33
© 2016 Journal compilation
http://mjbs.num.edu.mn
http://biotaxa.org./mjbs
Volume 14(1-2), 2016
Mongolian Journal of Biological
Sciences
ISSN 1684-3908 (print edition)
ISSN 2225-4994 (online edition)
MJBS
Original Ar� cle
http://dx.doi.org/10.22353/mjbs.2016.14.04
Key words:
Zygophyllum potaninii,
drought stress,
aquaporin, PIP2
Article information:
Received: 16 June 2016
Accepted: 11 Nov. 2016
Published online:
14 Nov. 2016
Correspondence*:
mn
Cite this paper as:
Characterization of an Aquaporin Gene ZpPIP2 from
Zygophyllum potaninii Maxim. (Zygophyllaceae)
Bayarmaa Gun-Aajav*, Munkhbileg Enkhbat and Oyuntsetseg Batlai
Department of Biology, School of Arts and Sciences, National University of Mongolia,
Ulaanbaatar 14201, Mongolia.
Abstract
Zygophyllum potaninii Maxim. is a medicinal plant, distributed in arid regions of
southern Mongolia. Full length of a cDNA clone, which was identifi ed as a stress
induced gene by suppression subtractive hybridization was obtained by 5’RACE-
PCR, and named ZpPIP2 as deduced amino acid sequence shows high homology
to plant aquaporin PIP2. ZpPIP2 is expressed in leaf and stem under normal
conditions, and it is accumulated in the root in response to drought stress in Z.
potaninii.
Bayarmaa, G., Munkhbileg, E. & Oyuntsetseg, B. 2016. Characterization of an
aquaporin gene ZpPIP2 from Zygophyllum potaninii Maxim. (Zygophylaceae).
Mong. J. Biol. Sci., 14(1-2): 33-38.
Introduction
Zygophyllum potaninii Maxim. is a medicinal
plant, widely distributed in arid regions of
southern Mongolia. In Mongolia this species
grows on debris tailings of mountains and hills,
the sandy-pebble bottom of dry river beds, stony
slopes of hills and pebble deserts (Fig. 1) in
Gobi-Altai, Zhungarian Gobi, Trans-Altai Gobi,
and Alashaa Gobi, and has acquired the ability
to adapt to arid environments (Nyambayar et al.,
2011).
Plants, being sessile, have evolved specifi c
acclimation and adaptation mechanisms to
respond to and survive short- and long-term
drought stresses. Analysis of these protective
mechanisms will contribute to our knowledge of
plant tolerance and resistance to stress (Harb et
al., 2010). Although drought responsive genes
have been studied intensively in model plants, it
is important to analyze drought-inducible genes
and their expression in drought-tolerant plants.
Plant respond to environmental stresses such
as drought by the induction of both regulatory
and functional sets of genes and relevant
genes have been identifi ed either by reference
to physiological evidence or by diff erential
screening (Krishnan & Pereira, 2008).
Aquaporins are water channel proteins of
intracellular and plasma membrane that play
a crucial role in plant water relations. Plants
contain a large number of aquaporin isoforms
with distinct cell type- and tissue-specifi c
expression patterns (Johansson et al., 2000).
Presently, 35 aquaporin encoding genes in
Arabidopsis thaliana (Johanson et al., 2001), and
34 members of the rice aquaporin gene family
have been identifi ed (Nguyen et al., 2013). On
Bayarmaa et al. Characterization of aquaporin gene ZpPIP234
the basis of sequence homology, aquaporins
in most plant species can be divided into four
subgroups. The plasma membrane intrinsic
proteins (PIP) (with two phylogenic subgroups,
PIP1 and PIP2), and the tonoplast intrinsic
proteins are the most abundant aquaporins in
the plasma membrane and vacuolar membrane
(Maurel, 2007).
Subtractive hybridization is a powerful
technique that enables researchers to identify
diff erentially regulated genes important for
cellular growth and diff erentiation (Diatchenko
et al., 1996). cDNA libraries prepared from
diff erent tissues exposed to various stress
conditions are valuable tools to obtain the
expressed and stress- regulated genes. This leads
to the identifi cation of genes that have known or
putative functions, and also to the discovery of
novel genes (Liu et al., 2013).
