Part:BBa_K4799990

pAtMRP3

AtMRP3 is a gene that codes for a protein called ATP-binding cassette transporter MRP3 in Arabidopsis thaliana. This protein is involved in the transport of heavy metals, such as cadmium, within the plant. It is part of a subfamily of ABC transporters called AtMRPs, which are thought to play a role in heavy metal detoxification and sequestration in vacuoles. AtMRP3 is specifically induced in the roots of Arabidopsis plants after exposure to cadmium, and its expression levels can be used as an indicator of heavy metal stress.

Usage and Biology

pAtMRP3

Design

Our project's final goal is to engineer plants that respond to cadmium in the soil by glowing. To achieve this, we will first test whether the gene expression levels regulated by this promoter will change in the presence or absence of cadmium. Prior to testing this in a vector that contains the full set of the fungal luminescent vector, we will first test the AtMRP3 promoter in the traditional and classical GUS reporter gene device. Because the full set of the fungal luminescent vector contains 4 different genes, and the total size of the vector exceeds 20Kb, it is more difficult to insert our promoter into this system. More information on the fungal luminescence gene system can be found at the 2022 iGEM project "Life Bulb" by the team “ubc-okanagan” at https://2022.igem.wiki/ubc-okanagan. So, we used transient infiltration of Nicotiana benthamina leaves and GUS staining to test whether this promoter can achieve the expected function.

If feasible, we will then integrate it into the luminescent reporter gene. We chose the AtMRP3 promoter based on literature research, please see the listed reference below.

Build

We will construct an Agrobacterium expression vector for pAtMRP3-GUS-NOS to test whether this promoter will respond to cadmium stimulation.

Figure 1. The simulation assembly of pAtMRP3 into a GUS expression vector.

• First, we search the gene information for AtMRP3 gene of Arabidopsis thaliana from GenBank database (AT3G13080) and designed primers to amplify the promoter region approximately 2 Kb upstream of the coding sequence. During the amplification of the promoter, we incorporated the homologous recombination arm sequences of the GUS vector, resulting in a promoter sequence with homologous arms.
• After performing homologous recombination, we cloned the construct into Escherichia coli, followed by single clone screening, bacterial liquid PCR, and sequencing verification.
• Once confirmed, we extracted the plasmid and transferred it into Agrobacterium competent cell (GV3101). Finally, we sprayed tobacco leaves with water and a solution containing 50 uM CdCl2, and then transiently infected the tobacco leaves with Agrobacterium carrying the pAtMRP3-GUS-NOS expression cassette. The response of the AtMRP3 promoter to Cd treatment was evaluated by performing GUS staining.

Test

Figure 2. Amplified AtMRP3 promoter about 1.6 Kb in length. Two replicate amplification at lane 11 and 12.

Figure 3. Sequencing verified pAtMRP3-GUS-NOS vector were transformed into GV3101 Agrobacterium Competent Cells.

Figure 4. PCR verification of the positive transformation for Agrobacterium monoclonal bacteria using GUS-seq-F and pAtMRP3-seq-R primer pairs (primer locations could be spot on Figure 1). Lane 1, marker; lanes 2，9，10，11，13 represent positive transformation.

Figure 5. GUS staining of tobacco leaves after transient infiltration.

Figure 6. GUS staining results of pAtMRP3-GUS-NOS. The Phosphate Buffer Solution (PBS) was used as null control. The unedited super promoter (mannopine synthase promoter) was used as positive control. AtMRP3-1 and AtMRP3-2 are two technical replicates. CdCl2 in the picture represents CdCl2 only spray on the tobacco leaves one day before transient infiltration. CdCl++ indicate both spray CdCl2 solution to the leaves like the second column and watering CdCl2 solution in to the soil three days before the infiltration.

In summary, pAtMRP3 will induce GUS gene expression with or without cadmium treatment, but if the tobacco was treated with a higher dose of cadmium, the expression level reduced compared to the pure water treatment.

Learn

Based on the results of this round of experiments, we have drawn the following conclusions and inspirations, which provide evidence for optimizing and improving subsequent experiments.

1. Based on the results, the GUS expression level decreased even further with cadmium treatment, and the discrimination ability of GUS was not high. Therefore, we plan to use an additional GFP expression vector reporter system to repeat the verification process.
2. Additionally, when the plants were sprayed with water, the promoter was highly expressed in the leaves. However, the expression level was reduced with cadmium treatment. To exclude the possibility that the high concentration of 50 μM cadmium used had damaged the plant cells, affecting the expression, we will consider increasing the concentration gradient in subsequent experiments. Furthermore, if Cd treatment really does reduce gene expression in the leaves, we can add a negative regulatory element or modify our luminescent plant reporting system, which normally emits light continuously but loses the ability of luminescence in the presence of heavy metal pollution.
3. Based on the experimental results for AtMRP3, we plan to continue searching for other available promoter sequences. This round of experiments builds up an effective testing system for finding heavy metal responding promoter.
4. To better understand the above results, we did more literature search and found literature suggesting that AtMRP3 gene can respond to cadmium only in the roots but is continuously expressed in the leaves. Therefore, it may be possible to use this promoter sequence in plants and use it to monitor heavy metal pollution in liquid culture systems or transparent gels.

References

• Zientara K, Wawrzyńska A, Łukomska J, López-Moya JR, Liszewska F, Assunção AGL et al. Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron. Journal of Biotechnology. 2009;139(3):258-263. https://doi.org/10.1016/j.jbiotec.2008.12.001
• BOVET, L., EGGMANN, T., MEYLAN-BETTEX, M., POLIER, J., KAMMER, P., MARIN, E., FELLER, U. and MARTINOIA, E. (2003), Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3. Plant, Cell & Environment, 26: 371-381. https://doi.org/10.1046/j.1365-3040.2003.00968.x

Sequence and Features

Assembly Compatibility:

• 10
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  Illegal SpeI site found at 915
  
• 12
  INCOMPATIBLE WITH RFC[12]

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• 21
  COMPATIBLE WITH RFC[21]
• 23
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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 915
  
• 1000
  COMPATIBLE WITH RFC[1000]