Investigation of Polymorphisms in Coding Region of OsHKT1 in Relation to Salinity in Rice

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Rice Science, 2016, 23(6): 334−338

Investigation of Polymorphisms in Coding Region of OsHKT1
in Relation to Salinity in Rice
PHAM Quynh-Hoa1, #, TRAN Xuan-An1, #, NGUYEN Thi-Nha-Trang1, TRAN Thi-Thuy-Anh1,
HOANG Hai-Yen1, NGUYEN Thi-Hong-Van1, TANG Thi-Hanh2, DO Thi-Phuc1
(1Faculty of Biology, VNU University of Science, Hanoi, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam; 2Faculty of Agronomy,
Vietnam National University of Agriculture, Ngo Xuan Quang Street, Trau Quy, Gia Lam, Hanoi, Vietnam; #These authors
contribute equally to this study)
Abstract: Rice (Oryza sativa) is sensitive to salinity, but the salt tolerance level differs among cultivars,
which might result from natural variations in the genes that are responsible for salt tolerance. High-affinity
potassium transporter (HKTs) has been proven to be involved in salt tolerance in plants. Therefore, we
screened for natural nucleotide polymorphism in the coding sequence of OsHKT1, which encodes the HKT
protein in eight Vietnamese rice cultivars differing in salt tolerance level. In total, seven nucleotide
substitutions in coding sequence of OsHKT1 were found, including two non-synonymous and five synonymous
substitutions. Further analysis revealed that these two non-synonymous nucleotide substitutions (G50T
and T1209A) caused changes in amino acids (Gly17Val and Asp403Glu) at signal peptide and the loop of
the sixth transmembrane domain, respectively. To assess the potential effect of these substitutions on the
protein function, the 3D structure of HKT protein variants was modelled by using PHYRE2 webserver. The
results showed that no difference was observed when compared those predicted 3D structure of HKT
protein variants with each other. In addition, the codon bias of synonymous substitutions cannot clearly
show correlation with salt tolerance level. It might be interesting to further investigate the functional roles of
detected non-synonymous substitutions as it might correlate to salt tolerance in rice.
Key words: polymorphism; salt stress; OsHKT1 gene; rice
Salinity can be stressful to most crop plants and result in severe
agricultural loss. In addition to hyperosmotic damage
(Tarczynski et al, 1993), elevated Na+ concentrations can
disrupt cellular processes by interfering with vital Na+-sensitive

enzymes (Tester and Davenport, 2003; Munns et al, 2006) and
affecting ion transport (Rains and Epstein, 1965; Schroeder
et al, 1994). Na+ uptake occurs via multiple Na+-permeable
channels/transporters under the saline conditions, and ion
toxicity is triggered when a high level of Na+ is accumulated in
the cytosol (Volkmar et al, 1999; Munns, 2002; Horie and
Schroeder, 2004). Therefore, it is very important for cells to
maintain a low concentration of cytosolic Na+ or to maintain a
low Na+/K+ ratio in the cytosol under NaCl stress. The most
important way to maintain a low cytosolic Na+ concentration is
to minimize the influx of Na+ into the cytosol (Horie and
Schroeder, 2004; Chinnusamy et al, 2005). Na+ influx can be

restricted by means of selective ion uptake. It has been
suggested that high-affinity potassium transporters (HKTs)
mediate a substantial Na+ influx in some species (Uozumi et al,
2000; Horie et al, 2001; Golldack et al, 2002; Garciadeblás et al,
2003).
While only one copy of HKT is present in Arabidopsis, 7–9
HKT genes (OsHKT1, OsHKT2, OsHKT3, OsHKT4, OsHKT5,
OsHKT6, OsHKT7, OsHKT8 and OsHKT9) have been
identified in rice (Oryza sativa) depending on cultivars
(Uozumi et al, 2000; Garciadeblás et al, 2003). These
functional genes encode proteins with distinct transport
activities, which might be expressed in various tissues and/or
organs (Jabnoune et al, 2009). It has been suggested that
OsHKT1 is a Na+ transporter (Horie et al, 2001; Maser et al,
2002; Garciadeblás et al, 2003) and OsHKT2 is a Na+/K+ cotransporter (Horie et al, 2001; Maser et al, 2002). OsHKT8 was

