Basic HTML Version
Legume Genomics and Genetics (online), 2010, Vol. 1, No.5, 24-29
http://lgg.sophiapublisher.com
28
extension at 72
℃
for 1 min, and a final extension at
72
℃
for 10 min. The PCR products were run on 1%
agarose gel and purified with Gel xtraction Kit (Taka
ra) according to the manufacturer’s protocol. The puri-
fied products were then cloned into the pMD18-T Easy
vector (Takara) and sequenced (Sangon, Shanghai).
3.4 Sequence analysis
The sequence analysis was performed using BioXM
version 2.6 program. Multiple Sequence alignment ob-
tained using the ClustalX ver 1.8 were displayed by
the GENEDOC. Molecular phylogenies were computed
using MEGA version 3.1. The TM prediction calcu-
lated with the hydrophobic values was completed by
the program TMPRED (http://www.ch.embnet.org/
software/TMPRED_form.html).
3.5 Semi-quantitative RT-PCR analysis
The gene special primers, A1: 5’-ACCCTCCTCTTC
CTCTACATC-3’ and A2: 5’-CAAGCACTGAGCCA
CCATAT-3’ were used for amplifying a fragment of
261 bp. Each reaction system contained 2.5 μL of 10×
PCR buffer with MgCl
2
, 0.5 μL of 10 μmol/L each
primer, 1.0 μL of 20 mmol/L dNTPs, 2 μL cDNA
samples and 0.5 μL Ex-
Taq
polymerase (Takara), and
18 μL double distilled water. The thermal cycle used
was as follows: 95
℃
for 5 min; 28 cycles of 95
℃
for
20 s, 55
℃
for 20 s, 72
℃
for 30 s, and a final 72
℃
for 10 min. As an internal control, a 226 bp PCR
fragment of peanut 18S rRNA gene was amplified
using primers 18S-F: 5’-ATTCCTAGTAAGCGCGA
GTCATCAG-3’ and 18S-R: 5’-CAATGATCCTTCC
GCAGGTTCAC-3’ (Wan et al., 2005). The PCR con-
ditions for amplifying 18S rRNA gene in semi-
quantitative RT-PCR assay are the same as those for
AhAQ1
gene, but the annealing temperature was changed
into 60
℃
. Three replicate PCR amplifications were
performed for eachsample.
Acknowledgements
This work was supported by the National High Technology Research and
Development Program of China (2006AA10A114), the National Key Basic
Research and Development Project of China (2007CB116212).
References
Baiges I., Schōffner A.R., Affenzeller M.J., and Mas A., 2002, Plant
aquaporins, Physiol. Plant, 115(2): 175-182
PMid:12060233
Chaumont F., Barrieu F., Wojcik E., Chrispeels M.J., and Jung R., 2001,
Aquaporins constitute a large and highly divergent protein family in
maize, Plant Physiol., 125(3): 1206-1215
PMid:11244102 PMCid:65601
Chaumont F., Moshelion M., and Daniels M.J., 2005, Regulation of plant
aquaporin activity, Biol. Cell, 97(10): 749-764
PMid:16171457
Johanson U., and Gustavsson S., 2002, A new subfamily of major intrinsic
proteins in plants, Mol. Biol. Evol., 19(4): 456-461 PMid:11919287
Johanson U., Karlsson M., Johansson I., Gustavsson S., Sjõvall S., Fraysse
L., Weig A.R., and Kjellbom P., 2001, The complete set of genes
encoding major intrinsic proteins in Arabidopsis provides a framework
for a new nomenclature for major intrinsic proteins in plants, Plant
Physiol., 126: 1358-1369
PMid:11500536
PMCid:117137
Katsuhara M., Koshio K., Shibasaka M., Hayashi Y., Hayakawa T., and
Kasamo K., 2003, Over-expression of a barley aquaporin increased the
shoot/root ratio and raised salt sensitivity in transgenic rice plants,
Plant and Cell Physiology, 44(12): 1378-1383
PMid:14701933
Kawasaki S., Borchert C., Deyholos M., Wang H., Brazille S., Kawai K.,
Galbraith D., and Bohnert H.J., 2001, Gene expression profiles during
the initial phase of salt stress in rice, The Plant Cell, 13(4): 889-905
PMid:11283343 PMCid:
135538
Lian H.L., Yu X., Ye Q., Ding X.S., Kitagawa Y., Kwak S.S., Su W.A., and
Tang Z.C., 2004, The role of aquaporin RWC3 in drought avoidance in
rice, Plant Cell Physiol., 45(4): 481-489
PMid:15111723
Luu D.T., and Maurel C., 2005, Aquaporins in a challenging environment:
molecular gears for adjusting plant water status, Plant, Cell and
Environment, 28(1): 85-96
Mahdieh M., Mostajeran A., Horie T., and Katsuhara M., 2008, Drought
stress alters water relations and expression of PIP-type aquaporin genes
in
Nicotiana tabacum
plants, Plant Cell Physiol., 49(4): 801-813
PMid:18385163
Maurel C., Reizer J., Schroeder J.I., and Chrispeels M.J., 1993, The
vacuolar membrane protein gamma-TIP creates waterspecific channels
in
Xenopus oocytes
, EMBO J., 12(6): 2241-2247 PMid:8508761
PMCid:413452
Murata K., Mitsuoka K., Hirai T., Walz T., Agre P., Heymann J.B., Engel A.,
and Fujiyoshi Y., 2000, Structural determinants of water permeation
through aquaporin-1, Nature, 407(6804): 599-605
PMid:11034202
Preston G.M., Carroll T.P., Guggino W.B., and Agre P., 1992, Appearance of
water channels in
Xenopus oocytes
expressing red cell CHIP28 protein,
Science, 256(5055): 385-387
PMid:
1373524
Sakurai J., Ishikawa F., Yamaguchi T., Uemura M., and Maeshima M., 2005,
Identification of 33 rice aquaporin genes and analysis of their ex-
pression and function, Plant Cell Physiol., 46(9): 1568-1577
PMid:16033806
Siefritz F., Tyree M.T., Lovisolo C., Schubert A., and Kaldenho R., 2002,
PIP1 plasma membrane aquaporins in tobacco: from cellular effects to
function in plants, The Plant Cell, 14: 869-876
PMCid:150688
van Hoek A.N., and Verkman A.S., 1992, Functional reconstitution of the
isolated erythrocyte water channel CHIP28, J. Biol. Chem., 267(26):
18267-18269 PMid:1526967
Wan X., and Li L., 2005, Molecular cloning and characterization of a
dehydration-inducible cDNA encoding a putative 9-cis-epoxycarotenoid
dioxygenase in
Arachis hygogaea
L., DNA Seq., 16(3): 217-223
PMid:16147878
Wang W., Vinocur B., and Altman A., 2003, Plant responses to drought,
salinity and extreme temperatures: towards genetic engineering for
stress tolerance, Planta, 218(1): 1-14
PMid:14513379
Yamada S., Komori T., Myers P.N., Kuwata S., Kubo T., and Imaseki H.,
1997, Expression of plasma membrane water channel genes under
water stress in Nicotiana excelsior, Plant Cell Physiol., 38(11):
1226-1231 PMid:9435139
Yamaguchi-Shinosaki K., Koizumi M., Urao S., and Shinozaki K., 1992,