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Bioinformatics Analysis of Arabidopsis thaliana and Oryza
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sativa AMT Family.
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ABSTRACT
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ammonium, a primary source of nitrogen. In this study, we compared six Arabidopsis thaliana
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AMTs and ten Oryza sativa AMTs in terms of the aspects of phylogeny tree, gene and protein
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information, exon/intron organization, prediction of trans-membrane helices, conserved domain
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and subcellular localization.
In most plant species, ammonium transporters (AMT) are responsible for mediating the
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Key words: ammonium transporters(AMT)， Arabidopsis thaliana, Oryza sativa, exon/intron,
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trans-membrane, conserved domain, subcellular localization.
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Introduction
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which are present in the soil as organic and inorganic complexes and compounds (Williams et al.,
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2001). It is ammonium transport (AMT) that absorbs these sources from the soil. AMT presents
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over the plasma membrane of root cells and incorporates into glutamine via glutamine synthetase
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(GS) that is in the cytoplasm and plastids (Kaiser et al., 2002).
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Many AMT genes have been identified and cloned from diverse plant species. Furthermore, the
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biochemistry and molecular biology of AMT in plants has been extensively studied (Loqué & von
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Wirén, 2004; Schjoerring JK et al., 2002). Previous studies on phylogenetic analyses of the AMT
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gene family showed that the AMT family could be subdivided into two subfamilies: the AMT1
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subfamily and the AMT2 subfamily. There were only one cluster in AMT1 subfamily and two
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clusters in AMT2 subfamily (Koegel et al., 2013; Loqué & von Wirén, 2004). Several plants’
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AMT1 subfamily members have been characterized in yeast and Arabidopsis thaliana, such as
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the AtAMT1;1, AtAMT1;2, AtAMT1;3 in Arabidopsis thaliana(Gazzarrini S et al., 1999;
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Ninnemann O et al., 1994), the LeAMT1;1, LeAMT1;2 and LeAMT1;3 in Lycopersicon
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esculentum (Lauter FR et al., 1996; von Wiren N et al., 2000b) and the OsAMT1;1, OsAMT1;2,
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OsAMT1;3 in Oryza sativa (Sonoda Y et al., 2003). Furthermore, the AMT2 subfamily with
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distinct biochemical features has been identified in several plants including Arabidopsis thaliana
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(Sohlenkamp C et al., 2002), Oryza sativa (Suenaga A et al., 2003) and Lotus japonicas
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(Simon-Rosin U et al., 2003.).
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Although plant physiologists have studies many of the specific AMT, available comprehensive
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information of AMT is still limited. For example, it is known that AMT1 cluster genes have a high
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affinity NH4+ -transport function (Loqué et al., 2006.; Yuan L. et al., 2007). Therefore, it is
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necessary to make a comprehensive comparison among the plant AMT subfamilies. In this study,
The main sources of nitrogen in plants are ammonium (NH4+ ), nitrate (NO3− ), and amino acids,
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we compared the commonness and individuality of AMT, predicted their structural types. In detail,
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we investigated the AMT families of Arabidopsis thaliana and Oryza sativa, in terms of
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phylogeny tree, gene and protein information, exon/intron organization, prediction of
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trans-membrane helices, conserved domain, and subcellular localization.
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Materials and Methods
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We downloaded the Arabidopsis thaliana and Oryza sativa AMT gene sequences form the Gene
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Database, and protein sequences from the UniProt Database. We used BioEdit (Kaiser et al., 2002)
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to do Multiple sequence alignment with 60% threshhold for shading. The full-length amino acid
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sequences of AMTs were aligned with ClustalW in MEGA6 software (Tamura et al., 2013). Then,
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we use the Neighbor-Joining (NJ) method and Poisson correction model to construct the
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phylogenetic tree. It carried out 1000 times Bootstrap method.
