重组毕赤酵母中Textilinin-1基因拷贝数的荧光定量PCR法检测

 目的 利用荧光定量PCR法检测重组毕赤酵母外源纤溶酶抑制剂Textilinin-1基因的拷贝数。方法 采用双标准曲线法，以甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）基因为内源参照基因，分别利用含有GAPDH和Textilinin-1基因的质粒，进行荧光定量PCR反应，建立标准曲线。对重组毕赤酵母基因组进行荧光定量PCR反应，通过标准曲线计算出外源Textilinin-1基因在毕赤酵母基因组中的拷贝数。结果 GAPDH和Textilinin-1基因的扩增效率分别95.04%和96.36%，两条标准曲线的决定系数均为0.999，且均具有良好的重复性，共检测10个单菌落，均得到Textilinin-1基因拷贝数为2。结论 建立的方法能够鉴定重组毕赤酵母中Textilinin-1基因的拷贝数。

Determination of copy number of Textilinin-1 gene in recombinant Pichia pastoris by fluorescence quantitative PCR

Objective  To detect the copy number of plasmin inhibitor gene Textilinin-1 in Pichia pastoris by fluorescence quantitative PCR. Method  The standard curves of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Textilinin-1 genes were generated with the plasmids containing GAPDH and Textilinin-1 genes, respectively, and the genomic DNA of Pichia pastoris transformants containing Textilinin-1 gene were analyzed by fluorescent quantitative PCR. The copy number of Textilinin-1 gene in each transformant was calculated according to the double standard curves. Results  The reaction efficiencies of GAPDH and Textilinin-1 genes were 95.04% and 96.36%, respectively. The coefficients of determination for standard curves of two genes were both 0.999, and both curves showed good reproducibility. Of the 10 transformants tested, the copy number of Textilinin-1 gene was 2. Conclusion  A fluorescence quantitative PCR method is successfully developed and it is useful for detecting the copy number of Textilinin-1 gene in recombinant Pichia pastoris.