Received: 25 February 2016; Accepted: 6 May 2016

Corresponding author: DO Thi-Phuc (; )
Copyright © 2016, China National Rice Research Institute. Hosting by Elsevier B
V This is an open access article under the CC BY-NC-ND license
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is an open access article under the CC BY-NC-ND license ( />( />Peer review under responsibility of China National Rice Research Institute
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PHAM Quynh-Hoa, et al. Polymorphism of OsHKT1 in Rice
recently shown to be a Na+ transporter that contributes to
increased salt tolerance by maintaining K+ homeostasis in the
shoot under salt stress (Ren et al, 2005; Rus et al, 2005). This
transporter is thought to be analogous to the function of
AtHKT1 in Arabidopsis, which is a Na+ transporter and plays a
crucial role in controlling cytosolic Na+ detoxification
(Berthomieu et al, 2003; Rus et al, 2005). Therefore, it is likely
that the HKT family plays an important role in Na+/K+
homeostasis in rice, and some of its members are evidently Na+
transporters.
Analyses of natural genetic polymorphism can provide
insight into the mechanisms of plant adaptation to
environmental conditions (Brady et al, 2005; Baxter et al,
2010). Although rice is considered to be a salt-sensitive species,
several varieties, such as Pokkali and Nona Bokra, display a
certain level of salt tolerance. Therefore, in this study, we
focused on the analysis of natural polymorphism in OsHKT1 in
eight Vietnamese rice cultivars, namely, Nep Non Tre, Chiem
Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep
Deo Dang, and several single nucleotide polymorphisms were
identified. However, further in silico analysis revealed no

difference in predicted 3D protein structures and in codon
usage bias of these variants in relation to salt tolerance.

MATERIALS AND METHODS
Rice materials
Seeds of nine rice cultivars were provided by Vietnam National
University of Agriculture (Hanoi, Vietnam), including
Nipponbare, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau,
Nep Oc, Ngoi, Dau An Do and Nep Deo Dang. The six
Vietnamese rice cultivars were collected from different costal
region in Vietnam (Nep Non Tre, Re Nuoc, Hom Rau, Ngoi
and Dau An Do were collected in Nam Dinh Province, while
Chiem Cu was collected in Quang Binh Province), and Nep
Deo Dang in Tuyen Quang Province and Nep Oc in Ha Noi
City. These cultivars showed differences in salt tolerance. The
highly salt tolerant cultivars are Nep Non Tre, Hom Rau and
Nep Oc, whereas the moderate salt tolerant cultivars are Chiem
Cu, Re Nuoc and Ngoi, and the sensitive cultivars are Dau An
Do and Nipponbare (Tran et al, 2015). The seedlings were
grown in soil for 14 d, then the leaves were collected and stored
at -80 °C for further analysis.
DNA extraction
The DNA extraction was performed by using the cetyl
trimethylammonium bromide (CTAB) method. About 200 mg
leaf powder was mixed with 500 µL CTAB buffer and incubated
at 65 °C for 20 min. Then 500 µL CI 24:1 (chloroform:
isoamylalcohol) was added and centrifuged at 14 000 r/min at
4 °C for 15 min. The supernatant was transferred into a new
tube, and the DNA was precipitated by cold isopropanol for 15
min. The DNA pellet was collected by centrifuging at 10 000

r/min at 4 °C for 5 min and washed with 70% ethanol. Finally,

335
after drying at room temperature, the pellet was dissolved in
Tris-EDTA buffer and kept at -20 °C. The quality and quantity
of extracted DNA were estimated by visualizing the band of
total DNA on ethidium bromide-stained 1% agarose gel in
comparison with the relative migration and intensity of the
standard 1 kb ladder (Fermentas).
Primer design and amplification of OsHKT1 by PCR
Four primer pairs were used for amplifying four flanking
fragments of OsHKT1 from genomic DNA, including F1-FW
(5′-CATACTCGTTGGCTCGTTGC-3′) and F1-RW (5′-ATCACA
GTGCTTGCCGAGTT-3′); F2-FW (5′-TGAAGCCAAGCAACCC
AGAA-3′) and F2-RW (5′-CAGCACCGAACAATGTGACC-3′);
F3-FW (5′-TGGCCTTATGGCTTCCTTGG-3′) and F3-RW (5′ATTGGCTTGATGCCCAGTGT-3′); F4-FW (5′-GGCATTTT
CACAGCTTGCCT-3′) and F4-RW (5′-GGCATTTTCACAGC
TTGCCT-3′) (Do and Nguyen, 2014). The PCR reaction
mixture consisted of genomic DNA (20–50 ng), Dream Taq
polymerase buffer (1×), MgCl2 (1.5 mmol/L), dNTPs mixture
(0.2 mmol/L), primers (0.4 µmol/L) and Dream Taq
polymerase (1 U). The PCR reaction was performed with a
thermocycle of 95 °C for 5 min, 35 cycles of 95 °C for 30 s,
56 °C for 30 s and 72 °C for 1 min, and 72 °C for 5 min. Then,
5 µL of PCR products were separated on 1% agarose gel for 28
min at constant 90 V in 1× Tris-acetate-EDTA buffer. If PCR
product showed only one bright single band on gel, it was
purified using GeneJET PCR Purification Kit (Thermo Fisher
Scientific) and sequenced on ABI PRISM 3730xl Genetic
Analyzer (Applied Biosystems, USA) at First BASE DNA

