Nephila pilipes venom works on DRG,which plays a foundation to further researchs about the functions of Nephila pilipes venom.Acid-sensing ion channels(ASICs)are a class of cation channels which are activated by extracellular acidification,and widely distributed in central and peripheral.we mainly analysis and study the present situation and progress of ASICs,and the feasibility and superiority characteristic in the experimental scheme of ASICs interaction between proteins,and also complete the construction of two yeast two-hybrid bait vectors,pGBKT7-ratAl a and pGBKT7-ratA2a,and fulfill their expressions and identifications.All of these build the experimental basis for the application of ASICs selection and proteins interaction.We found a peptide from tarantula Chilobrachys jingzhao,named JZTX-58.JZTX-58 is a 36-residue polypeptide.In order to study more about it,we decided to get the peptide by the molecular cloning and expression.We choose the eukaryotic secretion expression system which includes the vector of pVT102U/a and the Saccharomyces cerevisiae strain S78.By means of ion-exchang,HPLC and MALDI-TOFMS,JZTX-58 was purified and identified.With these expressions,we found the factors which influence the expression of toxin peptides are complicated and diversified.These experience will contribute to the expression of future.In summary,we constructed a cDNA library of Nephila pilipes,by using molecular biology,biochemistry,mass spectra,patch-clamp and bioinformactics,etc,we have investigated transcriptomes of the Nephila pilipes venom glands.Our investigation increases the knowledge of venom complexity and properties of venom components.These results have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms,provide a better understanding for molecular diversity,function and evolutionary relationship of spider peptide toxins.We made evaluation on a novel application of Sodium Laurate,a Novel Protease-and Mass Spectrometry-Compatible Detergent for Mass Spectrometry-Based Membrane Proteomics.we complete the construction of two yeast two-hybrid bait vectors,pGBKT7-ratA1a and pGBKT7-ratA2a,and fulfill their expressions and identifications.All of these build the experimental basis for the application of yeast two-hybrid screening for proteins that interact with ASICs.We also have cloned and expressed the novel genes JZTX-58,which provid toxin source for the function study of the genes in the future.