Selection and evaluation of reference genes for expression analysis of Cassi.

Area of Science:

• * Plant molecular biology and genetics.
• * Legume genomics and bioinformatics.

Background:

• * Cassia obtusifolia, a legume, possesses significant pharmaceutical, economic, and ecological value.
• * Robust gene expression studies require stable reference genes for accurate data normalization.
• * Previous research indicates a need for validated reference genes in C. obtusifolia.

Purpose of the Study:

• * To identify and validate stable reference genes for quantitative real-time PCR (qRT-PCR) in Cassia obtusifolia.
• * To evaluate the expression stability of ten candidate reference genes across diverse tissues and stress conditions.
• * To provide reliable reference genes for accurate gene expression normalization in C. obtusifolia.

Main Methods:

• * Quantitative real-time PCR (qRT-PCR) was employed to analyze the expression of 10 candidate reference genes.
• * Gene expression stability was assessed in 12 different C. obtusifolia tissues and under various stress treatments.
• * Expression data were analyzed using established algorithms: geNorm, NormFinder, and BestKeeper software.

Main Results:

• * Candidate reference gene expression varied significantly across different tissues and stress conditions.
• * CYP1, EF1α2, ACT2, and UBQ1 demonstrated the highest expression stability across all tested samples.
• * The reliability of the selected reference genes was confirmed through the detection of WRKY gene expression levels.

Conclusions:

• * CYP1, EF1α2, ACT2, and UBQ1 are recommended as suitable reference genes for normalizing gene expression in Cassia obtusifolia.
• * The choice of reference gene may vary depending on specific experimental conditions (tissue type or stress).
• * This study provides a crucial resource for accurate gene expression profiling in C. obtusifolia research.