巴斯德毕赤酵母不同生长阶段内参基因的筛选与验证

Identification and validation of reference genes for RT-qPCR normalization in Komagataella phaffii at different growth stages

摘要:

【背景】巴斯德毕赤酵母(Komagataella phaffii)是一种甲基营养型酵母，近年来作为生产重组蛋白和构建生物合成途径的细胞工厂受到广泛关注。实时荧光定量PCR (real-time quantitative PCR，RT-qPCR)是巴斯德毕赤酵母表达系统研究中一种快速、高效的基因表达水平检测技术，但需要进行归一化处理才能保证所得结果的可靠性。【目的】筛选并验证巴斯德毕赤酵母在不同生长阶段最稳定的内参基因用于精准归一化RT-qPCR的结果。【方法】通过转录组数据分析初步筛选出16个候选内参基因(rps8b、rpl35a、rpl10、eif5a、rpl19a、por1、rpl23b、0887、tif1、ole1、rpl14b、gs、sun、sdh2、trx1和ccp1)。通过RT-qPCR技术得到候选内参基因的C_t值，利用qBASE软件中的geNorm程序综合NormFinder算法评估内参基因的表达稳定性。【结果】通过geNorm分析得出精准归一化所需的最佳内参基因个数为2，最稳定的基因是rpl19a和tif1，NormFinder分析得到稳定性最高的内参基因为tif1。此外，利用甲酸脱氢酶编码基因fdh和乙醇脱氢酶甲醛脱氢酶双功能酶的编码基因afdh对候选内参基因进行验证。【结论】巴斯德毕赤酵母不同生长阶段的RT-qPCR进行精准归一化需要tif1和rpl19a这2个内参基因，为相关功能基因的表达定量提供了可靠的分析依据，补充了RT-qPCR分析中的内参基因，为巴斯德毕赤酵母不同生长阶段的基因表达调控及其应用研究提供了新的参考。

Abstract:

[Background] The methylotrophic yeast Komagataella phaffii (K. phaffii) or Pichia pastoris has received extensive attention as a cell factory to produce recombinant proteins and build biosynthetic pathways. Real-time quantitative PCR (RT-qPCR) is a rapid and efficient technique for detecting gene expression levels in the study of the K. phaffii expression system. However, normalization is required to ensure the reliability of the results obtained. [Objective] To screen and verify the most stable reference genes in K. phaffii at different growth stages for accurate normalization of RT-qPCR. [Methods] Sixteen candidate reference genes (rps8b, rpl35a, rpl10, eif5a, rpl19a, por1, rpl23b, 0887, tif1, ole1, rpl14b, gs, sun, sdh2, trx1, and ccp1) were preliminarily screened by transcriptome data analysis. After the C _t values of the candidate reference genes were detected by RT-qPCR, their expression stability was analyzed by applying qBASE geNorm combined with NormFinder. [Results] The optimal number of reference genes by geNorm analysis was 2, and the most stable genes were rpl19a and tif1. The NormFinder analysis demonstrated that the most stable gene was tif1. In addition, we validated the candidate reference genes using fdh encoding formate dehydrogenase and afdh encoding the bifunctional alcohol/formaldehyde dehydrogenase. [Conclusion] Two reference genes, tif1 and rpl19a, were required for accurate normalization of RT-qPCR at different growth stages of K. phaffii. This study provided a reliable basis for the analysis of expression quantification of related functional genes and enriched the reference genes in RT-qPCR analysis, facilitating the study of gene expression regulation and application at different growth stages of K. phaffii.