动物学研究 2007, Oct. 28(5): 457 464 CN 53-1040/Q ISSN 0254-5853 Zoological Research 两个蛇毒基因克隆及 cdna 序列多态性再分析 * 361005 摘要 :α-α-bungarotoxin cdna DNA α- 5 cdna cdna α- cdna α- cdna α- cdna RNA cdna 关键词 :α- ; ; RNA ; 中图分类号 :Q959.62; Q38; Q785 文献标识码 :A 文章编号 0254-5853(2007)05-0457-08 Cloning of Two Toxin Related Genes and Analysis of Their cdna Polymorphism LIN Lu-ping, LIN Qun, WANG Yi-quan * (School of Life Sciences, Xiamen University, Xiamen 361005, China) Abstract: Alpha-bungarotoxin is one of the post-synaptic neurotoxins, which widely exists in the Elapidae venom. The polymorphism of α-bungarotoxin s cdna sequences has been controversial in previous reports. In the present study, cloning of α-bungarotoxin gene from the genomic DNA, sequencing of five clones and analysis of their polymorphism were reported. Furthermore, we cloned and sequenced the nerve growth factor (NGF) gene from the same cdna pool as a standard, and compared the mutation rates among different sources of α-bungarotoxin cdnas, α-bungarotoxin genes and NGF cdna. The results indicated that there was no diversification in the genome or comparability in the mutative points between different sequences. Therefore, we speculate that the polymorphism of α-bungarotoxin cdna resulted neither from different transcripts, nor from RNA editing, but possibly from reverse transcript processes and gene-cloning processes. Key words: α-bungarotoxin; Polymorphism; RNA editing; Mutation α-α-bungarotoxin 1998 Liu et al 1998 cdna α- 16 mrna 10 2 α- Koesn et al,1988 V31 A31 Liu et al 1998 DNA α- cdna α- RNA Chang et al (1999) DNA 2 α- 2007-04-262007-08-20 30470938 30570208D0510002 * Corresponding author E-mail: wangyq@xmu.edu.cn 1982- Vanderbilt University
458 28 V31 A31 cdna 100 2 V31 A31 α- cdna V31 A31 RNA Qian et al (2000) V31 Wang et al 2005 cdna 12 α- 8 V31 A31 α- RNA RNA RNA editing RNA Benne et al 1986 RNA Sattelle et al, 2005 RNA (Barlati et al, 2005) Hoopengardner et al (2003) 16 mrna 1 mrna RNA α- (Wu et al, 2005) α- mrna α- cdna DNA α- cdna http://www.ensembl.org NGF α- cdna cdna α- cdna 1 材料与方法 1.1 材料 Elapidae Bungarus multicinctus RNA RNA Wang et al, 2005 cdna RNA DNA 1.2 方法 1.2.1 DNA RNA DNA 20 DNA ddh 2 O 4 RNA Trizol cdna Clotech SMART TM cdna 1.2.2 α- PCR GenBank α- DNA BgTX_genome_upper BgTX_genome_ lower α- 25 µl PCR 10 PCR Buffer 2.5 µl 10 mmol/l BgTX_genome_upper BgTX_genome_lower 0.5 µl 2.5 mmol/l dntps 4 µl 5 U/µL ExTaq 0.25 µl DNA 1 µlpcr 95 4 min 30 95 40 s 55 45 s 72 2 min 72 7 min 5 µl PCR 1.2% PCR 2 kb, TA pmd18 PCR M13-M4 M13-RV BgTX_sequencing BgTX_intronI-U BgTX_intronI-I BgTX_intron2 primer walking 1 BgTX_check_upper
5 : cdna 459 BgTX_check_lower α- PCR DNA α- CEQ-8000 Beckman-Coulter DNAStar- SeqMan 1.2.3 cdna 3 RACE mrna cdna RT-PCR PCR cdna NGFu SMART (Clotech) 3 RACE CDS Ⅲ /3 PCR cdna 25 µl PCR 10 PCR Buffer 2.5 µl 10 pmmol/l NGFu CDS Ⅲ/3 PCR 0.5 µl 2.5 mmol/l dntps 4 µl 5 U/µL ExTaq 0.25 µl cdna 1 µl PCR 95 4 min 30 95 40 s 55 45 s 72 40 s 72 2 min 5 µl PCR 1.2% PCR TA pmd18 PCR CEQ-8000 PolyA M13-M4 M13-RV 5 NGF_sequencing 1 DNAStar SeqMan 1.2.4 α- = /( ) Stapleton et al (2002) 1/1 000 1/15 000 2 2.1 α- cdna BgTX_genome_upper BgTX_genome_lower α- 2 kb 1A cdna NGFu CDSⅢ/3 PCR cdna 900 bp 500 bp 900 bp 1B 2.2 α- α- 5 2655 bp α- α- cdna 3 2 2A 5 α- α- 288 bp 1 58 bp N 20 1 Tab. 1 Information of theprimers used in gene cloning and sequencing Primers Sequences BgTX_genome_upper 5 -CCg gaa TTC AgA TCG CAA gat gaa AAC TC-3 BgTX_genome_lower 5 -gcg gga TCC TCA ACC Agg TCT CTg TTT C-3 BgTX_sequencing 5 -gat gaa AAA TgT TTC TgC TC-3 BgTX_intronI-U 5 -TTg gcc TTC CAT CCT TTT gaa ATg-3 BgTX_intronI-I 5 -TAg CCg AAC TAg CTg TTg TgT g-3 BgTX_intron2 5 -TTC ATC CAA TTT CCC TCA T-3 BgTX_check_upper 5 -CAA gaa gct CAC TCT gta ga -3 BgTX_check_lower 5 -TAT CAg CTC AAA AAA CCT TAA C-3 NGFu 5 -ATg TCC ATg CTg TgC TAC ACT CTg-3 CDS /3 PCR 5 -ATT CTA gag gcc gag gcg gc-3 NGF_sequencing 5 -AAg TTC CTg gac ACT gaa ga-3 M13-M4 5 -CAg CAC TgA CCC TTT Tgg gac CgC-3 M13-RV 5 -AgC gga TAA CAA TTT CAC ACA Agg-3
