Development of robust constitutive synthetic promoter using genetic resources of plant pararetroviruses

Abstract

With the advancement of plant synthetic biology, complex genetic engineering circuits are being developed, which require more diverse genetic regulatory elements (promoters) to operate. Constitutive promoters are widely used for such gene engineering projects, but the list of strong, constitutive plant promoters with strength surpassing the widely used promoter, the CaMV35S, is limited. In this work, we attempted to increase the constitutive promoter library by developing efficient synthetic promoters suitable for high-level gene expression. To do that, we selected three strong pararetroviral-based promoters from Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Horseradish latent virus (HRLV) and rationally designed and combined their promoter elements. We then tested the newly developed promoters in Nicotiana benthamiana and found a highly active tri-hybrid promoter, MuasFuasH17 (MFH17). We further used these promoter elements in generating random mutant promoters by DNA shuffling techniques in an attempt to change/improve the MFH17 promoter. We further evaluated the activity of the MFH17 promoter in Oryza sativa seedlings and studied the effect of as-1 elements present in it. Finally, we tested the efficacy and tissue specificity of the MFH17 promoter in planta by developing transgenic Nicotiana tabacum and Arabidopsis thaliana plants and found it highly constitutive and efficient in driving the gene throughout the plant tissues. Overall, we conclude that this tripartite synthetic promoter MFH17 is a strong, highly constitutive, and dual-species (dicot and monocot) expressing promoter, which can be a valuable addition to the constitutive plant promoter library for plant synthetic biology.

Keywords: DNA shuffling; as-1 element; figwort mosaic virus; horseradish latent virus; mirabilis mosaic virus; rational engineering.

Figure 1

Rational design of hybrid synthetic promoters. (A) The full-length transcript (Flt) promoter from Mirabilis mosaic virus (MMV-Flt), Figwort mosaic virus (FMV-Flt), and Horseradish latent virus (HRLV-Flt) was used as parent promoter in generating five promoter fragments, namely Fuas (195 bp) from FMV, Muas (259 bp) from MMV and H17uas (148 bp), H17 (250 bp) and H12 (500 bp) from HRLV, where Fuas, Muas, and H17uas were used as upstream activating sequence, and H17 and H12 was used as core promoter. (B) The five promoter fragments were used to develop ten hybrid promoters, namely H17uasH12 (654 bp), MuasH12 (765 bp), FuasH12 (701 bp), MuasFuasH12 (966 bp), FuasMuasH12 (966 bp), H17uasH17 (404 bp), MuasH17 (515 bp), FuasH17 (451 bp), MuasFuasH17 (716 bp), FuasMuasH17 (716 bp) and their transient promoter activity analysis was done in N. benthamiana along with Ctrl (VC), H12, H17, 35S, and 2X35S promoters. Their average GUS activity, respective SD, and statistical significance (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) are represented.

Figure 2

DNaseI-mediated DNA shuffling of MFH17 promoter. (A) Assay scheme. The Muas, Fuas, and H17 fragments from the MFH17 promoter were fragmented using the DNaseI enzyme and mixed. The fragments were then annealed using self-priming PCR, and PCR (with primers) was performed from the pool of annealed fragments. The PCR products were then cloned into pUC119 and sequenced. (B) Eight mutant clones were generated, viz. D1, D2, D3, D4, D5, D6, D7 and D8, out of which D1 and D2 were point mutant, D3, was both point and deletion mutants, D4, D6, D7 and D8 were deletion mutants and D5 was insertional mutant. All their promoter activity analysis were done in N. benthamiana along with MFH17 promoter and Ctrl (VC), and their average GUS activity, with respective SD and statistical significance (*p ≤ 0.05; **p ≤ 0.01), are represented.

Figure 3

Oligonucleotide-based DNA shuffling. (A) Assay scheme. Twelve pairs of 50 bp oligonucleotides from the MFH17 promoter were synthesized, containing AG as a 5’ overhang in the sense strand and TC as a 5’ overhang in the anti-sense strand. These oligos were annealed, phosphorylated, and ligated to one another. The pool of randomly ligated oligos was then cloned into the pUCH17min vector, and positive shuffled promoters were screened by sequencing. Lastly, the positive clones were sub-cloned into the pKYLXGUS vector and its promoter activity analysis was done. (B) Thirty shuffled promoters of MFH17 were screened, where only the Sh6 promoter showed moderate expression.

Figure 4

Transient promoter analysis. The promoters MFH17, 35S, and 2X35S with Ctrl (VC) were transiently expressed in rice seedlings, and its (A) histochemical staining and (B) fluorometric GUS assay were represented, where the asterisk symbol indicates the level of significance. Also, GFP was expressed under MFH17, 35S, and 2X35S promoters with Ctrl (VC) in (C) N. benthamiana leaves and photographed.

Figure 5

The as-1 elements mutation analysis in MFH17 promoter. (A) Schematic representation of positions of as-1 element pairs in MFH17 promoter with its native and mutated sequence. (B) The mutated promoter clones with single-pair mutation (M, F, and H), double-pair mutation (MF, MH, and FH), and triple-pair mutation (MFH) were represented, along with MFH17 promoter and Ctrl (VC), and their promoter activity analysis was done. The total average of MFH17 was taken as 100 units, and the averages of other promoters were calculated and represented accordingly.

Figure 6

Promoter activity analysis of MFH17, 35S, and Ctrl (VC) in Nicotiana tabacum cv. Samsun transgenic plants. (A) Histochemical staining of twenty-one-day-old seedlings, forty-five-day-old leaf, stem, root tissues, and ninety-day-old flowering tissues (ovary, style, anther, and filament). (B) Fluorometric GUS activity and (C) RT-PCR of GUS transcript level from twenty-one days old seedlings. (D) Fluorometric GUS activity analysis of MFH17 promoter in leaf, stem, and root of forty-five days plants. The asterisk symbol represents the level of significance (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Figure 7

Promoter activity analysis of MFH17, 35S, and Ctrl (VC) in Arabidopsis thaliana (ecotype Columbia) transgenic plants. (A) Histochemical staining of twenty-one-day-old seedlings, forty-five-day-old leaf, stem, root tissues, and seventy-day-old flowering tissues (inflorescence, flower, and pistil). (B) Fluorometric GUS activity and (C) RT-PCR of GUS transcript level from twenty-one days old seedlings. (D) Fluorometric GUS activity analysis of MFH17 promoter in leaf, stem, and root of forty-five days plants. The asterisk symbol represents the level of significance (*p ≤ 0.05; **p ≤ 0.01).