pGreenII 0800-LUC

Enhancers located in introns are highly abundant and play an important role in the regulation of gene expression in mammalian species.In contrast,the functions of plant intronic enhancers are largely unexplored,and only a few plant intronic enhancers have been reported.Here,the researchers performed genome-wide predictions of intronic enhancers in Arabidopsis using DNase I sequencing-based open chromatin signatures.941 and 1,271 candidate intronic enhancers were identified with potential functions in seedling and floral tissues,respectively.Transgenic lines containing homozygous deletions of predicted intronic enhancers associated with two different genes were created using CRISPR/Cas-based genome editing.Using these lines,the researchers demonstrate that deletion of candidate enhancers results in varying levels of transcriptional repression of homologous genes at specific developmental stages.

In this study,the pGreenII 0800-LUC vector purchased from Creative Biogene was involved in the Transient expression assay.Luciferase reporter vectors with three copies of the predicted SPL9 binding motif ATATGTACTTTA or the EDT1 binding motif CATTAAATTA were made using pGreenII 0800-LUC(Figure 1C).The researchers examined the ability of SPL9 or EDT1 to activate reporter gene expression in Nicotiana benthamiana leaves.Transient expression assays showed that the ATTTGTACTTTA sequence was strongly activated by SPL9(Fig.1D),whereas the CATTAAATTA sequence was activated by EDT1 at a modest but significant level(Fig.1E).Collectively,these results indicate that the predicted SPL9-and EDT1-binding motifs identified in the DHS7#1 and DHS7#2 enhancers are functional.

Figure 1.Functional assays of the SPL-and EDT1-binding motifs identified in the DHS7#1 and DHS7#2 enhancers.(Meng F,et al.,2021)