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The exon/intron organization of individual AMT genes was illustrated with the Gene Structure
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Display Server program (http://gsds1.cbi.pku.edu.cn/index.php) (Guo et al., 2007). The
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trans-membrane domains in each AMT protein were predict with TMHMM Server version 2.0
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(http://www.cbs.dtu.dk/services/TMHMM/)
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(http://www.ebi.ac.uk/interpro/) (Alex Mitchell et al., 2015) to discover each AMT protein
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conserved domain. PSORT II Prediction software (http://psort.hgc.jp/form2.html) (Emanuelsson O.
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et al., 2007.) was used to make each AMT family gene’s subcellular localization.
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(L.L.
et
al.,
1998).
We
used
InterPro
Results
Phylogenetic Tree Analyses of AMT Genes
A database search with the keywords AMT showed that there were 6 AMT proteins in Arabidopsis
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thaliana and 10 AMT proteins in Oryza sativa. The result of multiple sequence alignment is
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shown in Figure 1.
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The detailed information of AMTs was summarized as shown in Table 1. ACCESSION represents
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the accession number in the UniProt Database; Gene ID represents the Gene ID in the Gene
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Database; Chromosome represents the gene localization in Chromosome; GI represents the GI in
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Gene Bank;
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The distribution of gene on the chromosome is one of the decisive factors of functional features.
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In this study, the 6 members of Arabidopsis thaliana AMT distributed to number 1, 2, 3 and 4
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chromosome, and the 10 members of Oryza sativa AMT distributed to number 1, 2, 3, 4 and 5
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chromosome. They all characterize the scattered distribution.
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To investigate the evolutionary relationships among Arabidopsis thaliana and Oryza sativa AMT
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proteins, we used ClustalW to align full-length sequences of the 16 proteins, and constructed a
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phylogenetic tree with the Neighbor-Joining method using MEGA6 software (Figure 2).
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The results showed two subfamily and four clusters. Among the 16 AMT proteins, 8 proteins were
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in the AMT1 cluster, and the remaining AMT proteins were separately in clusters AMT2, AMT3,
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and AMT4. We named each of them includes the initials of the plant species and the cluster
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number. In cluster 1, they are AtAMT1-1, AtAMT1-2, AtAMT1-3, AtAMT1-4, AtAMT1-5,
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OsAMT1-1, OsAMT1-2, OsAMT1-3; In cluster 2, they are AtAMT2, OsAMT2-1, OsAMT2-2,
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OsAMT2-3; In cluster 3, they are OsAMT3-1, OsAMT3-2, OsAMT3-3; In cluster 4, there is only
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OsAMT4.
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Exon/Intron Organization Analyses of AMT Genes.
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It has been studied that members of the AMT1 subfamily was mostly intron-free (Salvemini et al.,
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2001); whereas members of AMT2 subfamily contained some introns in their gene sequences
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(Suenaga A et al., 2003).
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We get the same answer through analyzing the exon/intron structure of the 16 AMT genes in
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Arabidopsis thaliana and Oryza sativa (Figure 3 A). Genes in the AMT1 cluster all had only 1
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exon and null intron, while those in the AMT2 and AMT3 cluster have very different performance.
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AtAMT2 has 5 exons and 4 introns; OsAMT2-1 and OsAMT2-2 have 3 exons and 2 introns;
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OsAMT2-3 is the same as cluster1; OsAMT3-1 has 2 exons and 1 introns; OsAMT3-2 has 4 exons
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and 3 introns; OsAMT3-3 has 3 exons and 2 introns; Only OsAMT4 is in the cluster4, it has 3
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exons and 2 introns. Therefore, the substantial differences in gene structure may be resulted from
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differences in the size of exons and introns among the various genes.
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Trans-membrane Helices and Conserved Domain Prediction.
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We predicted the trans-membrane domains in each AMT protein using TMHMM Server
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(http://www.cbs.dtu.dk/services/TMHMM/). Details are summarized in Table 2 with visualization
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in Figure 3. The results indicate that all of them have 5 to 11 trans-membranes, and members in
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the same subfamily have similar trans-membrane helices (Figure 3 B). In addition, only
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N-terminus of AtAMT1-4 and AtAMT1-3 are on the cytoplasmic side of the membrane, others are
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out of the cytoplasmic side, while 15 out of 16 C-terminus are on the cytoplasmic side, except for
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OsAMT2-3. The length of encoded proteins ranged from 299 amino acids to 514 amino acids.
