Promoter cloning and analysis of the cold-stress response of PHD17 gene in rice

Objective This study aimed to clone promoter sequence of gene OsPHD17 in PHD （plant homeodomain） of rice and analyze its response to cold stress， so as to provide a theoretical reference for elucidating the cold-stress response mechanism of gene OsPHD17.Method Quantitative real-time PCR （qPCR） was employed to analyze expression pattern of gene OsPHD17 under cold stress treatment. Recognition elements of transcriptional factors of the promoter were predicted based on PlantRegMap database. The promoter sequence （length of 1160 bp upstream of the transcription start site） of gene OsPHD17 was cloned and inserted to vector pCAMBIA3301 to obtain plant expression vector P_OsPHD17-GUS. Transgenic Arabidopsis thaliana lines were generated via Agrobacterium tumefaciens-mediated transformation. Expression of GUS reporter genes under cold stress were detected by RT-PCR and qPCR to analyze the change in promoter transcriptional activity under cold stress conditions.Result The promoter of gene OsPHD17 included recognition elements of the transcriptional factors regulating growth and development （TALE， LBD， MIKC_MADS， G2-like， BBR-BPC） and recognition elements of the transcriptional factors regulating cold-stress response （ERF， C2H2， bHLH， bZIP， and NAC）. Expression of the gene OsPHD17 was significantly up-regulated at 3.0 hours of cold stress （P<0.05，the same below）； the expression peaked at 12.0 h with approximately 11.7-fold increase compared to the expression at 0 h， indicating a rapid cold-stress response of the gene OsPHD17. The cloned promoter sequence was 1160 bp in length， and transgenic Arabidopsis thaliana lines were successfully established. RT-PCR detection showed that T3 generation were homozygous with GUS genes expression of P_OsPHD17-GUS transgenic Arabidopsis thaliana lines， indicating that the promoter of gene OsPHD17 had transcriptional activity. Under cold stress of 4 ℃， the two homozygous T3 lines of Arabidopsis thaliana showed a highly significant （P<0.01） or significantly up-regulated relative expression of the GUS gene in the two lines compared to the CK group （untreated）， demonstrating an increase of transcriptional activity of promoter of gene OsPHD17.Conclusion The promoter of gene OsPHD17 that exhibits transcriptional activity is capable of modulating downstream gene expression. Transcriptional activity is markedly upregulated under cold stress conditions， and expression of gene OsPHD17 is significantly upregulated， thereby confirming the important regulatory role of gene OsPHD17 in cold-stress response of rice.