Taqman定量PCR技术检测转基因大豆中外源基因拷贝数

目的]采用Taqman定量PCR技术检测转基因杂交大豆中外源nos终止子基因的拷贝数。方法]以大豆凝集素基因为内参照基因,以非转基因大豆基因组DNA为内参照基因标准品,通过梯度稀释法分别求取了内参照基因和质粒DNA的Ct值与拷贝数对数值的相关性标准曲线方程,并通过将得到的Ct值代入标准曲线方程求取了样品的拷贝数。结果]内参照基因标准曲线方程为y=-3.422x＋35.201,R2=0.998;外源基因标准曲线方程为y=-3.348x＋34.890,R2=0.999。nos终止子基因及其下游边界序列在转基因杂交大豆中为单拷贝。结论]该研究为确定转基因大豆外源基因拷贝数提供了理论依据。

Detection of Exogenous Gene Copies in Transgenic Soybean by Taqman Quantitative PCR Technique

Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.