东亚钳蝎部分毒素基因组基因多序列比对分析
摘要
查阅文献并通过Entrez检索NCBI数据库(www.ncbi.nih.nlm.gov)，搜集目前已知的东亚钳蝎基因组基因序列，将获得的基因组基因序列存为FASTA格式的文本文件。逐个将基因组基因序列附加进入ClustalX1.83软件，运行多序列联配，生成多序列联配文件。利用MEGA软件，将由ClustalX1.83软件生成的多序列联配文件转换为MEGA文件，进行编辑后输出，构建neighbor joining(NJ)树。从结果来看，神经毒素结构与功能密切相关，Na~+通道神经毒素和K~+通道神经毒素构成了无根树的两个主要的树枝，Ca2~+通道神经毒素位于K~+通道神经毒素树枝的边缘，而Cl~-通道神经毒素隐藏在K~+通道神经毒素的树枝中。
以东亚钳蝎为原料，用蛋白酶K消化细胞，再用酚、氯仿除蛋白质，最后用乙醇沉淀DNA。以提取的东亚钳蝎DNA为模板，PCR扩增AGAP2成熟肽基因，将所获得的PCR产物进行电泳，将目的片段胶回收，双酶切后连于同样粘性末端的pET28a中，连接产物转化E．coli DH5α感受态细胞，涂板于卡那霉素的固体LB平板上，37℃培养过夜。挑选阳性重组菌落，经LB液体培养基培养12h后，提取质粒进行PCR扩增验证后，选出含有目的片段的阳性菌落，采用SSCP法筛选出有差异的菌落。测序后确定AGAP2的基因组基因序列，从琼脂糖宁焦点用和测序结果来看，AGAP2成熟肽编码区中没有内含子存在。这是基因组基因成熟肽编码区没有内含子的又一个例证。
The genomic sequences of the Buthus martensii Karsch toxins was collected and saved as Fasta format. Sequences were appended to the ClustalX1.83 software, did complement alignment, saved as '*.aln' file. Converted the '*.aln' file to MEGA format, and opened data, exported data, constructed phylogeny and did computer. The result shows that the sodium channel toxins on the one hand and the potassium channel toxins on the other hand are two distinct principle branches of the unrooted phylogenetic tree. Calcium channel toxins lies in the edge of the potassium channel toxins. Cl~- channel toxins are hiding in the potassium channel toxins' branch.
Genomic DNA was purified form the muscle tissue of the Buthus martensii Karsch scorpion. Cells from the muscle tissue were lysed by proteinase K and SDS, and the sample was gently extracted with phenol. DNA was precipitated by adding ammonium acetate and ethanol. Agarose gel electrophyrisis showed that the DNA's size was about 20kb. The genomic DNA of AGAP2 mature peptide was amplified under step down PCR reaction. PCR products were ligated into the pET28 vector. E.coli DH5αwas used for plasmid propagation. Recombinant clones were analyzed by PCR following agarose gel electrophoresis and SSCP. Compared with the amino acid sequence and the cDNA sequence of AGAP2, there is no intron in the mature peptide coding region.
以东亚钳蝎为原料，用蛋白酶K消化细胞，再用酚、氯仿除蛋白质，最后用乙醇沉淀DNA。以提取的东亚钳蝎DNA为模板，PCR扩增AGAP2成熟肽基因，将所获得的PCR产物进行电泳，将目的片段胶回收，双酶切后连于同样粘性末端的pET28a中，连接产物转化E．coli DH5α感受态细胞，涂板于卡那霉素的固体LB平板上，37℃培养过夜。挑选阳性重组菌落，经LB液体培养基培养12h后，提取质粒进行PCR扩增验证后，选出含有目的片段的阳性菌落，采用SSCP法筛选出有差异的菌落。测序后确定AGAP2的基因组基因序列，从琼脂糖宁焦点用和测序结果来看，AGAP2成熟肽编码区中没有内含子存在。这是基因组基因成熟肽编码区没有内含子的又一个例证。
The genomic sequences of the Buthus martensii Karsch toxins was collected and saved as Fasta format. Sequences were appended to the ClustalX1.83 software, did complement alignment, saved as '*.aln' file. Converted the '*.aln' file to MEGA format, and opened data, exported data, constructed phylogeny and did computer. The result shows that the sodium channel toxins on the one hand and the potassium channel toxins on the other hand are two distinct principle branches of the unrooted phylogenetic tree. Calcium channel toxins lies in the edge of the potassium channel toxins. Cl~- channel toxins are hiding in the potassium channel toxins' branch.