The expression of genes that is diff erentially
regulated during drought stress in Z. potaninii
was screened by suppression subtractive
hybridization (Bayarmaa et al., in preparation).
We performed forward and reverse subtraction
in order to identify genes that are up and down
regulated during drought stress, respectively. In
this study, a cDNA clone obtained from forward
subtractive hybridization that shows homology to
plant aquaporin PIP2 was studied. A full length
cDNA of ZpPIP2 was obtained and diff erential
expression in response to drought stress was
analyzed by RT-PCR.
Materials and Methods
Plant material. Z. potaninii seeds were
collected in 2011 in Shar Hulsny Bayanburd,
Bayankhongor aimag, Mongolia and kept at
4oC. The seeds were surface sterilized with 70 %
ethanol for 30 seconds, 4% sodium hypochloride
for 5 min, and rinsed three times with sterile
distilled water. Z. potaninii plants grown for 30
days under normal conditions were transferred
to MS media containing 200 mM mannitol
and incubated for 48 hours for drought stress
induction.
RNA isolation. Total RNA was isolated from
control (not stressed) and stress induced Z.
potaninii plants using Trizol (Life Technologies,
Ambion RNA). 100 mg of plant material was
homogenized with 1 ml of Trizol at room
temperature, incubated for 5 min at room
temperature. 0.2 ml of chloroform was added
to homogenized sample, mixed for 15 sec and
then centrifuged at 12000g, for 15 min, at 4°C.
Aqueous phase was transferred to new tube and
equal volume of isopropanol was added, and
then incubated for 10 min at room temperature,
centrifuged as above, washed with 75% ethanol
and dissolved in 50 μl of RNase free water.
5`RACE-PCR. First strand cDNA was
synthesized from 5 mg total RNA using
SMARTer® RACE 5’/3’ Kit (Invitrogen). 5`-
RACE and 3`-RACE ready cDNAs are obtained
using SMARTScribe Reverse transcriptase at
42oC for 90 minutes and kept at -20oC. 5`-RACE
was performed at following condition: 25 cycles
of denaturation at 94oC for 30 sec, annealing at
65oC for 30 sec and extension at 72oC for 3 min.
Nested PCR conditions were as following: 35
cycles of denaturation at 94oC 30 sec, annealing
at 55oC 30 sec and extension at 72oC for 1 min
with fi nal extension 72oC for 8 min.
RT-PCR analysis. RT-PCR was performed
with gene specifi c primers using One Step
All-in-one RT-PCR premix (MBiotech,
Figure 1. Zygophyllum potaninii. Dzungarian Gobi near Bulgan soum, Khovd aimag.
35Mongolian Journal of Biological Sciences 2016 Vol. 14 (1-2)
Korea). 1.0 μg total RNA was used for reverse
transcription and RT-PCR conditions were:
reverse transcription at 50°C for 30 min, RT
inactivation and pre-denaturation at 96°C for
3 min, followed by 35 cycles of denaturation
at 94oC for 30 sec, annealing at 53oC for 30
sec, and extension at 72oC for 30 sec., with
fi nal extension at 72oC for 7 min. For RT-
PCR following primers were used: F33F (5’-
GCCTGGGATGACCATTGGAT-3’), F33R (5’-
CTTTTGGAAAGACTTGTCACCA-3’). The
PCR products were visualized by 1.5% agarose
gel electrophoresis.
Results and Discussion
The expression of genes that is diff erentially
regulated during drought stress in Z. potaninii
was screened by suppression subtractive
hybridization, and F33 cDNA of length 263
bp was identifi ed from forward subtractive
hybridization (Bayarmaa et al., in preparation).
Full length of F33 was obtained by 5’RACE and
predicted protein encodes 245 amino acids (Fig.
