[Clone and expression of CpGAPDH gene in Codonopsis pilosula]

Area of Science:

• Plant molecular biology
• Biochemistry
• Genomics

Background:

• Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is crucial for carbohydrate metabolism and commonly used as a reference gene.
• Understanding polysaccharide biosynthesis requires reliable reference genes for gene expression analysis.

Purpose of the Study:

• To clone and characterize the GAPDH gene from Codonopsis pilosula (CpGAPDH).
• To evaluate the suitability of CpGAPDH as a reference gene for expression studies in Codonopsis pilosula.

Main Methods:

• Gene cloning based on transcriptome data and homology with potato GAPDH.
• Bioinformatic analysis for protein characteristics and subcellular localization.
• Prokaryotic expression and molecular weight verification.
• Quantitative analysis of transcript levels across different tissues and developmental stages.
• Stability assessment using statistical software (BestKeeper, geNorm, NormFinder, RefFinder).

Main Results:

• The CpGAPDH gene has a 1014 bp ORF encoding 337 amino acids, showing high similarity to other plant GAPDH genes.
• Bioinformatics predicted cytoplasmic localization and membership in the Gp_dh_N superfamily.
• Prokaryotic expression confirmed the recombinant protein's molecular weight.
• CpGAPDH transcript levels were stable across different tissues and developmental stages.
• Statistical analyses confirmed CpGAPDH as a stable reference gene.

Conclusions:

• CpGAPDH is a reliable and stable reference gene for Codonopsis pilosula.
• This finding supports future gene expression analyses related to polysaccharide synthesis in this species.