Genomic DNA was purified form the muscle tissue of the Buthus martensii Karsch scorpion. Cells from the muscle tissue were lysed by proteinase K and SDS, and the sample was gently extracted with phenol. DNA was precipitated by adding ammonium acetate and ethanol. Agarose gel electrophyrisis showed that the DNA's size was about 20kb. The genomic DNA of AGAP2 mature peptide was amplified under step down PCR reaction. PCR products were ligated into the pET28 vector. E.coli DH5αwas used for plasmid propagation. Recombinant clones were analyzed by PCR following agarose gel electrophoresis and SSCP. Compared with the amino acid sequence and the cDNA sequence of AGAP2, there is no intron in the mature peptide coding region.
引文
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2． Cao Zhijian 1,Luo Feng 1,Wu Yingliang,Mao Xin,Li Wenxin Genetic mechanisms of scorpion venom peptide diversification Toxicon 47(2006) 348-355
3． Deshane.,J.,Garner,C.C.,and Sontheiner,H.Chlorotoxin inhibints glioma cell invasion via matrix metalloproteinase-2 J Biol Chem.2003 Feb 7;278(6) :4135-44．
4． Wang W.X?Ji Y.H.,Scorpion venom induces glioma cell apoptosis in vitro and inhibits glioma tumor growth in vivo.Journal of Neuro-Oncology 2005,73:1-7
5． Cao Z,Mao X,Xu X,Sheng J,Dai C,Wu Y,Luo F,Sha Y,Jiang D,Li W.Adaptive evolution after gene duplication in alpha-KT x 14 subfamily from Buthus martensii Karsch.IUBMB Life.2005 Jul;57(7) :513-21
6． Zhu,S.Y.,Cao,Z.J.,Li,W.X.,2001． Two non-coding transcripts of scorpion toxins from Buthus martensii Karsch:evidence for NMD path existing in scorpion venom cell.Chinese Science Bulletin 48,1359-1363．
7． Cao,Z.J.,Dai,C.,Wu,Y.L.,Li,W.X.,2005． Splicing of scorpion toxin genes in the cultured HEK 293T cells system.JBMT,in press.
8． Xiong YM,Ling MH,Wang DC,Chi CW.The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch.Toxicon.1997Jul;35(7) :1025-31．
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14． Jing-Jiang Wu The gene clonging and sequencing of Bm12,a Chlorotoxin-like peptide from the scorpion Buthus martensi Karsch Toxicon38(2000) 661-8
15． Cao Zhijian,Xie Yun,Dai Chao,Zhu Shunyi,Yin Shijin,Wu Yingliang and Li Wenxin Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch?Peptides,2006 1235-40
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17． Zhu shunyi A naturally occurring non-coding fusion transcript derived from scorpion venom gland:implocation for the regulation of scorpion toxin gene expression FEBS letts 2001,508:241-4
18． Xiong YM,Lan ZD,Wang M,Liu B,Liu XQ,Fei H,Xu LG,Xia QC,Wang CG,Wang DC,Chi CW.Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.Toxicon.1999 37(8) :1165-80．
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24． Chun-guang Wang A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch,sequencing,gene cloning and structure determination toxicon 45(2005) 309-319
25. Xu X, Cao Z, Sheng J, Wu W, Luo F, Sha Y, Mao X, Liu H, Jiang D, Li W. Genomic sequence analysis and organization of BmKalphaTx11 and BmKalphaTx15 from Buthus martensii Karsch: molecular evolution of alpha-toxin genes. J Biochem Mol Biol. 2005 Jul 31;38(4):386-90.
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27. Chen-Qi Xu, Lin-Lin He, Bert Brone, Marie-France Martin-Eauclaire, Emmy Van Kerkhove, Zhuan Zhou and Cheng-Wu Chi A novel scorpion toxin blocking small conductance Ca~(2+) activated K~+ channel Toxicon, Volume 43, Issue 8, 2004, 961-971
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29. Huys I, Xu CQ, Wang CZ, Vacher H, Martin-Eauclaire MF, Chi CW, Tytgat J. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K~+ and HERG currents. Biochem J. 2004 Mar 15;378(Pt 3):745-52.
