转基因植物检测芯片体系的建立及转Xa21水稻的检测
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摘要
随着被批准释放转基因植物的增多,食品安全和环境风险引起了人们的争议和担心,为了保护消费者的知情权,许多国家制定并实施了转基因食品的标识法规。所以,对转基因植物及其产品的检测研究具有重要意义。通过中国、美国、欧盟、加拿大等相关转基因植物信息网站,搜集了目前转基因植物及其含有外源基因的批准释放情况,利用NCBI的GenBank数据库确定外源基因的基因序列,然后设计探针点制芯片。本研究制备了含有12种筛选型、35种目的基因型、5种物种特异型探针的寡核苷酸芯片,为了验证芯片体系的灵敏度和特异性,本试验利用转Xa21的水稻材料,结合基因组DNA随机扩增和klenow酶标记产物,然后与芯片杂交检测并扫描结果。结果表明,对于转Xa21的水稻基因组DNA所含有的筛选基因、种属相关基因和目的基因等,该实验体系能够进行很好的检测和鉴定。
With the number of transgenic plants authorized for release was increased rapidly, food safety and environment risk are being discussed, resulting in many countries and areas have issued GMO labeling regulations for protecting the consumers's authority. Thus, research for the detection of GMOs is necessary in order to verify compliance with labeling requirements. We have developed an array-based method to detect exogenous genes in genetically modified(GM) plants. Microarrays of 54 oligo-nucleotide probes involved 35 exogenous genes of GM plants permitted to grow in the field were assembled.The array was used to detect the exogenous gene xa21 from GM rice DNA. 1 ng of DNA was given a whole-genome amplification with phi29 polymerase followed by fluorescence labeling. Array hybridization showed the positive result.
With the number of transgenic plants authorized for release was increased rapidly, food safety and environment risk are being discussed, resulting in many countries and areas have issued GMO labeling regulations for protecting the consumers's authority. Thus, research for the detection of GMOs is necessary in order to verify compliance with labeling requirements. We have developed an array-based method to detect exogenous genes in genetically modified(GM) plants. Microarrays of 54 oligo-nucleotide probes involved 35 exogenous genes of GM plants permitted to grow in the field were assembled.The array was used to detect the exogenous gene xa21 from GM rice DNA. 1 ng of DNA was given a whole-genome amplification with phi29 polymerase followed by fluorescence labeling. Array hybridization showed the positive result.
引文
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[20]Xu X,Li Y,Zhao H,et al.Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray(J).J Agric Food Chem,2005,53(10):3789-94.
[21]Xu J,Miao H,Wu H,et al.Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray(J).Biosens Bioelectron,2006,22(1):71-7
[22]Leimanis S,Hernandez M,Fernandez S,et al.A microarray-based detection system for genetically modified(GM)food ingredients(J).Plant Mol Biol,2006,61(1-2):123-39
[23]Xu J,Zhu S,Miao H,et al.Event-Specific Detection of Seven Genetically Modified Soybean and Maizes Using Multiplex-PCR Coupled with Oligonucleotide Microarray(J).J Agric Food Chem,2007,55(14):5575-9
[24]Germini A,Mezzelani A,Lesignoli F,et al.Detection of genetically modified soybean using peptide nucleic acids(PNAs)and microarray technology(J).J Agric Food Chem,2004,52(14):4535-40
[25]Germini A,Rossi S,Zanetti A,et al.Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food(J).J Agric Food Chem,2005,53(10):3958-62
[26]Rossi S,Lesignoli F,Germini A,et al.Identification of PCR-amplified genetically modified organisms(GMOs)DNA by peptide nucleic acid(PNA)probes in anion-exchange chromatographic analysis.J Agric Food Chem.2007,55(7):2509-16.
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[28]Urbanek-Karlowska B,Sawilska-Rauteustrauch D,Jedra M,et al.Detection of genetic modification in maize and maize products by ELISA-test(J).Rocz Panstw Zakl Hig,2003,54(4):345-53
[29]严吉明,崔林开,叶华智,等,转Bt基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅱ.Bt杀虫晶体蛋白抗体的制备.四川农业大学学报,2005(3),280-284.
[30]Fantozzi A,Ermolli M,Marini M,et al.Van den Eede G.First application of a microsphere-based immunoassay to the detection of genetically modified organisms(GMOs):quantification of Cryl Ab protein in genetically modified maize (J). J Agric Food Chem, 2007,55(4): 1071-6.
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[32] Weber BL. Cancer genomics. Cancer Cell. 2002;1(1):37-47.
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[34] Tirkkonen M, Tanner M, Karhu R, et al. Molecular cytogenetics of primary breast cancer by CGH. Genes Chromosomes Cancer. 1998 ; 21(3): 177-84.
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[36] Albertson DG, Pinkel D. Genomic microarrays in human genetic disease and cancer. Hum Mol Genet.2003; 12 (Spec No 2) :R145-52.
[37] van Beers EH, Joosse SA, Ligtenberg MJ, et al. A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer. 2006; 94(2): 333-7.
[38] Weiss MM, Hermsen MA, Meijer GA, et al. Comparative genomic hybridisation. Mol Pathol. 1999; 52(5): 243-51.
[39] Little SE, Vuononvirta R, Reis-Filho JS, et al. Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA. Genomics. 2006 ;87(2):298-306.
[40] Stoecklein NH, Erbersdobler A, Schmidt-Kittler O, et al. SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am J Pathol. 2002 ; 161(1): 43-51.
[41] Cheung VG, Nelson SF. Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc Natl Acad Sci U S A. 1996; 93(25): 14676-9.
[42] Telenius H, Carter NP, Bebb CE, et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics. 1992; 13(3): 718-25.
[43] Obermann EC, Junker K, Stoehr R, et al. Frequent genetic alterations in flat urothelial hyperplasias and concomitant papillary bladder cancer as detected by CGH, LOH, and FISH analyses. J Pathol. 2003; 199(1): 50-7.
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