Taqman定量PCR技术检测转基因大豆中外源基因拷贝数

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

[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]为确定转基因大豆外源基因拷贝数提供了理论依据。

[ Objective ] It is to adopt Taqman quantitative PCR technique to detect the copies of fbreign nos terminator in transgenic hybrid soybean. [ Method] With endogenous reference gene of soybean lectin, and endogenous reference standard of gene complex DNA in non-GMO soybeans, the method of gradient dilution was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and rele- vance standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into standard curve equation. [ Result] The standard curve equation of endogenous reference gene isy = - 3. 422x ＋ 35.201 ,. R2 =0.998 ; and the standard curve equation of foreign 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 foreign gene copies in transgenic soybean.