Part:BBa_K4799502
pNtNRAMP2
NtNRAMP2 is a gene found in tobacco plants that encodes a protein involved in the transport of metal ions, including iron (Fe) and cadmium (Cd), from vacuoles to the cytoplasm.
NtNRAMP2 is an unexpected gain of this research. Since the research on NtNRAMP2 is not complete, the relevant introductions about this gene are replaced by a broader introduction to the NRAMP gene family. NRAMP (Natural Resistance-Associated Macrophage Protein) family members have been shown to be involved in the uptake and transport of metal ions in various plant species, from lower to higher plants. NRAMP proteins have 12 transmembrane domains and both N- and C- termini located intracellularly. NtNRAMP2 and NtNRAMP3 belong to the NRAMP gene family, and NtNRAMP3 has been experimentally proven to be responsible for the influx of Fe(2+), Mn(2+), Co(2+), Cu(2+), Ni(2+) and Cd(2+). Besides the response to metal ions，NtNRAMP3 also plays a general role in the regulation of the balance of several metals in plants grown under control conditions. As for NtNRAMP3 Promoter, it has two cis-regulatory elements which may involve in plant’s metal stress responses, MRE1 and IDE2([1]). Similar functions have been found for NRAMP2 in other plants. The MsNRAMP2 gene of Alfalfa (Medicago sativa L.) responds to excess iron ions, and MsNRAMP2 responds in different ways in different plant parts. MsNRAMP2 Promoter has core elements, cis-acting elements of hormone response and multiple MYB- binding sites, which has an important role in the regulation of iron homeostasis([2]). As for potato, StNRAMP2 improves the accumulation of Cd(2+) in potato roots, stems, and leaves, but inhibits the accumulation of Cd in tuber([3]). Plus, TcNRAMP2, which belongs to Theobroma cacao, phylogenetically similar to NtNRAMP2 (identified as TheccNRAMP2 in the figure 1), is also involved in transport of Cd(2+.)([4]). Since NtNRAMP3 belonging to the same gene family from the same plant and the same NRAMP2 (such as StNRAMP2 and TcNRAMP2) belonging to different plants both respond to Cd(2+), we are confident to suspect that NtNRAMP2 plays the similar or same role in the physiological mechanism of tobacco against metal ions.
Design considerations
In our biotechnology experimental design, we plan to initially attempt connecting our promoter pNtNRAMP2 separately with two different functional genes, GUS and GFP on two different carriers, to test whether our promoter can initiate the expression of these genes when the plant is exposed to a cadmium environment. We used transient infiltration of Nicotiana benthamina leaves to minimize the duration required for obtaining performance of pNtNRAMP2. If feasible, we will then integrate it into the luminescent reporter gene. We chose pNtNRAMP2 based on literature research, please see the listed reference below.
Test:
pNtNRAMP2-GFP can be constructed successfully and pNtNRAMP2-GUS can respond to cadmium 1.pNtNRAMP2-GFP can be constructed successfully Our promoter pNtNRAMP2 is designed to respond to cadmium stress. In order to locate the complete promoter sequence from the pNtNRAMP2 sequence, we amplified the first two thousand base pairs of the gene. We selected this specific range, as the promoter(s) of most genes would exist in the first two thousand base pair region.
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The PCR production of pNtNRAMP2-GFP is observed in the last lane on the right side, adjacent to the marker.
After locating the promoter, we construct sticky ends for the promoter to pair up with new vectors. This is accomplished by adding two different pairs homology arms to the 5’ end of the promoter sequence. One pair comes from the vector GFP (green fluorescent protein), and the other one comes from the vector GUS (beta-glucuronidase). Finally , we successfully constructed pNtNRAMP2-GFP.
P2P is a Petri dish coated with NtRAMP2-GFP recombinant Escherichia coli.
The electrophoresis results of homologous recombination of promoter pNtNRAMP2-GFP are marked at top of the figure above, we choose three tubes of those bacterial liquid with positive band for DNA sequencing, and the results show that the bacterial liquid sent for testing is homologous recombination.
2.pNtNRAMP2-GUS can respond to cadmium We used transient infiltration of Nicotiana benthaminaleaves and GUS staining to test whether this promoter can achieve the expected function.
| Name | |
| Groups | pNtNRAMP2-GUS |
| Water |  |
| CdCl2（50ng/μL） |  |
| No-treatment Control |  > |
| positive control |  |
Related information about NtNRAMP2 gene on NCBI:
The NtNRAMP2 gene ID is 107807466. There are NPAMP2 gene information for some other plants (such as Arabidopsis thaliana) in the database. However, for tobacco, the relevant information about the NtNRAMP2 gene is sparse and incomplete. NCBI shows that the chromosomal location of this gene in tobacco is unknown, with a total of 5 exons.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 952
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 952
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 952
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 952
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 952
- 1000COMPATIBLE WITH RFC[1000]
| None |