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目的:确定抗除草剂转基因大豆外源基因拷贝数和其插入位点侧翼序列。方法:采用绝对定量PCR法测定转EPSPS基因大豆中外源基因拷贝数,内参照基因标准曲线选用大豆凝集素(Lectin)基因为标准品,外源基因标准曲线以含EPSPS基因的阳性质粒为标准品。采用基因组步移技术和巢式PCR方法确定抗除草剂转基因大豆插入位点旁侧序列。结果:抗除草剂转基因大豆外源基因的拷贝数为1。CaMV35S上游扩增887bp,NOS下游扩增1 340bp。结论:明确了EPSPS外源基因在转基因大豆中为单拷贝,转基因大豆插入位点附近大豆基因组发生了DNA重排。
OBJECTIVE: To determine the copy number of the foreign gene and the flanking sequence of its insertion site of the herbicide-tolerant transgenic soybean. Methods: The copy number of exogenous gene in EPSPS transgenic soybean was determined by absolute quantitative PCR method. Lectin gene was selected as standard in the standard curve of reference gene. The positive gene of EPSPS gene was used as a standard . Genomic walking and nested PCR methods were used to determine flanking sequences of the herbicide-resistant transgenic soybean insertion site. Results: The copy number of the herbicide - resistant transgenic soybean was 1. 887bp upstream of CaMV35S and 1 340bp downstream of NOS. Conclusion: It is clear that the exogenous EPSPS gene is a single copy in transgenic soybean, and DNA rearrangement occurs in the soybean genome near the gene insertion site of transgenic soybean.