Simone Lawson
The use of constitutive promoters is essential in plant transformation and genetic engineering projects. They are involved in promoting the expression of transgenes, which can be used to confer resistance to diseases, biotic stress, or insects. Constitutive promoters are also used in the generation of genetically modified plants. One of the most widely used constitutive promoters is the 35S promoter, also known as CaMV35S, derived from the Cauliflower Mosaic Virus. However, the list of strong constitutive promoters is limited, and the continuous reuse of the same promoters can lead to issues such as homology-based gene silencing and impaired transgene expression. Therefore, there is a need to develop more well-characterized constitutive promoters, such as the recently identified MuasFuasH17 (MFH17) promoter, which has shown promising results in Nicotiana benthamiana plants.
| Characteristics | Values |
|---|---|
| Promoter type | Constitutive |
| Promoter function | Promotes gene expression in all tissues and throughout various developmental stages |
| Promoter structure | Consists of a core promoter region, proximal promoter region, and distal promoter region |
| Examples | CaMV35S, MFH17, AtSCPL30 (PD1-PD9) |
| Advantages | Widely used, well-studied, and experimentally validated; useful for gene engineering and synthetic biology applications |
| Disadvantages | Can hinder plant growth due to excessive energy consumption; may cause homology-based gene silencing with repeated use |
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What You'll Learn
The importance of constitutive promoters in plant research
A plant promoter consists of three essential regions: the core promoter region, where RNA polymerase binds and initiates transcription; the proximal promoter region, where various transcription factors bind to the cis-regulatory elements (CREs) to regulate promoter activity; and the distal promoter region, located away from the core region, which also regulates promoter activity. The differences in regulatory sequences within these regions give rise to distinct types of promoters, including constitutive, spatial/temporal-specific, and inducible promoters.
Constitutive promoters are distinguished by their ability to maintain control of genes throughout most of a plant's development. They are highly expressed in various tissues and developmental stages, making them valuable for the consistent production of proteins throughout a plant's lifecycle. This property has been leveraged in biotechnological applications to generate recombinant proteins, enzymes, and other biomolecules. For example, the strong constitutive promoter M24 was used to express the rat Par-4-SAC protein in transgenic tobacco plants, resulting in significant inhibition of tumour growth in a prostate cancer model.
However, the list of strong constitutive promoters is limited, and continuous reuse of the same promoters can lead to issues such as homology-based gene silencing and impaired transgene expression. This limitation has prompted the development of novel promoters, such as the MFH17 promoter, a synthetic promoter combining elements from three pararetroviral-based promoters, which has shown high activity and dual-species expression capability.
In conclusion, constitutive promoters are indispensable in plant research, particularly in genetic engineering and biotechnology applications. Their ability to drive gene expression in various tissues and developmental stages makes them valuable tools for producing proteins, enzymes, and biomolecules. However, the limited repertoire of strong constitutive promoters and the challenges associated with their reuse underscore the importance of developing new promoters with enhanced characteristics, such as the MFH17 promoter. These advancements in promoter engineering will undoubtedly contribute to further breakthroughs in plant research and biotechnology.
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The advantages of constitutive promoters over inducible promoters
Constitutive promoters are widely used for gene engineering projects in plant synthetic biology. They are also used in the generation of genetically modified plants. This is because constitutive promoters are always active and can promote the expression of transgenes with different purposes, such as disease resistance or biotic stress resistance.
Constitutive promoters are especially useful because they maintain control of the genes during most of the plant's development. The expression levels will depend on the type of cell with which they work. The 35S promoter derived from the CaMV is the most popular constitutive promoter for genetically modified crops.
However, the list of strong constitutive plant promoters with strength surpassing the widely used promoter, the CaMV35S, is limited. The continuous use of the same set of promoters in increasingly complex genetic engineering circuits can lead to homology-based gene silencing (HBGS) causing impairment of transgene expression.
Inducible promoters, on the other hand, are activated by hormones, chemicals, environmental conditions, and biotic or abiotic stresses. They can be switched from an OFF to an ON state. In the OFF state, the promoter is inactive because a bound repressor protein actively prevents transcription. Inducible promoters are essential tools for molecular and cell biology studies in bacteria and eukaryotes, allowing for control over the expression of genes of interest in terms of both timing and strength of expression.
Constitutive promoters have a wide range of applications, from expressing selective marker genes to consistent overexpression of proteins. They are also useful because they are not influenced by transcriptional factors.
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Examples of constitutive promoters
The Cauliflower Mosaic Virus 35S (35S) promoter is one of the most widely used constitutive promoters in plants. Variants of this promoter are often used in gene engineering projects. However, the continuous use of the same set of promoters can lead to homology-based gene silencing (HBGS) and impair transgene expression. Therefore, it is crucial to develop and identify more well-characterized, strong constitutive promoters for plant synthetic biology applications.
