用于筛查转基因作物成分的质粒标准分子的研制
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摘要
为弥补传统标准物质的缺乏,构建了一种可用于筛查转基因作物成分的质粒标准分子。首先,通过PCR扩增获得454 bp的RBCL基因、236 bp的CaMV35S启动子以及200 bp的NOS终止子目的片段,经两次重叠延伸PCR扩增将上述3个片段连接成849 bp的重组子。随后,将该重组子克隆至pMD18-T载体,经PCR扩增、测序分析后成功获得阳性重组质粒pMD-RBCL-CaMV35S-NOS。进一步的替代性研究显示,该重组质粒DNA与标准物质基因组DNA的检测分析结果一致。因此,该重组质粒标准分子可作为阳性标准物质用于转基因作物成分的初步筛查检测。
To make up for the lack of traditional reference materials, a plasmid reference molecule was constructed for screening the components of genetically modified crops. First, the 454 bp RBCL gene, 236 bp CaMV35 S promoter and 200 bp NOS terminator were amplified by PCR. Using overlap extension PCR technology the three amplified fragments were ligated to be a 849 bp recombinant. After cloning of the recombi-nant into p MD18-T vector, the positive recombinant plasmid pMD-RBCL-CaMV35 S-NOS was successfully confirmed by PCR amplification and sequence analysis. Further substitution studies showed that the analysis results of the recombinant plasmid DNA were identical with those of the genomic DNA of the standard substance. Therefore, the recombinant plasmid standard molecule can be used as a positive reference material for preliminary screening of the components of genetically modified crops.
To make up for the lack of traditional reference materials, a plasmid reference molecule was constructed for screening the components of genetically modified crops. First, the 454 bp RBCL gene, 236 bp CaMV35 S promoter and 200 bp NOS terminator were amplified by PCR. Using overlap extension PCR technology the three amplified fragments were ligated to be a 849 bp recombinant. After cloning of the recombi-nant into p MD18-T vector, the positive recombinant plasmid pMD-RBCL-CaMV35 S-NOS was successfully confirmed by PCR amplification and sequence analysis. Further substitution studies showed that the analysis results of the recombinant plasmid DNA were identical with those of the genomic DNA of the standard substance. Therefore, the recombinant plasmid standard molecule can be used as a positive reference material for preliminary screening of the components of genetically modified crops.
引文
[1] ISAAA. Brief 53:Global status of commercialized biotech/GM crops:2017[EB/OL]. http://www.isaaa.org/resources/publications/briefs/53/default.asp.
[2]王颢潜,陈锐,李夏莹,等.转基因产品成分检测技术研究进展[J].生物技术通报(WANG Hao-qian, CHEN Rui, LI Xiaying, et al. Advances in the detection technology of genetically modified products[J]. Biotechnology Bulletin), 2018, 34(3):31-38.
[3] SALISU I B, SHAHID A A, YAQOOB A, et al. Molecular approaches for high throughput detection and quantification of genetically modified crops[J]. Frontiers in Plant Science, 2017,8:1670
[4] CANKAR K, RAVNIKAR M, ZEL J, et al. Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the Ca MV35S screening assay for genetically modified organisms[J]. Journal of AOAC International, 2005, 88(3):814-822.
[5] WU Y, WANG Y, LI J, et al. Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods[J]. Scientific Reports, 2014, 4:7358.
[6] LIN C H, PAN T M. Perspectives on genetically modified crops and food detection[J]. Journal of Food and Drug Analysis, 2016,24(1):1-8.
[7]隋志伟,余笑波,王晶,等.转基因水稻TT51-1标准物质的研制[J].计量学报(SUI Zhi-wei, YU Xiao-bo, WANG Jing, et al.Development of genetically modified rice TT51-1 seed powder reference materials[J]. Acta Metrologica Sinica), 2012, 33(5):467-471.
[8] LU Y, XIAO S, YUAN M, et al. Using overlap-extension PCR technique to fusing genes for constructing recombinant plasmids[J]. Journal of Basic Microbiology, 2018, 58(3):273-276.
[9]高宏伟,相大鹏,覃文,等. GB/T 19495.3-2004,转基因产品检测—核酸提取纯化方法[S](GAO Hong-wei, XIANG Da-peng,QIN Wen, et al. Detection of genetically modified organisms and derived products. Nucleic acid extraction[S]). 2004-04-13.
[10] ALASAAD N, ALZUBI H, KADER A A. Data in support of the detection of genetically modified organisms(GMOs)in food and feed samples[J]. Data in Brief, 2016, 7:243-252.
[11]李亮,王晶,臧超,等.转基因水稻华恢1号检测用质粒标准分子研制[J].生物技术通报(LI Liang, WANG Jing, ZANG Chao,et al. Development of a reference plasmid for the quantitative detection of genetically modified rice Huahui 1[J]. Biotechnology Bulletin), 2013, 29(1):96-101.
[12] LI L, DONG M, AN N, et al. A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed[J]. BioMed Research International, 2015, 2015:948297.
[13]吴永彬,肖维威,张宝,等.转基因大豆、玉米、油菜和水稻品系特异性检测质粒标准分子的快速构建及应用[J].中国生物化学与分子生物学报(WU Yong-bin, XIAO Wei-wei, ZHANG Bao, et al. Construction and application of standard reference molecule for strain-specific detection in genetically modified soybean, maize, rape and rice[J]. Chinese Journal of Biochemistry and Molecular Biology), 2011, 27(8):775-782.
[14] XU C, LI L, JIN W, et al. Recombinase polymerase amplification(RPA)of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops[J]. International Journal of Molecular Sciences, 2014, 15(10):18197-18205.
