布莱克韦尔虫草实时荧光定量逆转录PCR内参基因的筛选

Screening of reference genes for real-time quantitative reverse transcription PCR in Cordyceps blackwelliae

摘要:

【背景】实时荧光定量逆转录PCR (real-time quantitative reverse transcription PCR, RT-qPCR)常用于分析基因表达，但选择合适的内参基因，对分析结果的稳定性非常重要。布莱克韦尔虫草是一种具有开发价值的虫草，研究其功能基因表达具有重要意义。【目的】筛选获得布莱克韦尔虫草不同生长阶段(液体发酵菌丝体、小麦粒培养基菌丝体及子实体)最稳定的内参基因。【方法】选取8个候选内参基因(18S rRNA、gapdh、ꞵ-tubulin、cyclophilin A、γ-actin、rpl10、tef1α和ubiquitin)，采用RT-qPCR技术进行扩增，结合geNorm、NormFinder、BestKeeper、ΔC_t等算法，评估这些基因在布莱克韦尔虫草不同生长发育阶段的表达稳定性。【结果】相比其他基因，γ-actin和18S rRNA在不同生长发育阶段的表达稳定性最好，可作为布莱克韦尔虫草基因表达分析的内参基因。【结论】本研究筛选出了布莱克韦尔虫草RT-qPCR的内参基因，为深入研究生长发育过程中的基因表达规律奠定了基础。

Abstract:

[Background] Gene expression is often studied by real-time quantitative reverse transcription PCR (RT-qPCR), the stability of which largely depends on the choice of reference genes. Cordyceps blackwelliae is a fungus of development potential, and it is thus vital to understand the expression patterns of functional genes in this fungus. [Objective] To identify the optimal reference genes that express stably across different developmental stages (mycelia from liquid culture, mycelia from wheat grain media, and fruiting bodies) of C. blackwelliae. [Methods] We utilized RT-qPCR to investigate the expression of eight reference genes (18S rRNA, gapdh, ꞵ-tubulin, cyclophilin A, γ-actin, rpl10, tef1α, and ubiquitin). The expression stability of these genes across different developmental stages was evaluated using geNorm, NormFinder, BestKeeper, and ∆C_t algorithms. [Results] The results indicated that γ-actin and 18S rRNA exhibited more stable expression than the other genes, making them suitable reference genes for the transcriptional analysis of functional genes in C. blackwelliae. [Conclusion] This study successfully identified appropriate reference genes for RT-qPCR in C. blackwelliae, aiding future research on gene expression dynamics.