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 [6].

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)[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. 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 [3].

Resources

1. W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
2. R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
3. 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.
4. N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
5. S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
6. T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for Pichia pastoris. ACS Synthetic Biology. 2014, 3 str. 188-191.