1 ATGGCTAAGGACATTGAAGTTGTCGAGCGTGGAGAGTTCTCAGGG
M A K D I E V V E R G E F S G
46 AAGGACTACACTGACCCACCTCCAGCACCATTTATTGACATGGAT
K D Y T D P P P A P F I D M D
91 GAACTGACCAAGTGGTCATTTTACAGAGCTCTTATTGCAGAGTTC
E L T K W S F Y R A L I A E F
136 ATAGCCACCTTATTGTTCCTATATGTTACTGTTTTGACTGTGATA
I A T L L F L Y V T V L T V I
181 GGTTACAAAGTTCAGACTGATGTCAACGCTGGTGGAGATGACTGT
G Y K V Q T D V N A G G D D C
226 GGTGGTGTTGGTATTCTTGGTATTGCTTGGGCCTTTGGGGGCATG
G G V G I L G I A W A F G G M
271 ATCTTCATTCTTGTTTACTGCACAGCTGGTATTTCGGGAGGACAT
I F I L V Y C T A G I S G G H
316 ATCAACCCGGCTGTGACCTTTGGGCTATTCTTGGCAAGAAAAGTG
I N P A V T F G L F L A R K V
361 TCTCTCACCAGGGCAGTGTTGTACATGGTTGCACAGTGCTTGGGA
S L T R A V L Y M V A Q C L G
406 GCAATCTGCGGTTGTGGGCTTGTGAAGGGATTCCAGCAGGCTTAC
A I C G C G L V K G F Q Q A Y
451 TACGATAGGTATGGTGGAGGGGCAAACATGATGGCTGATGGTTAC
Y D R Y G G G A N M M A D G Y
496 AATAATGGCACTGGATTGGGAGCTGAGATCATTGGTACCTTTGTT
N N G T G L G A E I I G T F V
541 CTTGTTTACACTGTCTTATCTGCTACTGATCCTAAAAGGAGTGCC
L V Y T V L S A T D P K R S A
586 AGAGACTCCCATGTTCCTGTATTGGCTCCACTTCCAATTGGGTTC
R D S H V P V L A P L P I G F
631 GCTGTGTTCATGGTACACCTTGCTACTATTCCAATTACGGGTACT
A V F M V H L A T I P I T G T
676 GGTATTAACCCTGCAAGAAGTTTTGGGGCAGCTGTTATCTACAAC
G I N P A R S F G A A V I Y N
721 AATGAAAAAGCCTGGGATGACCATTGGATTTTCTGGGTTGGACCT
N E K A W D D H W I F W V G P
766 TTCATTGGGGCACTTGCTGCTGCATTCTATCACCAGTTCATCCTG
F I G A L A A A F Y H Q F I L
811 AGGGCAGCAGCCATCAAGGCTCTTGGTTCATTCAGGAGCAACAAC
R A A A I K A L G S F R S N N
856 TAAGCATTCTCCTTCATGATTCATTTTTTAATGGTGACAAGTCTT
*
911 TCCAAAAGAATAATAGCAATTAAAAGTCTCGGTTT
Figure 2. ZpPIP2 cDNA nucleotide and deduced amino acid sequence. Nucleotide sequences obtained by
5`RACE-PCR are underlined. Putative start and stop codons are shown in bold letters.