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32. Jiqun S, Xiuling X, Zhijian C, Wanhong L, Yingliang W, Shunyi Z, Xianchun Z, Dahe J, Xin M, Hui L, Wenxin L, Teng W. Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK). J Biochem Mol Toxicol. 2004;18(4): 187-95.
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35. Alami M, Ouafik L, Ceard B, Legros C, Bougis PE, Martin-Eauclaire MF. Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus. Toxicon. 2001 Oct;39(10): 1579-85.
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53．F．M 奥斯伯等 精编分子生物学实验指南 (第四版) 科学出版社
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2． Cao Zhijian 1,Luo Feng 1,Wu Yingliang,Mao Xin,Li Wenxin Genetic mechanisms of scorpion venom peptide diversification Toxicon 47(2006) 348-355
3． Deshane.,J.,Garner,C.C.,and Sontheiner,H.Chlorotoxin inhibints glioma cell invasion via matrix metalloproteinase-2 J Biol Chem.2003 Feb 7;278(6) :4135-44．
4． Wang W.X?Ji Y.H.,Scorpion venom induces glioma cell apoptosis in vitro and inhibits glioma tumor growth in vivo.Journal of Neuro-Oncology 2005,73:1-7
5． Cao Z,Mao X,Xu X,Sheng J,Dai C,Wu Y,Luo F,Sha Y,Jiang D,Li W.Adaptive evolution after gene duplication in alpha-KT x 14 subfamily from Buthus martensii Karsch.IUBMB Life.2005 Jul;57(7) :513-21
6． Zhu,S.Y.,Cao,Z.J.,Li,W.X.,2001． Two non-coding transcripts of scorpion toxins from Buthus martensii Karsch:evidence for NMD path existing in scorpion venom cell.Chinese Science Bulletin 48,1359-1363．
7． Cao,Z.J.,Dai,C.,Wu,Y.L.,Li,W.X.,2005． Splicing of scorpion toxin genes in the cultured HEK 293T cells system.JBMT,in press.
8． Xiong YM,Ling MH,Wang DC,Chi CW.The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch.Toxicon.1997Jul;35(7) :1025-31．
9． Becerril B,Corona M,Mejia MC,Martin BM,Lucas S,Bolivar F,Possani LD.The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron.FEBS Lett.1993 Nov 29;335(1) :6-8．
10． Legros C,Bougis PE,Martin-Eauclaire MF.Genomic organization of the KTX2 gene,encoding a'short'scorpion toxin active on K+ channels.FEBS Lett.1997 Jan 27;402(1) :45-9．
11． Xiao F,Cao ZJ,Lu M,Jiang DH,Mao X,Liu H,Li WX.[Analysis of the genomic organization of a novel scorpion toxin BmTXK beta]Yi Chuan Xue Bao.2003 Jul;30(7) :663-7． Chinese.
