Part:BBa_K4213004
PHT1;5 from A.thaliana with a linker for reporter gene
AtPHT1;5 is an inorganic phosphate (Pi) transporter from Arabidopsis thaliana.
PHT1 Family
PHT1 transporters have 12 membrane spanning domains that are divided into two groups of six which are linked by a charged hydrophilic loop. It is estimated that the N- and C-terminal regions are located inside the cell, while the rest of the protein is located in the plasma membrane [1](Figure 1). The transporters are approximately 520 amino acids in size in Arabidopsis thaliana and are detected at 58kDa [2].
In addition to phosphorus, they have also affinity with phosphorus’ analogs, that is, with phosphite [4] and arsenate [5]. The PHT1 transporters are expressed primarily in plant roots and more specifically in the plasma membrane of cells of the root epidermis and the central cylinder [6]. Nevertheless, the transporters of this family have been shown to be expressed in parts of the shoot, too, such as leaves and flowers.
These properties, regarding their expression and localization, do not seem to change between monocots and dicots [7], [8].
Phosphate Transporter 5 from Arabidopsis thaliana
Plants with constitutive overexpression of AtPHT1;5 appeared to have an increased rate of phosphorus uptake in roots. At the same time, it has been observed that AtPHT1;5 overexpressors show increased total leaf area, floral stalk thickness and total leaf dry weight compared to wild-type or to atpht1;5 mutants. This trait is particular interesting because it implies that the Pi excess is located in these plant parts, which are easily removable for further exploitation. Also, root hairs show an increase in number and length, whereas the primary root does not appear to grow as much compared to wild-type plants. [9]
It should also be mentioned that the main role of AtPHT1;5 transporter is considered to be the regulation of Pi distribution between root and shoot rather than the Pi uptake [10].
Design
In our design we used the cds of each gene and, as it lacked the 5' UTR region that normally controls ribosome binding in eukaryotes, we checked that the nucleotides at positions +4 and +5 were G and C, respectively, so that the cds part of the Kozak frame observed in plants is present.
Also, due to the high degree of complexity of the cds of PHT1 according to IDT, we decided to do codon optimization for the model plant Nicotiana benthamiana, so that, firstly, we could order our sequences and, secondly, we would have better results in the primary experiments of agroinfiltration we would like to conduct. At the same time, however, we thought of ordering the actual cds sequences (from TWIST), too, to have a better chance of our system working and to be able to compare the results of these two conditions. Here, we present to you the non-codon optimized variant.
Downstream of the pht1 cds we also included a linker sequence, so that we can add, if needed, a reference gene.
References
[1] Raghothama, K. G. (1999). PHOSPHATE ACQUISITION. Annual Review of Plant Physiology and Plant Molecular Biology, 50(1), 665–693. doi:10.1146/annurev.arplant.50.1
[2] Bucher, M., Rausch, C., & Daram, P. (2001). Molecular and biochemical mechanisms of phosphorus uptake into plants. Journal of Plant Nutrition and Soil Science, 164(2), 209–217. doi:10.1002/1522-2624(200104)164:2<209::aid-jpln209>3.0.co;2-f
[3] Bayle V, Arrighi JF, Creff A, Nespoulous C, Vialaret J, Rossignol M, Gonzalez E, Paz-Ares J, Nussaume L. Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. Plant Cell. 2011 Apr;23(4):1523-35. doi: 10.1105/tpc.110.081067. Epub 2011 Apr 26. PMID: 21521698; PMCID: PMC3101552.
[4] Varadarajan DK, Karthikeyan AS, Matilda PD, Raghothama KG. Phosphite, an analog of phosphate, suppresses the coordinated expression of genes under phosphate starvation. Plant Physiol. 2002 Jul;129(3):1232-40. doi: 10.1104/pp.010835. OsPHT1;6
[5] Wu Z, Ren H, McGrath SP, Wu P, Zhao FJ. Investigating the contribution of the phosphate transport pathway to arsenic accumulation in rice. Plant Physiol. 2011 Sep;157(1):498-508. doi: 10.1104/pp.111.178921. Epub 2011 Jun 29. PMID: 21715673; PMCID: PMC3165895.
[6] Jia, H., Ren, H., Gu, M., Zhao, J., Sun, S., Zhang, X., Xu, G. (2011). The Phosphate Transporter Gene OsPht1;8 Is Involved in Phosphate Homeostasis in Rice. PLANT PHYSIOLOGY, 156(3), 1164–1175. doi:10.1104/pp.111.175240
[7] Nussaume, L. (2011). Phosphate import in plants: focus on the PHT1 transporters. Frontiers in Plant Science, 2. doi:10.3389/fpls.2011.00083
[8] Koyama, T., Ono, T., Shimizu, M., Jinbo, T., Mizuno, R., Tomita, K., … Sakka, K. (2005). Promoter of Arabidopsis thaliana phosphate transporter gene drives root-specific expression of transgene in rice. Journal of Bioscience and Bioengineering, 99(1), 38–42. doi:10.1263/jbb.99.38
[9] Nagarajan VK, Jain A, Poling MD, Lewis AJ, Raghothama KG, Smith AP. Arabidopsis Pht1;5 mobilizes phosphate between source and sink organs and influences the interaction between phosphate homeostasis and ethylene signaling. Plant Physiol. 2011 Jul;156(3):1149-63. doi: 10.1104/pp.111.174805. Epub 2011 May 31. PMID: 21628630; PMCID: PMC3135966.
[10]Smith AP, Nagarajan VK, Raghothama KG. Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis. Plant Signal Behav. 2011 Nov;6(11):1676-8. doi: 10.4161/psb.6.11.17906. Epub 2011 Nov 1. PMID: 22057342; PMCID: PMC3329334.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 868
Illegal EcoRI site found at 1452 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 868
Illegal EcoRI site found at 1452 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 868
Illegal EcoRI site found at 1452 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 868
Illegal EcoRI site found at 1452 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 868
Illegal EcoRI site found at 1452 - 1000COMPATIBLE WITH RFC[1000]
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