Bayarmaa et al. Characterization of aquaporin gene ZpPIP236
TM1
AtPIP2;2 MAKDVE--G-PEGFQTRDYEDPPPTPFFDADELTKWSLYRAVIAEFVATLLFLYITVLTV 57
AtPIP2;1 MAKDVEAVP-GEGFQTRDYQDPPPAPFIDGAELKKWSFYRAVIAEFVATLLFLYITVLTV 59
AtPIP2;4 MAKDLDVNESGP-PAARDYKDPPPAPFFDMEELRKWPLYRAVIAEFVATLLFLYVSILTV 59
ZpPIP2 MAKDIEVVE-RGEFSGKDYTDPPPAPFIDMDELTKWSFYRALIAEFIATLLFLYVTVLTV 59
ThPIP2;7 MAKDVEVQESAPEFTAKDYTDPPPAPLIDPEELTKWSFYRALIAEFIATLLFLYVTVLTV 60
****:: :** ****:*::* ** ** :***:****:*******:::***
TM2
AtPIP2;2 IGYKIQSDTKAGGVDCGGVGILGIAWAFGGMIFILVYCTAGISGGHINPAVTFGLFLARK 117
AtPIP2;1 IGYKIQSDTDAGGVDCGGVGILGIAWAFGGMIFILVYCTAGISGGHINPAVTFGLFLARK 119
AtPIP2;4 IGYKAQTDATAGGVDCGGVGILGIAWAFGGMIFVLVYCTAGISGGHINPAVTVGLFLARK 119
ZpPIP2 IGYKVQTDVNAGGDDCGGVGILGIAWAFGGMIFILVYCTAGISGGHINPAVTFGLFLARK 119
ThPIP2;7 IGYKVQSDTKAGGDDCGGVGILGIAWAFGGMIFILVYCTAGISGGHINPAVTFGLFLARK 120
**** *:*. *** *******************:******************.*******
TM3 TM4
AtPIP2;2 VSLIRAVLYMVAQCLGAICGVGFVKAFQSSYYDRYGGGANSLADGYNTGTGLAAEIIGTF 177
AtPIP2;1 VSLPRALLYIIAQCLGAICGVGFVKAFQSSYYTRYGGGANSLADGYSTGTGLAAEIIGTF 179
AtPIP2;4 VSLVRTVLYIVAQCLGAICGCGFVKAFQSSYYTRYGGGANELADGYNKGTGLGAEIIGTF 179
ZpPIP2 VSLTRAVLYMVAQCLGAICGCGLVKGFQQAYYDRYGGGANMMADGYNNGTGLGAEIIGTF 179
ThPIP2;7 VSLVRALLYMVAQCLGAICGCGLVKAFQKSYYNRYGGGANVLASGYNKGTGLGAEIIGTF 180
*** *::**::********* *:**.**.:** ******* :*.**..****.*******
TM5
AtPIP2;2 VLVYTVFSATDPKRNARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPARSFGAAVIY 237
AtPIP2;1 VLVYTVFSATDPKRSARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPARSFGAAVIY 239
AtPIP2;4 VLVYTVFSATDPKRNARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPARSFGAAVIY 239
ZpPIP2 VLVYTVLSATDPKRSARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPARSFGAAVIY 239
ThPIP2;7 VLVYTVFAATDPKRNARDSHVPVLAPLPIGFAVFMVHLATIPITGTGINPARSFGAAVIY 240
******::******.*********************************************
TM6
AtPIP2;2 NKSKPWDDHWIFWVGPFIGAAIAAFYHQFVLRASGSKSLGSFRSAANV---- 285
AtPIP2;1 NKSKPWDDHWIFWVGPFIGAAIAAFYHQFVLRASGSKSLGSFRSAANV---- 287
AtPIP2;4 NNEKAWDDQWIFWVGPMIGAAAAAFYHQFILRAAAIKALGSFGSFGSFRSFA 291
ZpPIP2 NNEKAWDDHWIFWVGPFIGALAAAFYHQFILRAAAIKALGSFRSNN------ 285
ThPIP2;7 NQDKAWDDHWLFWVGPFIGALAAAVYHQYVLRAAAIKALGSFRSNA------ 286
*:.* ***:*:*****:*** **.***::***:. *:**** *
Figure 3. Alignment of deduced amino acid sequence of ZpPIP2 with Arabidopsis thaliana AtPIP2;4
(AT5G60660), AtPIP2;2 (AT2G37170), AtPIP2;1 (AT3G53420) and Tarenaya hassleriana PIP2;7
(XP_010521765.1) aquaporins. Predicted transmembrane domains (TM1-TM6) are shaded in gray (TMHMM
2.0, http://www.cbs.dtu.dk/services/TMHMM/). The Asn-Pro-Ala (NPA) motifs and AEFXXT motif are shown
in bold letters. Sequences were aligned using CLUSTAL Omega program. Asterisk (*) indicates conserved
residues. Colon (:) indicates conservation between groups of strongly similar properties, and period (.) indicates
conservation between groups of weakly similar properties.
