Establishment of SYBR Green-base quantitative real-time PCR assay for determining transgene copy number in transgenic soybean
2011
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.
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