Part:BBa_K5160115
pTRV2-35S promoter - mCherry - NOS terminator
Overview
To shorten the time required for plant expression validation experiments, we employed the TRV virus for transient infection. By constructing the appropriate vector on the virus, we successfully integrated the target gene into the tomato genome. Simultaneously, we used the mCherry red fluorescent protein in the TRV virus-mediated transient expression for the following purposes:
1. Visual Marker: mCherry emits red fluorescence, allowing for intuitive observation of the virus infection process and its spread during experiments. Using equipment like fluorescence microscopes, researchers can trace the virus's transmission pathways in plant tissues, helping to identify the sites and rates of infection.
2. Monitoring Virus Vector Effectiveness: When mCherry is combined with the TRV virus vector, the fluorescence signal from mCherry can be detected to determine whether the virus vector has successfully infected plant cells. If mCherry fluorescence is observed in plant tissues, it indicates that the virus vector has entered the cells and begun expressing the fluorescent protein, thereby indirectly confirming the vector's effectiveness.
3. Assisting Gene Silencing Studies: In some gene silencing experiments based on the TRV virus, mCherry can be constructed alongside the target gene in the virus vector. By observing changes in mCherry fluorescence intensity, researchers can preliminarily assess whether the target gene has been successfully silenced. If the target gene is silenced, it may affect associated physiological processes, leading to changes in mCherry expression levels.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1328
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 66
Illegal BsaI site found at 1052
Illegal BsaI.rc site found at 1331
CaMV 35S Promoter
The CaMV 35S promoter refers to the 35S promoter from the Cauliflower Mosaic Virus (CaMV). This promoter directs the synthesis of 35S RNA during the infection of plants by CaMV and allows for efficient expression in various tissues of many dicotyledonous plants. As a constitutive promoter, the CaMV 35S promoter can initiate gene expression in all tissues. It offers a persistent and relatively constant level of RNA and protein expression but lacks spatiotemporal specificity. For detailed information on this part, see BBa_K788000.
mCherry
mCherry is a red fluorescent protein derived from mushroom coral, commonly used to label and trace certain molecules and cellular components. Compared to other fluorescent proteins, the advantage of mCherry is its ability to co-label with the most commonly used green fluorescent protein (GFP), along with its superior photostability compared to other monomeric fluorescent proteins. For detailed information on this part, see BBa_K5160005.
NOS Terminator
The nopaline synthase (NOS) terminator plays a crucial role in genetic engineering, especially in plant expression vectors. It ensures that the transcription of the target gene terminates at specific locations within the plant, thereby preventing unnecessary extension of gene expression and ensuring the accuracy and efficiency of gene expression. Thus, it is commonly used to terminate the transcription of the target gene in plant vectors, enhancing expression efficiency. For detailed information on this part, see BBa_P10401.
Source
35S Promoter
The CaMV 35S promoter is derived from the cauliflower mosaic virus (CaMV) and is used to initiate the expression of the target gene.
mCherry
mCherry is a red fluorescent protein originating from mushroom coral, commonly used for labeling and tracing specific molecules and cellular components.
NOS Terminator
The nopaline synthase terminator (NOS terminator) primarily originates from Agrobacterium tumefaciens. In genetic engineering, particularly plant genetic engineering, the NOS terminator is often used as a crucial element in plant expression vectors to mark the end position of target gene transcription.
Design consideration
During the design process, to avoid the influence of the virus itself on the experiment, we set up a control group by constructing the 35S promoter_mCherry_NOS terminator on the TRV2 virus.
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