One approach to expanding the number of available promoters is to engineer new ones by adding cis-elements to a 'minimal promoter region', often derived from 35S. For example, the synthetic promoter MUASCsV8CP, which has activity similar to the 2X35S promoter, was used to express Killer Protein 4 (KP4) in transgenic tobacco plants, increasing their tolerance to certain fungi. Another strong constitutive promoter, M24, was used to express the rat Par-4-SAC protein in transgenic tobacco plants, which significantly delayed tumour growth in a rat prostate cancer model.
Other examples of strong constitutive promoters include M12 and M24 from the Mirabilis mosaic virus (MMV), promoters from members of the ubiquitin and actin families, and promoters from the Figwort mosaic virus (FMV) and Horseradish latent virus (HRLV). By combining promoter elements from MMV, FMV, and HRLV, researchers developed a highly active tri-hybrid promoter, MuasFuasH17 (MFH17), which was found to be highly efficient in driving gene expression throughout plant tissues.
To address the limited number of strong promoters available for plant synthetic biology, researchers have developed toolkits such as PCONS, which provide a suite of constitutive promoters spanning a wide range of transcriptional levels. This allows for more nuanced and refined engineering endeavours and the optimization of synthetic transcriptional systems.
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The disadvantages of constitutive promoters
The use of constitutive promoters in plant research has some drawbacks. Firstly, there is a limited selection of well-characterised constitutive promoters available, such as the commonly used CaMV35S, and developing new promoters is crucial. This shortage poses challenges for researchers. Furthermore, reusing the same set of promoters in complex genetic engineering circuits can lead to homology-based gene silencing (HBGS) and impair transgene expression. This issue is addressed by developing more well-characterised constitutive promoters or using distinct strategies to engineer new promoters.
Another disadvantage of constitutive promoters is their non-specific expression. While they maintain control of genes during plant development, they can trigger problems at the cellular level in non-specific places within the plant. This issue is not present with inducible promoters, which offer robust and temporal expression controlled by specific stimuli. Inducible promoters are activated by factors like hormones, chemicals, environmental conditions, and biotic or abiotic stresses, and their performance is less affected by endogenous factors.
Constitutive promoters are widely used in plant transformation and genetic engineering projects. However, their constant expression can lead to issues in non-target tissues, highlighting the importance of specific promoters in certain contexts. The development of more well-characterised constitutive promoters is essential to support the increasing complexity of plant synthetic biology applications.
In summary, the disadvantages of constitutive promoters include a limited selection, the potential for homology-based gene silencing with repeated use, and non-specific expression that may cause problems in non-target tissues. These drawbacks emphasise the importance of developing new constitutive promoters and exploring alternative promoter types, such as inducible promoters, to overcome these challenges in plant research.
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The future of constitutive promoters in plant research
To address this challenge, researchers are exploring synthetic promoters by leveraging the genetic resources of plant pararetroviruses. These viruses have strong promoters, such as those found in the Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Horseradish latent virus (HRLV). By combining and engineering these viral promoter elements, scientists have developed synthetic promoters like MuasFuasH17 (MFH17), which has shown promising results in driving gene expression in both dicot and monocot plants.
The development of pipelines to identify and engineer constitutive promoters is another significant advancement. These pipelines aim to discover genes with stable expression across various plant tissues and developmental stages, expanding the repertoire of available promoters. The introduction of genome-orthogonal gRNA target sites allows for the creation of NOR logic gates, enabling the construction of more complex information processing circuits in plant synthetic biology.
Additionally, the identification of endogenous promoters in plants like Arabidopsis thaliana and their analysis through transient and stable expression assays contribute to the growing collection of constitutive promoters. The inclusion of CRISPR targets helps fine-tune transgene expression, making these promoters versatile tools for future applications.
In conclusion, the future of constitutive promoters in plant research holds promise with the development of robust synthetic promoters, the establishment of identification and engineering pipelines, and the exploration of endogenous promoters. These advancements will enable scientists to overcome the limitations of traditional promoters and unlock new possibilities in plant synthetic biology, metabolic engineering, and the creation of genetically modified plants with enhanced traits.
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Frequently asked questions
Constitutive promoters are regulatory elements that express genes in all tissues at all times. They are primarily used in plant transformation, promoting the expression of transgenes with different purposes, such as disease resistance or biotic stress.
Some examples of widely used constitutive promoters include the 35S promoter from the Cauliflower Mosaic Virus (CaMV35S), M12 and M24 from the Mirabilis Mosaic Virus (MMV), and promoters from members of the ubiquitin and actin families.
Constitutive promoters are advantageous because they are well-studied and have a wide range of applications in plant research and biotechnology. They have been instrumental in driving constitutive expression, helping elucidate plant gene functions, and increasing the understanding of plant processes. However, a disadvantage is that they may hinder plant growth due to excessive energy consumption and nutrient losses, as they drive constant gene expression regardless of necessity.
