SYBR Green实时定量PCR检测转基因大豆中外源基因拷贝数

采用SYBR Green I real-time PCR方法检测转基因大豆中外源基因35S边界基因的拷贝数,以大豆凝集素基因(Lectin)作为内参照基因,以转基因大豆基因组DNA为内参照基因标准品,初始浓度为0.43μg·μL-1,进行5倍梯度稀释得到内参照基因CT值与起始模板量的相关性标准曲线:y=-2.9915x...

Establishment of SYBR Green-base quantitative real-time PCR assay for determining transgene copy number in transgenic soybean

SYBR Green Ι real-time PCR was developed to determine copy number of exogenous 35S/plant gene in transgenic soybean.A conserved soybean housekeeping gene,Lectin was used as an internal contral and 5 times diluted soybean genome DNA was used as an initial template copy of Lectin.So y=-2.9915x+22.3321,R2=0.996,the standard curves of the cycle threshold (CT) relative to the log of each initial template copy of Lectin was obtained.In the same way,the standard curves of 35S/plant y=-3.4021x+17.7219,R2=0.999,was obtained using 5 times diluted plasmid DNA which contains 35S/plant gene.By SYBR Green Ι real-time the CT values of Lectin and 35S/plant gene in each transgenic soybean was got,the transgene copy number was calculated by comparing the initial template copy of 35S/plant to Lectin gene in the same transgenic soybean.It concluded that among four PCR putative transgenic plants:one had two copies,and the other had three copies,respectively.