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以棉花内源基因sad1、报告基因GUS、外源抗虫基因Cry1 Ab/Ac、筛选基因NPTⅡ、NOS终止子和CaMV35 S启动子为检测对象,设计的6对引物能扩增长度合适的基因片段且避免了引物二聚体。通过优化PCR扩增体系中不同引物终浓度的配比以及退火温度对多重PCR检测的影响,建立了棉花转基因成分6重PCR检测体系。结果表明:采用本研究建立的棉籽基因组DNA提取方法,该6重PCR检测体系能够有效地从少至1颗棉籽的少量样品里检测出棉花中的转基因成分。
Six pairs of primers were designed to amplify a suitable length of gene fragment using cotton endogenous gene sad1, reporter gene GUS, exogenous insect-resistant gene Cry1 Ab / Ac, screening gene NPTⅡ, NOS terminator and CaMV35 S promoter And primer dimers are avoided. By optimizing the ratio of the final concentration of different primers in the PCR amplification system and the effect of annealing temperature on the detection of multiplex PCR, a 6-fold PCR detection system of cotton transgenic components was established. The results showed that the six-fold PCR detection system could effectively detect the transgenic components of cotton from a few samples of as few as one cottonseed, using the cottonseed genomic DNA extraction method established in this study.