Expression and Stability Analysis of Seven Candidate Reference Genes in Different Tissues of Vaccinium dunalianum Wight

Purpose To select appropriate reference genes for gene expression analysis in different tissues of Vaccinium dunalianum Wight.

Methods Based on the transcriptome sequencing data of the third generation, seven candidate reference genes (PP2A-1, PP2A-2, EIF-4A-1, EIF-4A-2, 60S-1, 60S-2 and ARP-1) were selected. The expression level of candidate reference genes was detected by qRT-PCR in eight different tissues of V. dunalianum (tender leaves, mature leaves, flower buds, flowers, green fruits, red fruits, green fruit stems and red fruit stems). Firstly, geNorm, NormFinder and BestKeeper methods were used to analyze and evaluate the stability of gene expression, and then RefFinder method was used to comprehensively evaluate the optimal reference genes in different tissues of V. dunalianum. Finally, the tissue expression of phenylalanine ammonia-lyase gene (PAL) in chlorogenic acid synthesis pathway was analyzed to verify the reliability of the evaluation results of the appropriate reference gene.

Results In the different tissues of V. dunalianum, the expression stability of 60S-2 was the best, followed by PP2A-2 gene, and the expression stability of ARP-1, 60S-1 and EIF-4A-2 were in the middle, the expression stability of EIF-4A-1 was the worst. Using 60S-2 gene as the reference gene, the variation trend of PAL gene expression and chlorogenic acid content in different tissues was consistent, indicating that PAL gene was involved in the synthesis of chlorogenic acid in V. dunalianum.

Conclusion 60S-2 is the most stable reference gene for the seven candidate genes tested in different tissues of V. dunalianum, which provides the most appropriate reference gene for subsequent studies on the expression of other functional genes in different tissues of V. dunalianum.