Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

Povzeto po članku: Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.

Introduction

Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8].

Identification of candidate transcription factor binding sites (TFBSs)

All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

Construction of plasmids

Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be composed of any sequence). After ligation, no scar will exist between adjacent fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS terminator [3].

Growth of plant material, protoplast preparation and transfection

The following plant species - Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana and Hordeum vulgare were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

Results

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

Resources

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