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OsAMT4 has the shortest length, while AtAMT1-2 has the longest one. Interestingly, 14 out of the
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16
AMT
members
has
the
AMT
domain
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using
the
InterPro
software
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(http://www.ebi.ac.uk/interpro/), which can provides functional analysis of proteins by classifying
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them into families and predicting domains and important sites (Table 2).
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Subcellular Localization of AMT Genes
Subcellular localization of protein analysis showed that AMT members were mainly localized in
plasma membrane, vacuolar, endoplasmic reticulum membrane and Golgi body. Details were as
shown in Table 3. 15 out of 16 members were predicted in plasma membrane and only AtAMT1-1
was predicted in endoplasmic reticulum. The results implied that the AMT members were widely
distributed in the plant cells.
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Conclusion
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In this paper, we compared and summarized 6 AMTs in Arabidopsis thaliana, and 10 AMTs in
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Oryza sativa. Through the Phylogenetic Tree analysis, two clusters in Arabidopsis thaliana AMT
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family and four clusters in Oryza sativa AMT family were classified.
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The phylogenetic analysis and gene structure revealed that Genes in cluster 1 were well conserved
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in terms of exon/intron structure with similar numbers of introns and similar gene lengths.
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However, there were greater variations in gene structure among the cluster 2 and cluster 3. Further
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research is needed to investigate the internal mechanism, biologist should make greater efforts.
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In AMT gene family, extracellular N-terminus play a role for oligomer stability. We found that the
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majority of N-terminus of Arabidopsis thaliana and Oryza sativa ammonium transporters were
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outside the cell, except for AtAMT1-4 and OsAMT1-2. In addition, we analyzed proteins by
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predicting domains and important sites. These results suggested that the AMT gene family
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members are well conserved both in terms of gene structure and specific domain of AMT proteins.
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Results of Subcellular localization show us that, the vast majority of Arabidopsis thaliana and
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Oryza sativa AMTs are positioned in plasma membrane. Only AtAMT1-1 is mostly in
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endoplasmic reticulum. We need biology experiment to prove whether they are located in the
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predicted location.
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References
Alex Mitchell, Hsin-Yu Chang, Louise Daugherty, Matthew Fraser, Sarah Hunter, R. L., Craig
McAnulla, & Conor McMenamin, G. N., Sebastien Pesseat,Amaia Sangrador-Vegas,
Maxim Scheremetjew, Claudia Rato, Siew-Yit Yong. (2015). The InterPro protein
families database: the classification resource after 15 years. Nucleic Acids Res., D213–
D221.
Emanuelsson O., Brunak S., Von Heijne G., & H., N. (2007.). Locating proteins in the cell using
4
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
TargetP, SignalP and related tools. Nat. Protoc., 2(4): 953-971.
Gazzarrini S, Lejay L, Gojon A, Ninnemann O, Frommer WB, & N., v. W. (1999). Three
functional transporters for constitutive, diurnally regulated, and starvation-induced uptake
of ammonium into Arabidopsis roots. Plant Cell, 11: 937–948.
Guo, A. Y., Zhu, Q. H., Chen, X., & Luo, J. C. (2007). GSDS: a gene structure display server. Yi
Chuan, 29, 1023–1026. doi: doi: 10.1360/yc- 007-1023.
Kaiser, B., Rawat SR, Siddiqi MY, Masle J, & AD., G. (2002). Functional analysis of an
Arabidopsis T-DNA ‘knockout’ of the high-affinity NH4+ transporter AtAMT1;1. . Plant
Physiology, 130: 1263–1275.
Koegel, S., Ait Lahmidi, N., Arnould, C., Chatagnier, O., Walder, F., & Ineichen, K. (2013). The
family of ammonium transporters (AMT) in Sorghum bicolor: two AMT members are
induced locally, but not systemically in roots colonized by arbuscular mycorrhizal fungi.
