Structural and Functional Analysis of a Bidirectional Promoter from Gossypium hirsutum in Arabidopsis

Abstract

Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.

Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense

“…For characterization, the Pxy promoter sequence was cloned in pCSN44 vector in between two reporter genes (β-glucuronidase and EGFP) and the bidirectionality of the intergenic sequence was tested by assaying the gene products. This strategy was also adopted in plants such as rice and Arabidopsis, wherein two reporter genes (GUS and GFP) were cloned across the intergenic region 15,41 . In the current study, Pxy promoter sequence was inducible as there was no expression noticed in the zero hour time point irrespective of the media in which the transformant is grown.…”

“…A 1258 bp intergenic region between At4g35985 and At4g35987 genes of Arabidopsis contains a number of environmental stress-responsive and tissue-specific cis-regulatory elements which showed differential regulation of two genes under study 14 . A 1429 bp intergenic region between the genes Ghrack1 and Ghuhrf1 in Gossypium hirsutum was cloned and used for expressing reporter genes in Arabidopsis 15 . A BDP was built by arranging in tandem three copies of a modified enhancer from the promoter region of the Arabidopsis PLDZ2 gene, which strongly responds to phosphate starvation.…”

Plant Defenses Against Pests Driven by a Bidirectional Promoter

“…In this context and as many authors previously did, two reporter genes were fused to the promoter in both orientations, for a deep analysis of its function in planta . In previous studies, GUS and GFP genes were the reporters most commonly used but the GUS gene requires an enzymatic reaction to generate the reporter product (Mitra et al, 2009; Wang et al, 2016; Oropeza-Aburto et al, 2017; Yang et al, 2018). This step implies not only a cost and time-consuming but also a time lag between the two reporter quantification assays.…”

“…The deletion of the cis -acting elements contained in this promoter evidenced that the transcription polarity and differential strength were dependent on the presence of specific cis -motifs. Recently, the functional analysis of a bidirectional promoter (1,429 bp) shared by a gene pair from Gossypium hirsutum , Ghrack1 (receptor for activated C kinase1), and Ghhrf1 (E3 ubiquitin-protein ligase), has also showed an orientation dependence (Yang et al, 2018). Using series of expression vectors containing GUS and GFP reporter genes in forward and reverse orientations of the promoter to transform Arabidopsis and tobacco, they found differences on spatial and temporal gene expression controlled by the promoter orientation.…”

A Small Natural Light-induced Bidirectional Promoter of Rapeseed (Brassica Napus)

“…Here we report one of the shortest natural bidirectional and light induced promoters for Brassica napus, to our knowledge. Therefore, the promoter has broad application prospects in plant genetic improvement and genetic engineering [22,23].…”