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Schemes of constructs carrying BIP1 and different deleted versions fused with GFP and GUS reporter genes.
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Bidirectional promoters, which show great application potential in genetic improvement of plants, have aroused great research interest recently. However, most bidirectional promoters were cloned individually in the studies of single genes. Here, we initiatively combined RNA-seq data and cDNA microarray data to discover the potential bidirectional p...
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... Strategies have been developed to improve terpenoid production by re-targeting pathways between the cytosol and plastids [15,17,31,36,51], creating synthetic compartments [17,31], and targeting pathways to specific tissues [52][53][54][55][56][57][58]. Plants naturally compartmentalize terpenoid biosynthesis within cells as well as across different tissue types, plausibly to insulate specific routes from the competition with general metabolism, to utilize dedicated pools of precursor building blocks, and for their specialized roles. ...
... For example, an elegant system has been developed where a synthetic activator gene is placed under the control of an endosperm-specific promoter, which when expressed activates the expression of multiple downstream genes under the control of synthetic promoters responsive to the synthetic activator [100]. A simpler approach to the expression of multiple genes is the use of bidirectional promoters, which have been isolated from native genomic sequences [56,58,114,115] or created synthetically through fusing unidirectional promoters, like the CaMV 35S tailto-tail, i.e., CAT-3′-5′::5′-3′-AUG [52,116,117]. Bidirectional promoters have been well studied in microbial engineering [118,119] but have yet to be used more broadly for plant engineering. ...
Terpenoids represent the most diverse class of natural products, with a broad spectrum of industrial relevance including applications in green solvents, flavors and fragrances, nutraceuticals, colorants, and therapeutics. They are typically challenging to extract from their natural sources, where they occur in small amounts and mixtures of related but unwanted byproducts. Formal chemical synthesis, where established, is reliant on petrochemistry. Hence, there is great interest in developing sustainable solutions to assemble biosynthetic pathways in engineered host organisms. Metabolic engineering for chemical production has largely focused on microbial hosts, yet plants offer a sustainable production platform. In addition to containing the precursor pathways that generate the terpenoid building blocks as well as the cell structures and compartments required, or tractable localization for the enzymes involved, plants may provide a low input system to produce these chemicals using carbon dioxide and sunlight only. There have been significant recent advancements in the discovery of pathways to terpenoids of interest as well as strategies to boost yields in host plants. While part of the phytochemical field is focusing on the discovery of biosynthetic pathways, this review will focus on advancements using the pathway toolbox and toward engineering plants for the production of terpenoids. We will highlight strategies currently used to produce target products, optimization of known pathways to improve yields, compartmentalization of pathways within cells, and genetic tools developed to facilitate complex engineering of biosynthetic pathways. These advancements in Synthetic Biology are bringing engineered plant systems closer to commercially relevant hosts for the bioproduction of terpenoids.
... Bidirectional promoters (BiP) are more pertinent than unidirectional promoters, as they can control the expression of two genes simultaneously, thereby saving time from expression vector construction and piling multiple genes [5]. Moreover, the limited availability of unidirectional promoters with the same expression pattern has also encouraged the application of bidirectional promoters [92]. Bidirectional promoters have been designed in many plant species such as Arabidopsis [93], rice [94], melon [95], and Capsicum annum [96], all of which have been successfully reported. ...
Native/endogenous promoters have several fundamental limitations in terms of their size, Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in their insufficient transcriptional activity. Several customized synthetic promoters were designed and tested in plants during the past decade to circumvent such constraints. Such synthetic promoters have a built-in capacity to drive the expression of the foreign genes at their maximum amplitude in plant orthologous systems. The basic structure and function of the promoter has been discussed in this review, with emphasis on the role of the Cis-element in regulating gene expression. In addition to this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. This review also provides explicit information on the two major approaches for developing plant-based synthetic promoters: the conventional approach (by utilizing the basic knowledge of promoter structure and Cis-trans interaction) and the advancement in gene editing technology. The success of plant genetic manipulation relies on the promoter efficiency and the expression level of the transgene. Therefore, advancements in the field of synthetic promoters has enormous potential in genetic engineering-mediated crop improvement.
