Identification and Analysis of Reference and Tissue-Specific Genes in Bitter Gourd Based on Transcriptome Data

Accurate and standardized quantification of reverse transcription PCR (qRT-PCR) results relies on the use of a dependable reference gene. The precise control of transgene expression in terms of both spatial and temporal aspects necessitates the utilization of tissue-specific gene promoters. However, the identification of stable reference genes across various tissues, particularly in fruits at different ripening stages, as well as tissue-specific genes in bitter gourds, remains largely unexplored. In this study, we employed RNA-Seq-based transcriptome datasets obtained from nine tissues to comprehensively screen for new reference genes (NRGs) and tissue-specific genes. Through the utilization of five algorithms in conjunction with qRT-PCR analysis, we successfully identified two highly stable reference genes, namely HMG1/2 and PHOS32, from a pool of 11 NRGs and five traditional reference genes (TRGs). To validate their reliability, we performed expression pattern analysis of two genes associated with fruit ripening (McACO1 and McACO2) using HMG1/2 and PHOS32, as well as an unstable reference gene, HSCP2. Furthermore, we conducted qRT-PCR validation of 12 tissue-specific genes using HMG1/2 as the reference gene. This study not only contributes to the precise normalization of target genes in bitter gourd but also provides a solid foundation for regulating transgenes through the utilization of suitable tissue-specific promoters.