sequencing service (Singapore).
Sequence analysis
Gene sequences were analyzed using Bioedit (Hall, 1999) and
Multalin webserver (Corpet, 1988). The predicted peptide
sequences were analyzed using Expasy webserver
( />Modelling OsHKT1 protein in rice
The 3D model of OsHKT1 protein was predicted and analyzed
by using PHYRE2 program (Kelley et al, 2015). PHYRE2 is a
web-based tool to predict protein structure. The predicted
structure of protein in PDB file created by PHYRE2 was
visualized by Discovery studio 4.5 visualizer.

RESULTS AND DISCUSSION
Amplification of OsHKT1 from nine rice cultivars
The leaves of 14 d rice seedlings were collected for genomic
DNA extraction. The extracted DNA was used as template for
the amplification of OsHKT1 in the PCR assay. The specific
primers were designed to amplify four amplicons that covered
the complete genomic sequence of OsHKT1. As shown in
Fig. 1, the PCR products were specific and with the correct size.
Thus, the OsHKT1 gene was successfully amplified in all the


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Rice Science, Vol. 23, No. 6, 2016

Fig. 1. PCR products using four pairs of primers.
A, PCR product of 499 bp using primer pair 1 (F1); B, PCR product of 828 bp using primer pair 2 (F2); C, PCR product of 603 bp using primer
pair 3 (F3); D, PCR product of 668 bp using primer pair 4 (F4).

M, 1 kb ladder marker; Lanes 1 to 9, Nipponbare, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep Deo Dang,
respectively.

investigated rice cultivars.
Polymorphism in nucleotide sequence of OsHKT1 gene
Amplified DNA fragments were purified, and sequenced and
allowed the detection of seven nucleotide substitutions when
compared with the reference sequence in the database
(Nipponbare allele) (Fig. 2). Among the seven detected
nucleotide substitutions, five were synonymous and two were
non-synonymous substitutions. The detected five synonymous
substitutions were A360G (Nep Non Tre, Dau An Do, Chiem
Cu and Nep Oc), A645G (Chiem Cu), A708G (Nep Non Tre,
Dau An Do, Chiem Cu and Nep Oc), G744A (Nep Non Tre,
Dau An Do, Chiem Cu and Nep Oc), and G1440A (Nep Non
Tre). Interestingly, the two non-synonymous substitutions were
present only in Hom Rau which was classified as salt tolerant
cultivar (Tran et al, 2015). These two non-synonymous substitutions
(G50T and T1209A) lead to change in amino acid of the
corresponding protein variant (G17V and D403E, respectively)
(Fig. 2). Among those detected single nucleotide polymorphisms
(SNPs), only one SNP at position 50 (G50T) is reported in
RiceVarMap SNPs database ( />Prediction of potential subsequence change caused by
nucleotide substitution on protein structure and protein
synthesis rate
The protein structure of OsHKT1 transporter was predicted by

A

B

Fig. 2. Polymorphism in OsHKT1 sequence.
A, Schematic representation of polymorphism locations in the
protein. The position of changes in amino acid are indicated with a star;
MPM, transmembrane-pore loop-transmembrane domain. B,
Nucleotide sequence of OsHKT1 was compared among the cultivars
with the Nipponbare sequence. Non-synonymous substitutions are
indicated in dark grey, and the encoded amino acids are listed above.
Synonymous substitutions are indicated in light grey.
G17V means that glycine is replaced by valine; D403E means that
aspartic acid is replaced by glutamic acid.


