抗环斑病毒转基因番木瓜55-1的PCR检测_药学论文.docVIP

抗环斑病毒转基因番木瓜55-1的PCR检测_药学论文 抗环斑病毒转基因番木瓜55-1的PCR检测_药学论文 【关键词】 ,转基因；番木瓜55-1； 　　摘要： 目的 建立对抗环斑病毒转基因番木瓜55-1进行品系鉴定的检测方法。方法 采用改良十六烷基三甲基溴化铵(CTAB)法及试剂盒方法进行番木瓜基因组DNA提取，根据番木瓜管家Papain基因和抗环斑病毒转基因番木瓜55-1品系的外源结构基因(35S-GUS)和调控基因(NOS-35S)序列设计合成特异性检测引物，采用PCR技术对抗环斑病毒转基因番木瓜55-1进行品系鉴定。结果 内源Papain基因PCR扩增结果表明，改良CTAB法及试剂盒方法都可用于番木瓜种籽及果肉的DNA提取。实验中合成的引物及建立的PCR反应体系、反应参数能特异性地扩增转基因番木瓜55-1的外源基因35S-GUS序列和NOS-35S序列。该方法的绝对检测低限为815×10-2ng，相对检测低限为014％。结论 本检测方法有较高的稳定性，能有效地对转基因番木瓜55-1进行品系鉴定，该方法的检测灵敏度可完全满足转基因食品标识管理定性检测的需要。 　　关键词： 转基因；番木瓜55-1； 　　PCRPCR for event-specific detection of transgenic virus resistant papaya 55-1 　　Abstract： Objective PCR assay was developed for event-specific detection of transgenic virus resistant papaya 55-1.Methods A developed Cetyl Trimethyl Ammonium Bromide(CTAB) method and Qiagen DNeasy plant mini kit were used to extract DNA from papaya seed and fruit.Specific primers for PCR detection of papaya 55-1 were designed to amplify endogenous papain gene sequence and external segments,35S-GUS and NOS-35S,and corresponding detection methods were developed.Results Results of PCR amplification of papain gene indicated that both the developed CTAB method and Qiagen DNeasy plant mini kit were useful for DNA extraction from papaya seed and fruit.Results of amplification of the external segments (35S-GUS and NOS-35S) indicated that the developed method was useful to detect the transgenic virus resistant papaya line 55-1,and the absolute and relative limit of detection (LOD)of the method got to 8.15×10-2 ng and 0.14% respectively.Conclusion The developed PCR method can detect transgenic virus resistant papaya 55-1 specially and detective sensitivity can satisfy the need of qualitative detection for genetically modified foods. 　　Key words： genetically modified;papaya 55-1;PCR 　　番木瓜属热带亚热带重要水果，为有效控制番木瓜环斑病毒(PRSV)对番木瓜产业的威胁，1998年世界上第1个转基因番木瓜品系――抗环斑病毒转基因番木瓜55-1在美国和加拿大获准商业种植〔1，2〕。从此转基因番木瓜55-1以其独特的抗病优势被广泛种植，每年都有大量转基因番木瓜流入国际水果市场。本研究采用改良十六烷基-三甲基-溴化铵(CTAB）法及试剂盒方法进行番木瓜基因组DNA提取，根据番木瓜管家Papain基因和抗环斑病