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为获得进口转基因玉米筛查检测策略,并根据策略建立多重PCR方法,对15个进口转基因玉米分子特征进行分析。结果表明,将P-Ca MV 35S、T-NOS等2个基因组合作为检测策略可全部检出15个进口转基因玉米至少1次;将P-Ca MV 35S、P-ract1、T-NOS、bar、pat、PMI等6个基因组合作为检测策略,除MON810检出1次外,其他每个转化体可检出至少2次。同时,利用获得的筛查检测策略建立多重PCR体系,对2个基因组合的策略优化,结果表明适宜退火温度为58℃,适宜引物终浓度(μmol·L-1)配比为0.2∶0.2;对6个基因组合的策略优化,结果表明适宜退火温度为60℃,引物终浓度配比为0.2∶0.1∶0.1∶0.1∶0.1∶0.05。采用已知样品对多重PCR体系进行验证,图谱显示与转化体分子特征完全一致。该研究结果将为进口转基因玉米筛查检测提供参考。
In order to obtain a screening test strategy for imported maize and to establish a multiplex PCR method according to the strategy, the molecular characteristics of 15 imported maize maize were analyzed. The results showed that 15 genetically modified (GM) maize plants could be detected at least once by using two gene combinations of P-Ca MV 35S and T-NOS as detection strategies. The P-Ca MV 35S, P-ract1, T-NOS and bar , Pat, PMI and other six genes as a detection strategy, in addition to MON810 detected one time, the other each transformant can be detected at least twice. At the same time, the multiplex PCR system was established by using the screening and screening strategy, and the strategy of two gene combinations was optimized. The results showed that the suitable annealing temperature was 58 ℃, and the suitable primer concentration (μmol·L-1) was 0.2: 0.2. The strategy optimization of the six gene combinations showed that the optimum annealing temperature was 60 ° C and the final primer concentration was 0.2: 0.1: 0.1: 0.1: 0.1: 0.05. Multiplex PCR systems were validated using known samples and the chromatograms showed exactly the same molecular characteristics as the transformants. The results of this study will provide a reference for the detection of imported GM maize.