畜牧兽医学报2011，Vol.42Issue(5)：742-746,5.
TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数
Development of a Real-time PCR for Determination of Foreign Gene Copy Number in Genome of Pichia pastoris
摘要
Abstract
A TaqMan real-time PCR assay for rapid determination of the foreign gene copy number in genome of recombinant P. pastoris strains was developed using gp90 gene of reticuloendotheliosis virus as target gene and Glyceraldehydes-3-phosphate dehydrogenase (GAP) gene of P. pastoris as reference gene. The double standard curves of reference gene and gp90 gene were mapped and the genomic DNA of P. pastoris transformants containing gp90 gene was analyzed by realtime PCR, then the gp90/reference ratio in the samples was calibrated as the gp90 gene copy numbers integrated into the genome of P. pastoris. Using this method, one copy of the foreign gene integrated into the yeast genome can be detected sensitively and had good reproducibility.Sixty recombinant P. pastoris transformants were detected by this real-time PCR method. It was found that 38 recombinant strains contained one copy of gp90 gene and 22 contained multiple copies. This study suggests that the real-time quantitative PCR is a good tool for detecting of foreign gene copy number in P. pastoris transformants and it will be useful for screening of P. pastoris transformants with high level productivity.关键词
TaqMan荧光定量PCR/毕赤酵母/基因拷贝数/GAP基因分类
生物科学引用本文复制引用
李凯,高宏雷,高立,祁小乐,高玉龙,徐延伟,王笑梅..TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数[J].畜牧兽医学报,2011,42(5):742-746,5.基金项目
现代农业肉鸡产业技术体系建设(nycytx-42-G3-01) （nycytx-42-G3-01）
