The BrAFP1 promoter drives gene-specific expression in leaves and stems of winter rapeseed (Brassica rapa L.) under cold induction

BrAFP1(antifreeze protein in winter turnip rape) effectively limits recrystallization and growth of ice crystals. The BrAFP1 expression level determines whether the freezing-induced damage to winter turnip rape plants is avoided. This study analyzed the activity of the BrAFP1 promoters of several varieties at various cold tolerance levels. We cloned the BrAFP1 promoters from five winter rapeseed cultivars. The multiple sequence alignment revealed the presence of one inDel and eight single-nucleotide mutations (SNMs) in the promoters. One of these SNMs (base mutation from C to T) at the –836 site away from the transcription start site (TSS) enhanced the transcriptional activity of the promoter at low temperature. The promoter activity was specific in cotyledons and hypocotyls during the seedling stage and was referential in stems, leaves, and flowers but not the calyx. This consequently drove the downstream gene to be specifically expressed in leaves and stems, but not in roots at low temperature. The truncated fragment GUS staining assays revealed that the core region of the BrAFP1 promoter was included in the 98 bp fragment from the –933 to –836 site away from the TSS, which was necessary for transcriptional activity. The LTR element of the promoter significantly enhanced expression at low temperatures and suppressed expression at moderate temperatures. Moreover, the BrAFP1 5′-UTR intron bound the scarecrow-like transcription factor and enhanced expression at low temperature.