Reference genes selection for Calotropis procera under different salt stress conditions

Area of Science:

• Plant Biology
• Molecular Biology
• Biotechnology

Background:

• Calotropis procera exhibits high tolerance to salt and drought, making it a valuable genetic resource for biotechnology.
• Accurate gene expression quantification using real-time quantitative polymerase chain reaction (qPCR) relies on stable reference genes (RGs).
• Evaluating RG stability is crucial for reliable gene expression analysis, especially under stress conditions.

Purpose of the Study:

• To assess the stability of ten candidate reference genes in Calotropis procera leaves and roots under varying salt stress levels and collection times.
• To identify the most suitable RGs for normalizing gene expression data in C. procera subjected to salinity stress.
• To validate the expression of target genes using the identified stable RGs.

Main Methods:

• Ten candidate reference genes were evaluated for stability in C. procera leaves and roots under different NaCl concentrations (50, 100, 200 mM) and time points (30 min to 45 days).
• Gene expression stability was analyzed using three algorithms (geNorm, NormFinder, BestKeeper).
• The expression of three target genes (NAC78, CNBL4, ND1) was validated using the most stable RGs.

Main Results:

• High agreement was observed among the three algorithms for the top four most stable reference genes.
• CYP23 emerged as the most stable reference gene across all tested experimental conditions (Leaf100, Root50, Root200).
• The expression of target genes NAC78, CNBL4, and ND1 was successfully validated in stressed leaf and root samples.

Conclusions:

• This study provides the first comprehensive selection of stable reference genes for gene expression studies in Calotropis procera under salinity stress.
• CYP23 is recommended as a reliable reference gene for qPCR normalization in C. procera, enhancing the accuracy of gene expression studies.
• The findings highlight the importance of carefully evaluating reference gene stability under diverse experimental conditions to avoid misinterpretation of gene expression data.