转基因棉花中棉41多重PCR体系的建立

Establishment of An Exogenous Detecting System of Genetically Modified Cotton CCRI 41

利用多重PCR建立转基因棉花中棉41外源基因的检测体系。多重PCR技术的关键在于多重PCR反应条件的优化,本文从多重引物组合、PCR反应体系、PCR反应条件这三个方面对转基因棉花多重PCR反应进行优化,建立了扩增效率较高的中棉41的六重PCR反应体系,优化后的体系为HotstarTaq DNA聚合酶用量为1.25 U/50μL,镁离子的终浓度为3.5 mM,dNTP的终浓度为400μM,BSA(10mg/mL)的加入量为2μL,选取58℃为反应扩增的退火温度。利用优化的多重PCR体系特异性地检测到转基因棉花中棉41中的外源基因启动子CaMV 35S、终止子TNOS、报告基因NPTII和抗虫基因Cry1Ac。

In this paper,an exogenous genes detection system of genetically modified cotton was established by multiplex PCR.The system could amplificate heterologous gene of genetically modified cotton by multiple PCR technology in a high throughput way for specific detection.The optimization of the conditions is a key point of multiplex PCR.In order to optimize the multiplex PCR system,we did the experiments from multiple primer combinations,polymerase chain reaction system and polymerase chain reaction condition three aspects,establishing the high efficiency amplification of six sequence of genetically modified cotton CCRI 41.The optimal dosage for Hotstar Taq DNA polymerases was 1.25 U/50 μl,magnesium ions concentration was 3.5 mM,dNTP concentration was 400 μM,BSA（10 mg/ml）to the final volume of 2 μl,and the annealing temperature was 58℃.Using the optimized multiplex PCR system,exogenous genes of genetically modified cotton like promoter CaMV 35S,terminator TNOS,report NPT II genes and insect-resistant genes Cry1Ac were detected.