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*:

g.bayarmaa@num.edu.

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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