Part:BBa_K4213021
PHT1;6 from O. sativa (optimized & domesticated) - linker
OsPHT1;6 is an inorganic phosphate (Pi) transporter from Oryza sativa.
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 6 from Oryza sativa (OsPHT1;6)
The main functions of the OsPHT1;6 transporter are the Pi uptake, its translocation and transport within the plant. In fact, under Pi-deficient conditions, its expression levels increase significantly in roots and shoots [9]. This transporter appears to be expressed mainly in the cortical and epidermal cells of the primary and lateral roots.
Rice lines overexpressing the OsPHT1;6 have shown 75% and 73% greater Pi uptake rate in comparison with wild-type plants under Pi sufficient and -deficient conditions, respectively.
These overexpressors showed increased biomass and enhanced Pi accumulation rates in various plant tissues under hydroponic and soil culture conditions (Figure 2).
The ratio shoot:root was also increased (Figure 3), indicating that the overexpressed OsPHT1; 6 transports the Pi overabundance to the upper part of the plant thus increasing the possibility of removing these phosphorus-laden parts through for example pruning.
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.
In fact, in the case of OsPHT1;6 we had to change the nucleotide and amino acid sequence in order to form the Kozak frame. More specifically, we changed the nucleotides G and A at positions +5 and +6 with C and T to have cytosine at the +5 position and at the same time change the amino acid from glycine to alanine. We chose alanine because together with glycine are the simplest amino acids, with a neutral charge and non-polar. The difference between them is that alanine is hydrophilic, so it is probably located in the cytoplasm, while glycine is hydrophobic, so it is probably located in the intramembrane part of the protein. Nevertheless, as this glycine is the first of the hydrophobic amino acids and therefore the beginning of the intramembrane part, we hope that this will not cause a big problem in the functionality of the transporter.
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 you the 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.
[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] Ai P, Sun S, Zhao J, Fan X, Xin W, Guo Q, Yu L, Shen Q, Wu P, Miller AJ, Xu G. Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. Plant J. 2009 Mar;57(5):798-809. doi: 10.1111/j.1365-313X.2008.03726.x. Epub 2008 Nov 22. PMID: 18980647.
[10] Zhang, F., Wu, XN., Zhou, HM. et al. Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants. Plant Soil 384, 259–270 (2014). https://doi.org/10.1007/s11104-014-2168-8
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 512
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 577
Illegal PstI site found at 512 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 986
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 512
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 512
- 1000COMPATIBLE WITH RFC[1000]
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