[15] DATUKISHVILI N, KUTATELADZE T, GABRIADZE I, et al.New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 2015,6:757.
[16] HUSSAIN H, CHONG N F. Combined overlap extension PCR method for improved site directed mutagenesis[J]. BioMed Research International, 2016, 2016:8041532.
[17] XIAO Y H, PEI Y. Asymmetric overlap extension PCR method for site-directed mutagenesis[J]. Methods in Molecular Biology,2011, 687:277-282.
[18] LI G, DONG B X, LIU Y H, et al. Gene synthesis method base on overlap extension PCR and DNAWorks program[J]. Methods in Molecular Biology, 2013, 1073:9-17.
[19] DONG B, MAO R, LI B, et al. An improved method of gene synthesis based on DNA works software and overlap extension PCR[J]. Molecalar Biotechnology, 2007, 37(3):195-200.
[20] KARKHANE A A, YAKHCHALI B, RASTGAR J F, et al. A nested-splicing by overlap extension PCR improves specificity of this standard method[J]. Iran Journal of Biotechnology, 2015,13(2):56-59.
[21] THORNTON J A. Splicing by overlap extension PCR to obtain hybrid DNA products[J]. Methods in Molecular Biology, 2016,1373:43-49.
[2]王颢潜,陈锐,李夏莹,等.转基因产品成分检测技术研究进展[J].生物技术通报(WANG Hao-qian, CHEN Rui, LI Xiaying, et al. Advances in the detection technology of genetically modified products[J]. Biotechnology Bulletin), 2018, 34(3):31-38.
[3] SALISU I B, SHAHID A A, YAQOOB A, et al. Molecular approaches for high throughput detection and quantification of genetically modified crops[J]. Frontiers in Plant Science, 2017,8:1670
[4] CANKAR K, RAVNIKAR M, ZEL J, et al. Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the Ca MV35S screening assay for genetically modified organisms[J]. Journal of AOAC International, 2005, 88(3):814-822.
[5] WU Y, WANG Y, LI J, et al. Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods[J]. Scientific Reports, 2014, 4:7358.
[6] LIN C H, PAN T M. Perspectives on genetically modified crops and food detection[J]. Journal of Food and Drug Analysis, 2016,24(1):1-8.
[7]隋志伟,余笑波,王晶,等.转基因水稻TT51-1标准物质的研制[J].计量学报(SUI Zhi-wei, YU Xiao-bo, WANG Jing, et al.Development of genetically modified rice TT51-1 seed powder reference materials[J]. Acta Metrologica Sinica), 2012, 33(5):467-471.
[8] LU Y, XIAO S, YUAN M, et al. Using overlap-extension PCR technique to fusing genes for constructing recombinant plasmids[J]. Journal of Basic Microbiology, 2018, 58(3):273-276.
[9]高宏伟,相大鹏,覃文,等. GB/T 19495.3-2004,转基因产品检测—核酸提取纯化方法[S](GAO Hong-wei, XIANG Da-peng,QIN Wen, et al. Detection of genetically modified organisms and derived products. Nucleic acid extraction[S]). 2004-04-13.
[10] ALASAAD N, ALZUBI H, KADER A A. Data in support of the detection of genetically modified organisms(GMOs)in food and feed samples[J]. Data in Brief, 2016, 7:243-252.
[11]李亮,王晶,臧超,等.转基因水稻华恢1号检测用质粒标准分子研制[J].生物技术通报(LI Liang, WANG Jing, ZANG Chao,et al. Development of a reference plasmid for the quantitative detection of genetically modified rice Huahui 1[J]. Biotechnology Bulletin), 2013, 29(1):96-101.
[12] LI L, DONG M, AN N, et al. A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed[J]. BioMed Research International, 2015, 2015:948297.
[13]吴永彬,肖维威,张宝,等.转基因大豆、玉米、油菜和水稻品系特异性检测质粒标准分子的快速构建及应用[J].中国生物化学与分子生物学报(WU Yong-bin, XIAO Wei-wei, ZHANG Bao, et al. Construction and application of standard reference molecule for strain-specific detection in genetically modified soybean, maize, rape and rice[J]. Chinese Journal of Biochemistry and Molecular Biology), 2011, 27(8):775-782.
[14] XU C, LI L, JIN W, et al. Recombinase polymerase amplification(RPA)of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops[J]. International Journal of Molecular Sciences, 2014, 15(10):18197-18205.
[15] DATUKISHVILI N, KUTATELADZE T, GABRIADZE I, et al.New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 2015,6:757.
[16] HUSSAIN H, CHONG N F. Combined overlap extension PCR method for improved site directed mutagenesis[J]. BioMed Research International, 2016, 2016:8041532.
[17] XIAO Y H, PEI Y. Asymmetric overlap extension PCR method for site-directed mutagenesis[J]. Methods in Molecular Biology,2011, 687:277-282.
[18] LI G, DONG B X, LIU Y H, et al. Gene synthesis method base on overlap extension PCR and DNAWorks program[J]. Methods in Molecular Biology, 2013, 1073:9-17.
[19] DONG B, MAO R, LI B, et al. An improved method of gene synthesis based on DNA works software and overlap extension PCR[J]. Molecalar Biotechnology, 2007, 37(3):195-200.
[20] KARKHANE A A, YAKHCHALI B, RASTGAR J F, et al. A nested-splicing by overlap extension PCR improves specificity of this standard method[J]. Iran Journal of Biotechnology, 2015,13(2):56-59.
[21] THORNTON J A. Splicing by overlap extension PCR to obtain hybrid DNA products[J]. Methods in Molecular Biology, 2016,1373:43-49.