460 28 1 α- PCR A 3 RACE PCR B Fig. 1 Agarose gel electrophoresis of PCR amplification products of α-bungarotoxin gene (A) and 3 RACE PCR amplification products of nerve growth factor gene from the venom cdna(b) M (bp)( DL2000 marker); 1 α- (α-bungarotoxin gene); 2 cdna(nerve growth factor cdna) 2 105 bp 4 N 33 3 128 bp 41 1 V31 1 2 1790 bp 538 bp 5 α- 13 2B α- cdna Wang et al 2003 1 A31 DNA 5 BgTX_check_upper BgTX_check_ lower DNA α- 508 bp 2A PCR PCR 5 PCR α- 2.3 3 RACE 3 RACE 6 3 RACE 891 bp 732 bp 6 11 ( 3A) 2 4 3B 6 cdna 2 24 581 G 5 2.4 α- PCR 5 5 cdna6 5 Wang et al(2005) 18 12 α- cdna Liu et al (1998) 50 16 α- Wang et al (2003) 7 cdna 2 α- cdna 2 α-a 5 α-b Fig. 2 α-bungarotoxin gene organization, distribution of mutation sites and indication of verified region A and mutation sites of five clones with α-bungarotoxin gene B
5 : cdna 461 3 A 6 cdna B Fig. 3 Indication for the coding region and non-coding region and distribution of mutation sites of Bungarus multicinctus nerve growth factor gene A and mutation sites of six clones for nerve growth factor cdna B Gene Mutation number 2 Tab. 2 Results of mutation rates of several genes Sequenced clones Amino acid number bp Length of sequence (bp) Mutation rate Mutation rate with deducted reversal error α- α-bungarotoxin genome 13 5 1 2655 0.00098 N/A α- cdna α-bungarotoxin cdna 16 18 8 472 0.00188 0.00088-0.00181 a cdna NGF cdna 11 6 2 891 0.00206 0.00106-0.00199 α- cdna α-bungarotoxin cdna 19 50 10 222 0.00171 0.00071-0.00164 b cardiotoxin-like gene 7 7 1 505 0.00198 0.00098-0.00191 Wang et al (2005) (a) Liu et al (1998) (b) The mutation rates calculated according to sequences from Wang et al (2005) (a) and Liu et al (1998) (b). α- cdna cdna 2.5 α- cdna α- cdna RNA α- Wang et al (2005) 12 cdna 3 9 Liu et al (1998) 16 cdna NCBI 3 cdna C1 Y17057 C2 Y17058 K X91990 29 4 A31 3 V31 22 α- cdna 28 157 T-C V31 A31 Wang et al (2005) 12 cdna Liu et al (1998) 211 C Wang et al (2005) cdna A Liu et al (1998) cdna T Wang et al (2005) cdna Liu et al (1998) cdna ( 4) 2.6 PCR α- α- cdna cdna α- cdna Liu et al, 1998 cdna 1/1000 TAKARA ExTaq 30 1 kb DNA 2 3
462 28 4 α cdna Fig.4 Alignment of exons of α bungarotoxin genomic DNA and cdna from Wang et al (W-1 W-12), Liu et al (L-R1 L-R16) and another α bungarotoxin cdna sequences from GenBank (C1, C2, K) Identical sequences are omitted. The identical nucleotides with the first line are indicated by dots and deleted nucleotides by hyphens. Wang et al(2003, 2005) rtaq 30 1 kb DNA 5 6 PCR PCR 3 α-α- cdna cdna PCR 3 讨论 3.1 α- 银环蛇毒素 cdna 多态性不是由于基因组不同转录本造成 DNA 2.65 kb α- V31 Wang et al (2003) cdna α- A31 Chang et al (1999) Wang et al (2005) α- α- V31 A31 α- 13 PCR 5 PCR α- 5 α- α- cdna α- cdna 3.2 造成 α- 银环蛇毒素 cdna 多态性的原因分析 Stapleton et al (2002) cdna 0.00106 0.00199 Wang et al (2005) α- cdna 0.00088 0.00181 Liu et al (1998) α- cdna 0.00071 0.00164 Wang et al (2003) cdna 0.00098 0.00191 cdna α- cdna cdna Liu et al (1998) α- cdna DNA α- 0.00098 cdna cdna cdna 3 α- α- cdna
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