Systematic Identification of Suitable Reference Genes for Quantitative Real-Time PCR Analysis in Melissa officinalis...

Area of Science:

• Plant Molecular Biology
• Biochemistry
• Genomics

Background:

• Melissa officinalis L. possesses valuable pharmacological properties, but its biosynthetic pathways remain underexplored.
• Accurate gene expression studies, particularly using quantitative real-time PCR (qRT-PCR), are essential for understanding these pathways.
• The reliability of qRT-PCR hinges on the selection of stable, experiment-specific reference genes for normalization.

Purpose of the Study:

• To identify and validate optimal reference genes for gene expression normalization in Melissa officinalis.
• To assess the stability of ten candidate reference genes under various abiotic stress conditions and across different plant organs.
• To provide a foundation for reliable gene expression studies in M. officinalis and related species.

Main Methods:

• Quantitative real-time PCR (qRT-PCR) was employed to analyze the expression stability of ten candidate reference genes.
• Gene expression data were analyzed using multiple statistical algorithms: BestKeeper, comparative ΔCt, geNorm, and NormFinder.
• RefFinder was utilized to integrate results and rank the candidate genes for overall stability.

Main Results:

• The combination of elongation factor 1-alpha (EF-1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the most stable and suitable reference gene pair for normalizing gene expression in M. officinalis.
• The gene HLH71 exhibited the least stability and is not recommended for normalization in this species.
• Validation using phenylalanine ammonia lyase (PAL) gene expression under elicitor treatments confirmed the reliability of the recommended reference gene combination.

Conclusions:

• The study provides validated reference genes (EF-1α and GAPDH) crucial for accurate gene expression studies in Melissa officinalis.
• These findings will enhance the reliability of future research on M. officinalis's biosynthetic pathways and pharmacological properties.
• The recommended reference genes are applicable across various experimental conditions, including abiotic stress and different plant organs.