实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数

Determination of Copy Number of Foreign Gene in Genome of Pichia pastoris by Real-time Fluorescent Quantitative PCR

目的建立实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数的方法。方法采用双标准曲线法,分别利用含有GAP基因和绿色荧光蛋白(GFP)基因的质粒,进行实时荧光定量PCR反应,建立标准曲线。将含有外源GFP基因的毕赤酵母基因组进行实时荧光定量PCR,通过标准曲线计算出外源GFP基因在毕赤酵母基因组中的拷贝数。结果毕赤酵母GAP基因Ct值与起始拷贝数对数的回归方程为:y=-3.612x+39.28,GFP基因Ct值与起始拷贝数对数的回归方程为:y=-3.544x+37.99,GAP基因和GFP基因的反应效率分别为0.89和0.90,两条标准曲线的相关系数均为0.999,且均有良好的重复性。检测10个转入GFP基因的毕赤酵母菌株,筛选到9个单拷贝整合GFP基因的菌株和1个多拷贝整合GFP基因的菌株。结论已建立了检测毕赤酵母基因组中外源基因拷贝数的实时荧光定量PCR法,该方法能够筛选出不同外源基因拷贝数的毕赤酵母菌株。

Objective To develop a method for determination of the copy number of foreign gene in genome of Pichia pastoris by real-time fluorescent quantitative PCR. Methods The standard curves of GAP and GFP genes were generated with the plasmids containing GAP and GFP genes respectively, and the genomic DNA of P. pastoris transformants containing GFP gene was analyzed by real-time fluorescent quantitative PCR. The copy number of GFP gene in each transformant was calculated with the Ct value of P. pastoris genomic DNA and the standard curve. Results The regression equation of Ct value and logarithm of original copy number of GAP gene was y = -3. 612x ＋ 39. 28, while that of GFP gene was y = -3. 544x ＋ 37. 99. The reaction efficiencies of GFP and GAP genes were 0. 89 and 0. 90 respectively. However, both the correlation coefficients of standard curves of the two genes were 0. 999, and both the curves showed good reproducibility. Of the ten P. pastoris transformants tested, nine contained one copy and one contained multiple copies of GFP gene. Conclusion A real-time fluorescent quantitative PCR method was successfully developed, which might be used for screening of P. pastoris transformants containing various copies of foreign genes.