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.

Keywords: Gossypium hirsutum; bidirectional promoter; cloning; stable expression; transient expression.

Figure 1

Schematic representation of the organization of the Ghrack1 and Ghuhrf1 genes on chromosome D09 of Gossypium hirsutum. The 1429 bp of the intergenic region and the core 1073 bp promoter are marked. In total, 226 bp and 130 bp 5′-UTRs were upstream of Ghuhrf1 and Ghrack1, respectively.

Figure 2

Semiquantitative PCR and qRT-PCR (quantitative real-time PCR) data of the transcript levels of the Ghrack1 and Ghuhrf1 genes in different tissues of Gossypium hirsutum. (a) Semiquantitative PCR survey of various cotton tissues for the detection of Ghrack1 and Ghuhrf1 transcripts. Ghsad1 was used as a control. (b,c) Relative transcript abundance of two Gossypium hirsutum genes, that is, Ghrack1 (a) and Ghuhrf1 (c), detected in various cotton tissues by qRT-PCR. The data represent the relative expression of the Ghrack1 and Ghuhrf1 transcripts ± SD of three biological replicates in each tissue (n = 3). Asterisks and double asterisks indicate significant deviations from the root at p < 0.05 and p < 0.01, respectively, using the Student’s t-test for comparisons between the root and other tissues separately for both genes. R (root), L (leaf), A (anther), S (stigma), 0 dpa (0 dpa fiber), 5 dpa (5 dpa fiber), 7 dpa (7 dpa fiber), 14 dpa (14 dpa fiber), 21 dpa (21 dpa fiber), 26 dpa (26 dpa fiber), and 28 dpa (28 dpa fiber).

Figure 3

Sequence analysis of the cloned promoter GhZU. The putative cis-acting elements in both orientations of the promoter GhZU determined by the softwares PLACE and PlantCARE.

Figure 4

Transient expression of the gus gene and gfp gene in tobacco leaves, using the epidermis infection method. (a) Schematic diagram of the promoter-reporter gene constructs GFP::GUS, 35S::GUS, GhZUf::GUS, and GFP::GhZU::GUS, used for the transient assay in the N. benthamiana leaf using the pCambia1305 vector. (b) Schematic diagram of the promoter-reporter gene constructs GFP::GUS, 35S::GFP, GhZUr::GFP, and GFP::GhZU::GUS, used for the transient assay in the N. benthamiana leaf using the pCambia1305 vector. Below each construct, a representative assay of transient gus expression detected histochemically, transient gfp expression detected based on fluorescence imaging via the Agrobacterium infiltration assay in N. benthamiana leaf, and the respective promoter, no promoter, 35S promoter, Ghuhrf1 promoter (GhZUf), and Ghrack1 promoter (GhZUr) activities are shown.

Figure 5

Detection of the copy number of transgenic Arabidopsis thaliana using the droplet digital PCR method. (a) Internal reference gene; (b) gus gene; (c) thirty selected single-copy gus gene individuals. The gray signal in the map represents the micro-droplets that had not been amplified by PCR, and the system was considered a negative signal; the blue signal shows the FAM fluorescence signal, and the green signal shows the HEX fluorescence signal representing the PCR amplification. The system was considered a positive signal.

Figure 6

Localization of GUS and GFP in vegetative and reproductive tissues of GFP::GUS, 35S::GUS, and GFP::GhZU::GUS transgenic Arabidopsis plants. (a–h) Histochemical GUS localization data of GFP::GUS (up), 35S::GUS (middle), and GFP::GhZU::GUS (down) in vegetative and reproductive tissues of Arabidopsis plants. (a) Root region; (b) two-week-old Arabidopsis seedlings; (c) young leaf; (d) old leaf; (e) developing silique; (f) mature silique; (g) inflorescence; and (h) flower. Histochemical GUS images of GFP::GUS (up), 35S::GUS (middle), and GFP::GhZU::GUS (down) are shown. (i–m) Confocal laser-scanning microscopic analysis of gfp expression under GFP::GUS and GFP::GhZU::GUS in Arabidopsis plants. (i) Root, (j) young leaf trichomes, (k) old leaf trichomes, (l) trichomes, and (m) flower. Green fluorescence images of GFP::GUS (up) and GFP::GhZU::GUS (down) are shown.

Figure 7

GUS and GFP expression in various tissues of transgenic Arabidopsis plants generated for the constructs GFP::GhZU::GUS. (a,b) Relative expression (transcript) of two reporter genes, that is, gus (a) and gfp (b), detected in various Arabidopsis tissues by qRT-PCR. The data represent the relative expression of the gus and gfp transcripts ± SD of three biological replicates of each tissue (n = 3). Asterisks and double asterisks indicate significant deviations from the root at p < 0.05 and p < 0.01, respectively, using Student’s t-test for comparisons between the root and other tissues separately for both genes. (c,d) Relative expression (protein) of two reporter genes, that is, gus (c) and gfp (d), detected in various Arabidopsis tissues by protein hybridization. R (root), YL (young leaf), OL (old leaf), DS (developing silique), MS (mature silique), and F (inflorescence).