12． Delabre ML,Pasero P,Marilley M,Bougis PE.Promoter structure and intron-exon organization of a scorpion alpha-toxin gene.Biochemistry.1995 May 23;34(20) :6729-36．
13． Peng F,Zeng XC,He XH,Pu J,Li WX,Zhu ZH,Liu H.Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxin. Eur J Biochem.2002 Sep;269(18) :4468-75．
14． Jing-Jiang Wu The gene clonging and sequencing of Bm12,a Chlorotoxin-like peptide from the scorpion Buthus martensi Karsch Toxicon38(2000) 661-8
15． Cao Zhijian,Xie Yun,Dai Chao,Zhu Shunyi,Yin Shijin,Wu Yingliang and Li Wenxin Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch?Peptides,2006 1235-40
16． Jiqun S,Molecular cloning and analysis of BmKBT genomic sequence form scorpion Buthus martensii Karsch Cinese Journal of Biochemistry and molecular biology 2004,20:491-6
17． Zhu shunyi A naturally occurring non-coding fusion transcript derived from scorpion venom gland:implocation for the regulation of scorpion toxin gene expression FEBS letts 2001,508:241-4
18． Xiong YM,Lan ZD,Wang M,Liu B,Liu XQ,Fei H,Xu LG,Xia QC,Wang CG,Wang DC,Chi CW.Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.Toxicon.1999 37(8) :1165-80．
19． Luo F,Zeng XC,Hahin R,Cao ZJ,Liu H,Li WX.Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch:Gaining insight into evolutionary mechanism.Peptides,Volume 26,Issue 12,December 2005,Pages 2427-2433
20． Lan ZD,Dai L,Zhuo XL,Feng JC,Xu K,Chi CW.Gene cloning and sequencing of BmK AS and BmK AS-1,two novel neurotoxins from the scorpion Buthus martensi Karsch.Toxicon.1999 37(5) :815-23
21． Zeng XC,Peng F,Luo F,Zhu SY,Liu H,Li WX.Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides:a new subfamily of venom peptides(alpha-KTx14) and genomic analysis of a member.Biochimie.2001 Sep;83(9) :883-9．
22． Zeng XC,Luo F,Li WX.Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch:trans-splicing may play an important role in the diversification of scorpion venom peptides.Peptides.2006 Apr;27(4) :675-81．
23． Dai L,Wu JJ,Gu YH,Lan ZD,Ling MH,Chi CW.Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels.Biochem J.2000 Mar 15;346 Pt 3:805-9．
24． Chun-guang Wang A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch,sequencing,gene cloning and structure determination toxicon 45(2005) 309-319
25. Xu X, Cao Z, Sheng J, Wu W, Luo F, Sha Y, Mao X, Liu H, Jiang D, Li W. Genomic sequence analysis and organization of BmKalphaTx11 and BmKalphaTx15 from Buthus martensii Karsch: molecular evolution of alpha-toxin genes. J Biochem Mol Biol. 2005 Jul 31;38(4):386-90.
26. Wu JJ, Dai L, Lan ZD, Chi CW. Genomic organization of three neurotoxins active on small conductance Ca~(2+)-activated potassium channels from the scorpion Buthus martensi Karsch. FEBS Lett. 1999 Jun 11;452(3):360-4.
27. Chen-Qi Xu, Lin-Lin He, Bert Brone, Marie-France Martin-Eauclaire, Emmy Van Kerkhove, Zhuan Zhou and Cheng-Wu Chi A novel scorpion toxin blocking small conductance Ca~(2+) activated K~+ channel Toxicon, Volume 43, Issue 8, 2004, 961-971
28. Zeng XC, Zhu ZH, Li WX, Zhu SY, Peng F, Mao X, Liu H. Molecular cloning and genomic organization of a K(+) channel toxin from the Chinese scorpion Buthus martensii Karsch. Toxicon. 2001 Feb-Mar;39(2-3):407-10.
29. Huys I, Xu CQ, Wang CZ, Vacher H, Martin-Eauclaire MF, Chi CW, Tytgat J. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K~+ and HERG currents. Biochem J. 2004 Mar 15;378(Pt 3):745-52.
30. Cao Z, Zhu S, Li W, Jiang D, He X, Liu H. Genomic organization of BmTXKbeta and BmTXKS2, two scorpion venom peptides from Buthus martensii Karsch. IUBMB Life. 2001 May;51(5):305-8.
31. Zhu SY, Li WX, Zeng XC. Precursor nucleotide sequence and genomic organization of BmTXKS1, a new scorpion toxin-like peptide from Buthus martensii Karsch. Toxicon. 2001 Sep;39(9):1291-6.
32. Jiqun S, Xiuling X, Zhijian C, Wanhong L, Yingliang W, Shunyi Z, Xianchun Z, Dahe J, Xin M, Hui L, Wenxin L, Teng W. Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK). J Biochem Mol Toxicol. 2004;18(4): 187-95.
33. Ji YH, Wang WX, Ye JG, He LL, Li YJ, Yan YP, Zhou Z. Martentoxin, a novel K~+-channel-blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization. J Neurochem. 2003 Jan;84(2):325-35.
34. S. Zhu, B. GaoMolecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii FEBS Letters 580 (2006) 5979 - 5987
35. Alami M, Ouafik L, Ceard B, Legros C, Bougis PE, Martin-Eauclaire MF. Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus. Toxicon. 2001 Oct;39(10): 1579-85.
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