Isolation and identification of five cold-inducible promoters from Oryza sativa

Five promoters of the cold-inducible rice genes were isolated. The quantitative and qualitative expression analyses in the high generation transgenic rice suggest that the genes are stably induced by low temperature. Cold-inducible promoters are highly desirable for stress-inducible gene expression in crop genetic engineering. In this study, five rice genes, including OsABA8ox1, OsMYB1R35, OsERF104, OsCYP19-4, and OsABCB5, were found to be transcriptionally induced by cold stress. The promoters of these five genes were isolated, and their activities were identified in various tissues of transgenic rice plants at different growth stages both before and after cold stress. Histochemical staining, quantitative fluorescence assays, and GUSplus gene expression assays in corresponding promoter-GUSplus transgenic rice plants confirmed that the five promoters were cold-inducible with different expression patterns and strengths. The OsABA8ox1 and OsERF104 promoters had very low background expression; in contrast, the OsMYB1R35 promoter had higher basal activity in the roots, and OsCYP19-4 promoter activity was preferentially high in leaves and flowers of untreated transgenic lines. The OsABCB5 promoter had the highest basal activity among the five promoters. After cold induction, the activities of the OsABA8ox1, OsMYB1R35, and OsABCB5 promoters were high in both roots and leaves, slightly lower than that of the constitutively expressed OsActin1 promoter but comparable to that of the AtRD29A promoter. During the cold treatment time course, the activities of OsABA8ox1 and OsABCB5 promoters were quickly up-regulated in the early period and peaked at 24 h, after which the induction level gradually decreased until 48 h. The activities of the OsMYB1R35 and OsCYP19-4 promoters increased under stress in a time-dependent manner, while OsERF104 promoter activity began to increase at 4 h and then decreased strongly. Furthermore, activities' analysis in T₃, T₄, and T₅ homozygous progeny of single-copy plants revealed that five promoters maintained their activities at comparable levels with no evidence of silencing under cold stress. Overall, the five cold-inducible rice promoters described herein could potentially be used in crop biotechnology.