... Bidirectional promoters (BiP) are more pertinent than unidirectional promoters, as they can control the expression of two genes simultaneously, thereby saving time from expression vector construction and piling multiple genes [5]. Moreover, the limited availability of unidirectional promoters with the same expression pattern has also encouraged the application of bidirectional promoters [92]. Bidirectional promoters have been designed in many plant species such as Arabidopsis [93], rice [94], melon [95], and Capsicum annum [96], all of which have been successfully reported. ...
Native/endogenous promoters have several fundamental limitations in terms of their size,Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in theirinsufficient transcriptional activity. Several customized synthetic promoters were designed andtested in plants during the past decade to circumvent such constraints. Such synthetic promotershave a built-in capacity to drive the expression of the foreign genes at their maximum amplitude inplant orthologous systems. The basic structure and function of the promoter has been discussed inthis review, with emphasis on the role of the Cis-element in regulating gene expression. In additionto this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. Thisreview also provides explicit information on the two major approaches for developing plant-basedsynthetic promoters: the conventional approach (by utilizing the basic knowledge of promoterstructure and Cis-trans interaction) and the advancement in gene editing technology. The success ofplant genetic manipulation relies on the promoter efficiency and the expression level of thetransgene. Therefore, advancements in the field of synthetic promoters has enormous potential ingenetic engineering-mediated crop improvement
... Rice is one of the most important food crops in the world [26,27]. The application of strong specific promoters is very important for rice genetic improvement [28]. In this study, a green tissue-specific promoter was obtained through the screening of the T-DNA capture line. ...
Plant promoters play a vital role in the initiation and regulation of gene transcription. In this study, a rice protein/gene of unknown expression, named Os8GSX7, was gained from a rice T-DNA capture line. The semi-quantitative RT-PCR analysis showed that the gene was only expressed in root, glume, and flower, but not in stem, leaf, embryo, and endosperm of japonica rice. The GUS activity analysis of the GSX7R promoter showed that it was a reverse green tissue expression promoter, except in endosperm. The forward promoter of GSX7 cannot normally drive the expression of the foreign GUS gene, while the reverse promoter of GSX7 is a green tissue-specific expression promoter, which can drive the expression of the foreign GUS gene. The region from −2097 to −1543 bp was the key region for controlling the green tissue-specific expression. The regulatory sequences with different lengths from the 2097 bp reverse sequence from the upstream region of the Os8GSX7 were fused with the GUS reporter gene and stably expressed in rice. Furthermore, transgenic rice plants carrying Cry1Ab encoding Bacillus thuringiensis endotoxin, regulated by GSX7R, were resistant to yellow stem borer. The analysis suggested that 10 light responsive elements of tissue-specific expression were found, including ACE, Box4, CAT-box, G-Box, G-box, GATA motif, GC motif, I-box, Sp1, and chs-unit1 M1. In addition, the results of 5′ and 3′ deletions further speculated that ACE and I-box may be the key elements for determining the green tissue-specific expression of GSX7R promoter.
... In monocot species, certain constitutive promoters of plant origin, such as rice actin promoter and maize ubiquitin promoter (Beringer et al., 2017), have been isolated and are often used for transformation of grasses (Wang et al., 2016). In dicot plants, although a number of endogenous constitutive promoters have been isolated, they are not widely used or tested in other species, particularly in legumes. ...
Transcriptional regulation is a dynamic process and the first level of gene expression control, largely governed by
the gene promoters and their contributing cis-regulatory elements (CREs), the binding of which to the regulatory
proteins and transcription factors (TFs) lead to the activation or repression of the genes. Strong constitutive
promoters that drive higher expression levels have become a valuable tool in the genetic engineering of plants.
