ORA47 binding cis-element prediction

To find genes directly regulated by ORA47, the P35S:ORA47-GR transgenic line was grown in the normal condition, treated with DEX and as well cycloheximide (CYC), prevented secondary transcriptional regulation downstream of ORA47 regulation by blocking protein synthesis. Gene expression patterns were then analyzed by microarray. To exclude genes that were induced by DEX, CYC, and the transgenic plant background, seven-day-old ORA47-GR transgenic T2 and WT seedlings were separated into two subgroups: (1) 35S:ORA47-GR transgenic T2 and WT seedlings treated with 50 μM CYC and 20 μM DEX, and WT plants were used as control. (2) 35S:ORA47-GR transgenic T2 seedlings treated with 50 μM CYC and 20 μM DEX, and ORA47-GR seedlings treated with 50 μM CYC only were used as controls. Seedlings were treated with CYC for 1 h followed by DEX treatment for 2 h before harvesting. Total RNA of each group was isolated and prepared for microarray assay. There were two independent replicates for each group. The common up-regulated genes obtained from these two datasets were considered as the putative ORA47 direct downstream genes. The one kb promoter regions of the 100 most highly up-regulated genes (fold change greater than 4.1) were analyzed by the eTFBS program.