PHAM Quynh-Hoa, et al. Polymorphism of OsHKT1 in Rice

337
B

A

Fig. 3. 3D ribbon model of OsHKT1 protein visualized by Discovery studio 4.5 visualizer.
A, 3D ribbon model of Ni-OsHKT1 protein visualized from the top that shows the pore. Four residues in Ser-Gly-Gly-Gly ion selection motif are
labeled and Na+ ion is indicated as a black round circle at the center of the pore. B, Visualization of Ho-OsHKT1 transporter from the side that shows
the zoom in D403E variance position (green) (G17V substitution belongs to the signal peptide so they are not modeled in 3D structure of OsHKT1
transporter).

using PHYRE2 webserver. It showed that OsHKT1 contains
eight potential transmembrane domains and the two
polymorphisms G17V and D403E, were placed in the signal
peptide and the loop of the sixth transmembrane domain,

respectively (Fig. 2-A). The 3D model of OsHKT1 transporter
showed the presence of three glycine residues (Gly243, Gly367
and Gly468) and one serine residue (Ser87) forming a selective
filter for Na+/K+ ion (Fig. 3-A). The 3D structure prediction
showed that the amino acid substitutions caused no influence
on the structure of OsHKT1 protein (Fig. 3). That might be due
to the fact that the substituted amino acids have the same
charge properties and bulk to former amino acids, and also the
position of the altered amino acid did not appear in the filter
region. Our finding is in agreement with previous results
reported by Oomen et al (2012). In that study, the same
polymorphism was also detected in other rice cultivars and the
site-directed mutagenesis on wild-type OsHKT1 protein
indicated that transport activity of variant transporter was not
significantly different to that of wild-type (Oomen et al, 2012).
Although synonymous substitutions did not alter the amino
acid sequence, they can effect on translation efficiency via
codon usage bias (Yu et al, 2015). Among five synonymous
substitutions, the two at position 360 and 1440 caused clear
change in codon usage frequency (Table 1). SNP at position
360 probably increased the translation rate because the usage
frequency of the substituted codon was 3-fold higher than the

former one. However, this substitution appeared in both tolerant
(Nep Non Tre and Nep Oc) and sensitive (Dau An Do and Chiem
Cu) cultivars. In contrast, SNP at position 1440 was only present
in the tolerant cultivar Nep Non Tre, and probably reduced the
translation rate. With our current data, it is quite difficult to
point out the relationship between nucleotide polymorphism in
the coding region of OsHKT1 and the salt tolerance level of the

investigated rice cultivars. It might be helpful to explore the
nucleotide polymorphism in the promoter region of OsHKT1,
which plays role in the regulation of gene expression.

CONCLUSIONS
We have successfully amplified and sequenced the OsHKT1
from eight Vietnamese rice cultivars and Nipponbare (control).
Our data revealed seven single nucleotide substitutions
compared to the reference sequence of the rice database. From
these, two substitutions led to amino acid substitutions which
occurred in salt tolerance cultivar Hom Rau. Thus, it is
deserved to further research on the functional role of the
detected non-synonymous substitutions. Based on the
topological model of OsHKT1 protein, the two detected aminoacid variants were placed in signal peptide and the loop of the
sixth transmembrane, respectively. Our data suggests that they
probably do not have a significant impact on the structure of
OsHKT1 transporter. Furthermore, among the different

Table 1. Changes in codon usage bias by nucleotide substitutions in nine rice cultivars.
Position of
Former Usage frequency of Substituted
Usage frequency of Amino
Cultivar
nucleotide variance codon former codon (‰)
substituted codon (‰) acid
codon
360
CUA
7.7
CUG

21.0
Leu Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
645
UCA
12.4
UCG
12.3
Leu Chiem Cu
708
ACA
11.6
ACG
11.4
Thr Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
744
ACG
11.4
ACA
11.6
Thr Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
1440
AAG
32.3
CAA
13.5
Lys Nep Non Tre
Values in bold meant that the substituted nucleotide caused an increase in codon usage frequence.


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Rice Science, Vol. 23, No. 6, 2016

investigated rice cultivars, two out of five detected synonymous
substitutions showed an effect on the protein synthesis rate in
terms of codon frequent usage. However, a correlation with salt
tolerance level was not found.

ACKNOWLEDGEMENTS
This work was financially supported by the Vietnam National
University, Hanoi, Vietnam (Grant No. QG.14.22). Many
thanks to Dr. SANG Nguyen Van for his helpful suggestions and
comments, and to Dr. ANTONIO Carla for her great help in
English improvement.

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