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为了鉴定携带单拷贝外源基因的转基因烟草植株,以烟草核基因组上已知的单拷贝内源基因(RNR2)为内参,转基因烟草植株基因组DNA为模板,在同一PCR反应体系中扩增内源基因(RNR2)和外源目的基因(NPTⅡ)。反应产物在琼脂糖凝胶上电泳,获得了预期大小的两条特异性扩增条带。经ImageJ软件捕捉分析两条目的条带的灰度比,当T1代转基因烟草植株中外源基因与内源基因的扩增条带灰度比为1时,所检测植株即为单拷贝外源基因的转基因烟草植株。孟德尔经典遗传学方法证实了上述检测结果高度可信。
In order to identify transgenic tobacco plants carrying a single copy of the foreign gene, a single copy of the known endogenous gene on the nuclear genome of tobacco (RNR2) was used as an internal reference, genomic DNA of the transgenic tobacco plants was used as a template, and the endogenous gene was amplified in the same PCR reaction system Gene (RNR2) and exogenous gene (NPTII). The reaction product was electrophoresed on an agarose gel to obtain two specific amplification bands of the expected size. The gray ratio of the two bands was captured and analyzed by ImageJ software. When the gray ratio of the exogenous gene to the endogenous gene in T1 transgenic tobacco plants was 1, the detected plants were single copy of the exogenous gene Of transgenic tobacco plants. Mendelian classical genetic method confirmed the above test results highly credible.