无选择标记转基因大白菜的检测

Detection of Transgenic Chinese Cabbage with Nonselective Marker Gene

通过对转基因大白菜进行TuMV-C4病毒摩擦接种鉴定、ELSIA检测、提取转基因大白菜DNA进行PCR检测,并通过PCR-Southern杂交、RT-PCR检测转基因大白菜,构建了针对无选择标记转基因大白菜的系统检测方法。试验结果表明:转基因大白菜通过摩擦接种TuMV-C4病毒抗病性鉴定,188株表现为抗性,初步筛选率为19.46%;进行ELISA检测抗病植株中TuMV含量,有63株明显低于非转化植株,筛选率达到26.25%;对抗TuMV植株直接进行目的基因PCR检测,有25株呈阳性,筛选率达到13.30%。经PCR-Southern杂交检测和RT-PCR检测,表明转基因大白菜中不仅整合了Nib基因,而且获得了表达。这种逐级递进的检测系统可以高效地获得无选择标记的转基因植株。

A systematic detection method for nonselective marker transgenic Chinese cabbage was constructed by the identification of TuMV-C4 virus friction inoculation of transgenic Chinese cabbage,ELSIA detection,extraction and PCR detection of the transgenic Chinese cabbage DNA,PCR-Southern hybridization and RT-PCR detection of the transgenic Chinese cabbage.The results showed that 188 strains of transgenic Chinese cabbage were resistant to TuMV-C4 virus inoculated by friction,and the preliminary screening rate was 19.46%.Secondly,the content of TuMV in resistant plants was detected by ELISA,and 63 of them were significantly lower than those of nontransformed plants.The screening rate was 26.25%,and then the target gene PCR was detected directly against TuMV plants,25 of them were positive and the screening rate was 13.30%.Finally,PCR-Southern hybridization and RT-PCR analysis were carried out,which proved that Nib gene was not only integrated but also expressed in transgenic Chinese cabbage.This stepbystep detection system can efficiently obtain nonselective marker transgenic plants.