New Phytol., 198, 853–865. doi: 10.1111/nph.12199
L.L., E., Sonnhammer, Gunnar von Heijne, & Krogh, A. (1998). A hidden Markov model for
predicting transmembrane helices in protein sequences. . 6:175-182.
Lauter FR, Ninnemann O, Bucher M, Riesmeier JW, & WB., F. (1996). Preferential expression of
an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.
Proceedings of the National Academy of Sciences, USA, 93: 8139–8144.
Loqué, D., & von Wirén, N. (2004). Regulatory levels for the transport of ammonium in plant
roots. J. Exp. Bot., 55, 1293–1305. doi: 10.1093/jxb/erh147
Loqué, D., Yuan, L., Kojima, S., Gojon, A., Wirth, J., & Gazzarrini, S. (2006.). Additive
contribution of AMT1; 1 and AMT1; 3 to high-affinity ammonium uptake across the
plasma membrane of nitrogen-deficient Arabidopsis roots. Plant J., 48, 522–534. doi:
10.1111/j.1365-313X.2006.02887.x.
Ninnemann O, Jauniaux JC, & WB., F. (1994). Identification of a high affinity NH4+ transporter
from plants. EMBO Journal, 13: 3464–3471.
Salvemini, F., Marini, A., Riccio, A., Patriarca, E. J., & Chiurazzi, M. (2001). Functional
characterization of an ammonium transporter gene from Lotus japonicus. . Gene, 270,
237–243. doi: 10.1016/S0378-1119(01)00470-X
Schjoerring JK, Husted S, Mack G, & M, M. (2002). The regulation of ammonium translocation
in plants. Journal of Experimental Botany, 53: 883–890.
Simon-Rosin U, Wood C, & MK., U. (2003.). Molecular and cellular characterisation of LjAMT2;
1, an ammonium transporter from the model legume Lotus japonicus. Plant Molecular
Biology, 51: 99–108.
Sohlenkamp C, Wood CC, Roeb GW, & MK., U. (2002). Characterization of Arabidopsis
AtAMT2, a high-affinity ammonium transporter of the plasma membrane. . Plant
Physiology, 130: 1788–1796.
Sonoda Y, Ikeda A, Saiki S, von Wiren N, Yamaya T, & J., Y. (2003). Distinct expression and
function of three ammonium transporter genes (OsAMT1; 1–1; 3) in rice. Plant and Cell
Physiology, 44: 726–734.
Suenaga A, Moriya K, Sonoda Y, Ikeda A, von Wiren N, Hayakawa T, . . . T., Y. (2003).
Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants.
Plant and Cell Physiology, 44: 206–211.
Tamura, K., Glen Stecher, Daniel Peterson, Alan Filipski, & Sudhir Kumar, e. a. (2013). MEGA6:
5
183
184
185
186
187
188
189
190
191
192
193
194
Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol., 30(12): 2725–
2729.
von Wiren N, Lauter FR, Ninnemann O, Gillissen B, Walch-Liu P, Engels C, . . . WB, F. (2000b).
Differential regulation of three functional ammonium transporter genes by nitrogen in
root hairs and by light in leaves of tomato. Plant Journal, 21: 167–175.
Williams, L. E., & Miller, A. J. (2001). Transporters responsible for the uptake and partitioning of
nitrogenous
solutes.
Annu.
Rev.
Plant
Biol.,
52,
659–688.
doi:
10.1146/annurev.arplant.52.1.659
Yuan L., L. D., Kojima S., Rauch S., Ishiyama K., & E., I. (2007). The organization of
high-affinity ammonium uptake in Arabidopsis roots depends on the spatial arrangement
and biochemical properties of AMT1-type transporters. . Plant Cell Online, 19, 2636–
2652. doi: 10.1105/tpc.107.052134.
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Table 1 Database information of members of family of AtAMT and OsAMT.
