巴斯德毕赤酵母基因组外源基因整合数的高通量定量分析

High-throughput quantitative analysis for integration number of exogenous gene in Pichia pastoris genome

目的探索一种巴斯德毕赤酵母表达体系外源基因整合数的高通量定量分析方法.方法采用基因工程技术构建pPIC9K-HG质粒作为定量标准品,建立毕赤酵母基因组外源基因整合的高通量定量PCR分析方法,用灵敏度、特异性等对方法进行评价,将之初步用于hA20基因毕赤酵母转化子外源基因整合数的分析.同时对3种酵母基因组制备方法进行了比较.结果方法灵敏度达10^3拷贝,线性范围在10^4～10^9,平均变异系数6.62%,特异性100%.14株hA20基因毕赤酵母转化子外源基因整合数分析显示,成功收获外源基因整合数为单拷贝、3拷贝、10拷贝、13拷贝、17拷贝、50拷贝的阳性转化子.酵母基因组制备经典法、煮沸法和酶法阳性率分别为85.71%、28.57%和85.71%,经χ^2检验证实,酶法与经典法相比提取酵母基因组的效率一致（P＞0.05）;煮沸法与经典法相比提取酵母基因组的效率存在显著性差异（P＜0.05）.结论成功建立了巴斯德毕赤酵母基因组外源基因整合数高通量定量分析方法,方法学评价指标满意,可广泛用于不同外源基因整合之阳性菌株的大批量分析.酶法制备酵母基因组具有可靠、简单、迅速等优点,适合大规模提取酵母基因组.

Objective Here we establish a high-troughput quantitative method for identifying the integration number of exogenous gene in Pichia pastoris genome. Methods pPIC9K-HG was constructed by genetic engineering. After sequencing, pPIC9K-HG was taken as the standard for quantitative analysis. A highthroughput method was set up for detecting the integration number of exogenous gene in Pichia pastoris genome and evaluated with methodological indexes such as sensitivity,specificity, etc. Copies of hA20 in integrants were analyzed. The three methods for isolating the yeast genome were compared. Results The sensitivity was 10^3 and the specificity was 100%.The linearity was between 10^4 and 10^9.The average of CV achieved 6. 62%. The positive rate of the three methods （classics, boiled and enzymatic） was 85.71%,28.57% and 85.71% respectively.There was no significant difference between the classics and the enzymatic by x^2 test, but there was significant difference between the classics and the boiled .We obtained single copy, 3 copies, 10 copies, 13 copies, 17 copies and 50 copies positive strain with hA20. Conclusion A high-throughput quantitative method was successfully established to determinate the copy number of heterologous gene inserted in yeast genome. Methodological indexes suggested that it meet the whole analysis request.The enzymatic method to prepare yeast genome is effective, simple and rapid, and can prepare yeast genome on large scale.