Recently, the search for strong plant-derived constitutive promoters has expanded to several monocot and dicot
species. In this study, we isolated a novel promoter of ~850 bp located in the upstream of a house keeping Cucumis
sativus polyubiquitin gene (Ub) with an open reading frame (ORF) of 1374 bp. The conserved domain results of the
Ub gene revealed the presence of a typical Ub like superfamily conserved domain and a k27 lysine residue involved
in the chain linkage of Ub genes. The bioinformatics analysis of the cis-regulatory elements (CREs) using plant
CARE and PLACE showed 22 functional classes in the Ub gene promoter, many of which are involved in the binding
of transcription factors (TFs) for the expression during abiotic and biotic stresses. Therefore, it would be an ideal
choice of plant-derived promoter for driving higher levels of transgene expression in dicots especially in cucurbits
which needs further validation by gene expression and functional characterization studies.
... Bidirectional promoters are well studied in prokaryotes 3 , humans 4,39,40 , plants 41 , yeasts 5,11 and insects 7 . However, there are only two reports on identification of bidirectional promoters from filamentous fungi. ...
... 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. ...
Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5′ ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.
... To create the mini 35s-enhancer-Cas9-Csy4 system (pGEL051), the Arabidopsis enhancer (Additional Data: Figure S4) Zhu et al., 2015) was amplified from Arabidopsis Col-0, cloned into XbaI and PvuII-linearized pGEL050 plasmids by T4 ligase. To generate the OsBiP1-Cas9-tRNA system (pGEL052) and OsBiP1-Cas9-Csy4 system (pGEL053), the OsBiP1 promoter fragment (Additional Data: Figure S5) (Wang et al., 2016) was amplified from Oryza sativa Geng/Japonica cultivar P r o v i s i o n a l 5 Nipponbare and cloned into pGEL038 and pGEL039 by Gibson assembly, respectively. For creating nuclease expression vector, sgRNAs were synthesized as duplexed oligonucleotides (Table S1). ...
... While we recognized that stronger BiP systems could be engineered by using stronger enhancers or multiple copies of enhancers, we reasoned that utilization of a plant endogenous BiP system may represent a straightforward strategy for improvement. Based on RNAseq and cDNA microarray data analysis, a recent study identified a constitutive bidirectional promoter of high expression in rice, OsBiP1, which drives expression of Os02g42314 at the 5' end and Os02g42320 at the 3' end (Wang et al., 2016). We decided to test OsBiP1 in our study and compared its expression strength by positioning a GFP reporter gene at the 3' end of this BiP. ...
... First, rational P r o v i s i o n a l design-based approach could be used to engineer BiP systems with improved expression strength and stability in plants, as was done in E. coli and yeast (Elison et al., 2018;Yang et al., 2013). Second, aided by genomic and transcriptome data sets, many endogenous BiP systems could be identified and tested, as was done in rice (Wang et al., 2016). Third, different 3'-UTR and terminator sequences could be tested for tuning the expression of Cas9 and guide RNAs as well as tissue specificity (Lianoglou et al., 2013). ...
CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9–93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.
... As the development of crop genetic improvement and functional genomics research, crop traits such as stress resistance, nutrient utilization, yield and quality are attracting more and more attention. Simultaneously, several lines of candidate genes for crop trait improvement were emerged, and their effective application depends on their efficient expression at specific sites, growth stages or signal stimulation (Wang et al. 2016). ...
Bidirectional green‐tissue specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green‐tissue specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequences selection and the bidirectional green‐tissue specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activity in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiency specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters and we successfully created four bidirectional green‐tissue specific synthetic promoters. It is the first report on bidirectional green‐tissue specific promoters that could be efficiently applied in genetic engineering. This article is protected by copyright. All rights reserved.
... These promoters control of the expression of gene pairs usually involved in the same or in related physiological processes (Mitra et al., 2009;Singh et al., 2009;Oropeza-Aburto et al., 2017). Most of the known bidirectional promoters have been found during the research of single genes, but the development of sequencing technologies and bioinformatics tools discovered that bidirectional nuclear gene promoters are very abundant in eukaryotes, including plants (Wang et al., 2009(Wang et al., , 2016Dhadi et al., 2013). In a first search of bidirectional promoters in the Arabidopsis genome, Wang et al. (2009) identified near 2,500 gene shared intergenic regions, enriched in regulatory elements essential for transcription. ...