Figure 4. ZpPIP2 expression analysis. Reverse transcription was conducte d with 1.0 μg total RNA from leaf,
stem and root tissues. RT-PCR conditions were: reverse transcription at 50°C for 30 min., reverse transcriptase
inactivation and pre-denaturation at 96°C for 3 min., followed by 35 cycles of denaturation at 94oC for 30 sec.,
annealing at 53oC for 30 sec., and extension at 72oC for 30 sec., with fi nal extension at 72oC for 7 min. The PCR
products were visualized by 1.5% agarose gel electrophoresis. Actin was used as a control. M-100 bp marker;
1-leaf, 3-stem and 5-root from control Z. potaninii plant. 2-leaf, 4-stem, 6-root samples from drought stressed Z.
potaninii for 48 hours with 200 mM mannitol.
ZpPIP2
Actin
М 1 2 3 4 5 6bp
200-
700-
37Mongolian Journal of Biological Sciences 2016 Vol. 14 (1-2)
2). Similarity search revealed high homology
to plant aquaporins. The highest homology was
found with Tarenaya hassleriana aquaporin
PIP2-7-like protein with 89% identity over the
entire length.
Aquaporins belong to a highly conserved
family of membrane proteins known as major
intrinsic protein. The common characteristics
of major intrinsic proteins are that it possesses
six alpha helical transmembrane helices
(TM1–TM6), fi ve inter-helical loops (LA-
LE), an AEF (Ala-Glu-Phe) or AEFXXT
motif in the N-terminal domain and two highly
conserved NPA (Asp-Pro-Ala) motifs (Afzal
et al., 2016). Sequence analysis of ZpPIP2
identifi ed six putative transmembrane domains
(Fig. 3). Multiple sequence alignment of
ZpPIP2 deduced amino acid sequence with T.
hassleriana ThPIP2-7 (XP_010521765.1), and
A. thaliana AtPIP2-1 (AT3G53420), AtPIP2;2
(AT2G37170), AtPIP2-4 (AT5G60660)
sequences revealed that it is highly conserved
in transmembrane domains and identifi ed two
highly conserved NPA motif and AEFXXT
motif in TM1, which is shown in bold letters
(Fig. 3). Based on the sequence homology to
plant aquaporin, PIP2, F33 clone was named as
ZpPIP2 for Zygophyllum potaninii PIP2.
Expression of ZpPIP2 in the control and
drought stressed leaf, stem and root mRNAs
were analyzed. Drought stress was induced
by incubating the four weeks old Z. potaninii
for 48 hours on MS media containing 200 mM
mannitol. ZpPIP2 mRNA is accumulated in
leaf and stem, and the expression is not aff ected
by the drought stress. In root, there is no
accumulation of ZpPIP2 mRNA in control plant
and its expression is up-regulated by drought
stress (Fig. 4). Tissue specifi c expression pattern
and diff erential regulation in response to drought
stress of aquaporin PIP genes was analyzed in
some model and agronomical important plants,
such as rice (Sakurai et al., 2005; Nguyen et al.,
2013), Arabidopsis (Jang et al., 2004), barley
(Hove et al., 2015) and coff ee (Santos et al.,
2013). Sakurai et al. (2005) investigated organ-
specifi c expression of rice aquaporin genes,
which identifi ed in the rice genome database,
and higher levels of the mRNA of two aquaporin
PIP genes (OsPIP2;7, OsPIP2;8) were detected
in leaf blades than in roots in the 21 and 56
day old rice plants. The expression profi le of
aquaporin genes in diff erent organs and growth
stages should provide information on their
physiological roles. Also, in rice, four aquaporin
genes were found to be up-regulated by water
defi ciency in the root, suggesting that these genes
might be involved in the regulation of water
uptake from the soil (Nguyen et al., 2013).
The function of aquaporin genes during
water stress is still unclear. In this research,
we identifi ed aquaporin PIP2 cDNA from Z.
potaninii with diff erential expression during
drought stress.
Acknowledgements
The research was funded by the Asia
Research Center at the National University
of Mongolia, and the Korean Foundation for
Advanced Studies.
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