TaqMan定量PCR检测水稻中外源基因含量

Detection of Foreign Gene Content in Transgenic Rice by TaqMan Quantitative PCR

[目的]采用TaqMan定量PCR技术检测水稻中外源基因含量。[方法]采用TaqMan定量PCR技术以编码磷脂酶D基因PLD为内标准基因,检测转基因水稻中外源TT51-1的含量。利用DNA梯度稀释法分别求内标准基因PLD和TT51-1的C_t值与拷贝数对数值的标准曲线方程,并将得到的C_t值代入标准曲线方程求取样品中外源基因的拷贝数。[结果]内标准基因PLD标准曲线方程为y=-3.403x+39.939,R^2=0.993;TT51-1基因标准曲线方程为y=-3.35x+37.37,R^2=0.991,转基因含量为1.21%,不确定度为0.45。[结论]TaqMan定量PCR技术可以用于确定转基因作物外源基因含量。

[Objective] Foreign gene content in transgenic rice was detected by TaqMan quantitative PCR.[Method]Taqman quantitative PCR technique was adopted to detect exogenous TT51-1 gene content in transgenic rice with PLD as endogenous reference gene. The method of gradient dilution was used to separately calculate Ctvalue of endogenous reference gene and relevance 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 was y =-3. 403 x ＋ 39. 939,R^2= 0. 993; the standard curve equation of TT51-1 gene was y =-3. 35 x ＋ 37. 37,R^2= 0. 991. The content of transgene was 1. 21%,the uncertainty was 0. 45. [Conclusion]TaqMan quantitative PCR technology can be used to determine the foreign gene content of transgenic crops.