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Legume Genomics and Genetics (online), 2010, Vol. 1, No.5, 24-29
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Figure 4 Phylogenetic tree analysis of AhAQ1 and PIP-type pro-
teins in other plants
Note: The tree was constructed by neighbor-joining method with
MEGA (ver 3.1) program; Branch numbers represent per-
centage of bootstrap values in 1000 sampling replicates and the
scale indicates branch length; The corresponding proteins are
listed as follows: maize, ZmPIP1.2 (AAD29676), ZmPIP2.1 (AAK
26758), barley, HvPIP1.1 (BAF41978), HvPIP2.1 (BAA23744),
Arabidopsis
, AtPIP1.1 (X75881), AtPIP2.3 (D13254), Brassica,
BoPIP1b1 (AAG23179), BoPIP3 (AAG30607), olive, OePIP1.1
(DQ202708), Oe-PIP2.1 (DQ202709), tobacco, NtPIP1.1 (AF4
40271) and NtPIP2.1 (AF440272)
Figure 5 Tissue Expression pattern of
AhAQ1
under salt stress.
Note: A~C:
AhAQ1
expression in roots, stems and leaves after
0 h, 1 h, 6 h, 12 h and 24 h treatment with 250 mmol/L NaCl;
18S
rRNA
was used as the loading control of mRNA
plant tolerance to water stress is complex. AhAQ1 in-
duced by salt stress in different tissues of peanut might
confer the membrane permeability to water transport in
water-deficient condition (Yamada et al., 1997).
Peanut is one of the most important oil crops in the
world. Although many aquaporin genes were iden-
tified in different plants, but up to now there was no
report on aquaporins in peanut. Severe salt stress leads
to dramatic suppression of plant growth and develop-
ment and causes a large loss of productivity during re-
cent years. Our analysis suggests that the expression
of AhAQ1 might function in plants’ response against
salt stress. Further detailed investigations on this gene
will provide comprehensive information of peanut salt
stress tolerance and will show us a new way for mole-
cular breeding leading to improve stress tolerance of
agricultural crops.
3 Materials and methods
3.1 Plant materials and stress treatments
peanut seeds (
Arachis hypogaea
L. cv. Huayu28) were
sown in sand and soil mixture (1:1), grown in a grow-
th chamber with 16 h of light at 26
℃
, followed by 8 h
of darkness at 22
℃
, which were watered daily. The
plants at 3–leaf-stage were removed from the soil care-
fully to avoid injury and placed into a container with
250 mmol/L NaCl for 24 h. The roots
，
stems and lea-
ves of the treated seedlings were sampled separately
and stored at −80
℃
at once at different time intervals
after treatment.
3.2 RNA isolation and first strand cDNA synthesis
Total RNAs were extracted from various peanut tissues
by using Trizol reagent (Tiangen) according to the
manufacturer's protocol. The RNAs was sequentially
treated with DNase I (Takara) at 37
℃
for 15 min in
order to remove the remaining genomic DNA. The
first strand cDNA was synthesized with 2 μg of puri-
fied total RNAs using the RT-PCR system (Promega)
following the manufacturer's protocol.
3.3 Cloning of
AhAQ1
To amplify the coding region of
AhAQ1
, two primers
were designed. sense primer: 5’-CACACTTACCCAT
CTCATCAC-3’, antisense primer: 5’-CAATAATCA
AGCACTTGCATT-3’. 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, 1 μL cDNA
samples and 0.5 μL Ex-
Taq
polymerase (Takara), and
19 μl double distilled water. PCR was performed on a
DNA amplification machine (Effendorf) for an initial
denaturizing at 95
℃
for 5 min, 28 cycles of dena-
turing at 95
℃
for 30 s, annealing at 55
℃
for 30 s,
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