Evaluation of reference genes and expression patterns of CONSTANS-LIKE genes in Tetrastigma hemsleyanum under different photoperiods

CONSTANS-LIKE (COL) genes are a key signalling molecule that regulates plant growth and development during the photoperiod. Our preliminary experiments showed that the photoperiod greatly influence the formation of Tetrastigma hemsleyanum root tubers. In this study, we examined the oscillation patterns and expression characteristics of COL genes in leaves of T. hemsleyanum under different photoperiod conditions. Six genes were selected as candidate reference genes for further analyses: (1) 18S ribosomal RNA (18S rRNA); (2) alpha-tubulin (TUBA); (3) 30S ribosomal RNA (30S rRNA); (4) TATA binding protein (TBP); (5) elongation factor 1 alpha (EF-1 alpha); and (6) RNA polymerase II (RPII). The geNorm, NormFinder, and BestKeeper software programs were used to evaluate expression stability. Two ThCOL genes were screened in the T. hemsleyanum transcriptome library, and their expression patterns under different photoperiod conditions were analysed using quantitative reverse transcription PCR. The genes EF-1 alpha, TUBA, and 18S rRNA were used to analyse the expression profiles of CONSTANS genes (ThCOL4 and ThCOL5) under different photoperiods. The expression peaks of ThCOL4 and ThCOL5 appeared at different times, demonstrating that their oscillation patterns were influenced by the photoperiod. We speculate that these two ThCOL genes may be involved in different biological processes.