NAME
ACCESSION
Gene ID
Chromosome
GI
AtAMT1-1
P54144
826983
4
240256243
AtAMT1-2
Q9ZPJ8
842786
1
240254421
AtAMT1-3
Q9SQH9
822018
3
240255695
AtAMT1-4
Q9SVT8
828988
4
240256243
AtAMT1-5
Q9LK16
822017
3
240255695
AtAMT2
Q9M6N7
818409
2
240254678
OsAMT1-1
Q7XQ12
4336365
4
297603645
OsAMT1-2
Q6K9G1
4330008
2
297600179
OsAMT1-3
Q6K9G3
4330007
2
297600179
OsAMT2-1
Q84KJ7
4339064
5
297605017
OsAMT2-2
Q8S230
4327434
1
297598437
OsAMT2-3
Q8S233
4327433
1
297598437
OsAMT3-1
Q84KJ6
4324937
1
297598437
OsAMT3-2
Q851M9
4334717
3
297602023
OsAMT3-3
Q69T29
4329628
2
297600179
OsAMT4
Q10CV4
Unclear
3
Unclear
197
7
198
199
Figure 1 Multiple sequence alignment.
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200
201
202
Figure 2 Phylogenetic tree of proteins encoded by AMT genes.
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203
204
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Figure 3 Gene structure (A) and Trans-membrane Helices(B).
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Table 2 Protein information of members of family of AtAMT and OsAMT.
NAME
AMT
Length
Trans-membrane
domain
N-terminus
C-terminus
Number
AtAMT1-1
198-223
501
9
N-out
C-in
AtAMT1-2
211-236
514
11
N-out
C-in
AtAMT1-3
202-227
498
10
N-in
C-in
AtAMT1-4
206-231
504
10
N-in
C-in
AtAMT1-5
201-226
496
9
N-out
C-in
AtAMT2
180-205
475
11
N-out
C-in
OsAMT1-1
191-216
498
11
N-out
C-in
OsAMT1-2
191-216
496
11
N-in
C-in
OsAMT1-3
193-218
498
9
N-out
C-in
OsAMT2-1
187-212
486
11
N-out
C-in
OsAMT2-2
200-225
501
11
N-out
C-in
OsAMT2-3
196-221
497
10
N-out
C-out
OsAMT3-1
498
11
N-out
C-in
OsAMT3-2
203-228
unclear
479
11
N-out
C-in
OsAMT3-3
195-220
480
11
N-out
C-in
OsAMT4
unclear
299
5
N-out
C-in
207
208
11
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Table 3 Subcellular localization of AtAMT and OsAMT.
NAME
Plasma
vacuolar
membrane
endoplasmic
Golgi
cytoplasmic
mitochondrial
reticulum
AtAMT1-1
-
11.1 %
44.4 %
-
22.2 %
11.1 %
AtAMT1-2
60.9 %
13.0 %
13.0 %
4.3 %
-
-
AtAMT1-3
47.8 %
17.4 %
21.7 %
4.3 %
-
-
AtAMT1-4
56.5 %
13.0 %
21.7 %
4.3 %
-
-
AtAMT1-5
52.2 %
17.4 %
17.4 %
4.3 %
-
-
AtAMT2
65.2 %
8.7 %
17.4 %
4.3 %
-
-
OsAMT1-1
60.9 %
13.0 %
17.4 %
4.3 %
-
-
OsAMT1-2
60.9 %
8.7 %
21.7 %
4.3 %
-
-
OsAMT1-3
60.9 %:
13.0 %
13.0 %
4.3 %
-
-
OsAMT2-1
60.9 %
8.7 %
21.7 %
4.3 %
-
-
OsAMT2-2
60.9 %
8.7 %
17.4 %
4.3 %
-
-
OsAMT2-3
73.9 %
4.3 %
13.0 %
4.3 %
-
-
OsAMT3-1
60.9 %
8.7 %
17.4 %
4.3 %
-
-
OsAMT3-2
69.6 %
8.7 %
13.0 %
4.3 %
-
-
OsAMT3-3
65.2 %
-
21.7 %
-
-
8.7 %
OsAMT4
47.8 %
8.7 %
34.8 %
4.3 %
-
-
210
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