... 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 plant defense responses to pests results in the synchronized change of a complex network of interconnected genes and signaling pathways. An essential part of this process is mediated by the binding of transcription factors to the specific responsive cis-elements within in the promoters of phytophagous-responsive genes. In this work, it is reported the identification and characterization of a bidirectional promoter that simultaneously co-regulate two divergent genes, At5g10300 and At5g10290, upon arthropod feeding. Computational analysis identified the presence of cis-elements within the intergenic region between two loci, mainly from the DOF but also from the AP2/ERF, Golden 2-like and bHLH families. The function of the bidirectional promoter was analyzed using two enhanced variants of the GFP and CherryFP reporter genes, in both orientations, in transient tobacco and stably transformed Arabidopsis plants. Promoter activity was tested in response to feeding of Tetranychus urticae and Pieris brassicae, as well as wounding, flagellin and chitin treatments. Using RT-qPCR assays and confocal microscopy, it was shown that all treatments resulted in the induction of both reporter genes. Furthermore, our findings revealed the asymmetric character of the promoter with stronger activity in the forward than in the reverse orientation. This study provides an example of a bidirectional promoter with a strong potential to be used in plant biotechnology in pest control that requires stacking of the defense genes.
... Cette co-régulation permet également de ne faire intervenir qu'un seul facteur de transcription pour plusieurs gènes, comme il a été démontré dans le cas de la synthèse du thalianol et du marneral (Field et al., 2011;Hurst et al., 2004). A ce jour, plusieurs promoteurs bidirectionnels ont été décrits chez différentes plantes telles que Arabidopsis thaliana ( Wang et al., 2009b), le riz ( Wang et al., 2016) ou le melon ( Wang et al., 2008). ...
Les plantes sont soumises durant leur vie à de nombreux stress environnementaux. Face à ces contraintes, les végétaux ont développé au cours de l'évolution différentes stratégies. La plus emblématique est la mise en place du métabolisme spécialisé, représenté par une grande diversité chimique et fonctionnelle. Bien que ce métabolisme soit de plus en plus étudié ces dernières années, de nombreuses lacunes persistes à son propos, liées notamment (i) à la complexité des modifications métabolomiques engendrées par la perception de stress, (ii) aux coûts et avantages que ces métabolites imputent à la plante les accumulant, et (iii) aux voies métaboliques menant à cette diversité de composés. Pour appréhender ces différentes problématiques, nous avons adopté une stratégie combinant des approches de phytochimie, de biologie moléculaire et de génétique. Dans un premier temps, nous avons étudié les changements métaboliques globaux engendrés par l’application de deux stress environnementaux, l’ozone et la blessure mécanique, sur une plante modèle au laboratoire, le panais, en fonction du temps. Les résultats de ces travaux nous ont permis d’identifier 40 métabolites différentiellement accumulés dans ces conditions, dont certaines furocoumarines. Par la suite, nous avons focalisé notre étude sur ces molécules en évaluant leurs profils d’accumulation, en condition de stress par blessures mécaniques, par la biais d’analyses différentielles. A partir de ces données, nous avons initié la recherche et l'identification de gènes candidats potentiellement impliqués dans cette voie à partir de plusieurs banques transcriptomiques et génomiques de panais. La fonction des gènes sélectionnés a été évalué par des approches d'expression hétérologue dans la levure. En parallèle de ces travaux, nous avons développé une stratégie destinée à mieux comprendre le coût métabolique de la synthèse de métabolites spécialisés. Pour ce faire, nous avons adapté aux furocoumarines une technique de clonage multigénique permettant de transférer dans une plante, et en une seule opération, plusieurs gènes impliqués dans la même voie de biosynthèse. Cette méthode nous a permis d'initier la génération de lignées stables ayant intégré les deux premiers gènes de la voie. Ces plantes seront comparées à des plantes sauvages et permettront ainsi d’étudier les coûts métaboliques et physiologiques de l’introduction de cette nouvelle voie de biosynthèse ainsi que ses bénéfices en termes de défense de la plante.
