全 文 ：棉 花 学 报 Cotton Science 2012，24（5）：435~443
盐胁迫下棉属野生种旱地棉（Gossypium aridum）差异表达基因的
cDNA-AFLP分析
刘章伟，冯 娟，范昕琦，徐 鹏，张香桂，沈新莲 *
（江苏省农业科学院经济作物研究所 / 农业部长江下游棉花与油菜重点实验室，南京 210014）
摘要：以一个耐盐的二倍体野生种旱地棉和对盐敏感的陆地棉栽培种苏棉 12 号为材料，运用 cDNA-AFLP 技
术， 比较两个材料分别在盐胁迫前后的表达情况， 获得了 25 个仅在旱地棉盐胁迫下特异表达的转录片段
（TDF）。 将这些片段进行电子克隆，延伸后的序列进行 BLAST 分析，结果显示 23 个转录片段推断的氨基酸序
列与已知的蛋白同源，这些盐诱导表达的基因主要涉及离子转运、活性氧清除、细胞信号传导、细胞分裂、转录
调节、膜保护、渗透调节等功能蛋白。 从 23 个差异表达的转录片段中选择 9 个进行实时定量 PCR（qRT-PCR）
分析，结果表明这些基因在盐胁迫后表达显著增强，而且多数在 12~24 h 达到高峰。 这些 cDNA 克隆是开展棉
花耐盐性分子基础研究的重要资源。
关键词：棉属； 盐胁迫； cDNA-AFLP；转录片段(TDFs)；实时定量 PCR (qRT-PCR)
中图分类号：S562.035.3 文献标志码：A
文章编号：1002-7807（2012）05-0435-09
cDNA-AFLP Analysis of Differentially-Expressed Genes in Response to Salt Stress in
Gosspyium aridum
LIU Zhang-wei, FENG Juan，FAN Xin-qi, XU Peng, ZHANG Xiang-gui, SHEN Xin-lian*
（Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/ Key Laboratory of Cotton and Rapeseed（Nanjing），
Ministry of Agriculture, Nanjing 210014, China）
Abstract: In this study, we used a salt-tolerant diploid G. aridum species and the salt-sensitive G. hirsutum cultivar Sumian 12 to
investigate differential expression in the presence of salt stress. Using cDNA-AFLP, 25 transcript-derived fragments(TDFs) were
isolated and confirmed to be present only in salt tolerant species G. aridum under salt-stressed conditions. BLAST analysis with
sequences assembled using an in-silico approach demonstrated that 23 of the cDNA fragments had homology to known proteins.
The up-regulated genes were mainly involved in ion transport, ROS scavenging, cell signaling, cell division, transcription regula-
tion, membrane protection, and penetration regulation. Quantitative real-time PCR(qRT-PCR) was used to analyze the expres-
sion patterns of nine of the 23 TDFs at different stages of salt stress in the tolerant species G. aridum. Their expression showed a
significant increase and reached a peak between 12–24 h of stress. These TDFs may serve as useful resources for future re-
search on molecular mechanisms of salt stress response in cotton.
Key word: Gossypium; salt stress; cDNA-AFLP; transcript-derived fragments (TDFs); quantitative real-time PCR (qRT-PCR)
CLC Number: S562.035.3 Document Code: A
Article ID: 1002-7807(2012)05-0435-09
收稿日期：2012-02-17
作者简介：刘章伟（1987-），男，硕士，liuzw_1987@126.com；* 通讯作者，shenxinlian@yahoo.com.cn
基金项目：转基因生物新品种培育重大专项(2011ZX08005-004-002)；江苏省农业科技自主创新基金 (CX(11)1201)
Salinity and drought are the main abiotic
stresses severely limiting plant growth and develop-
ment, and salt tolerance is consequently one of the
important areas of research in plant science. Over
time, plants have evolved unique salt tolerance
mechanisms, which can be broadly classified as ion
homeostasis, osmotic homeostasis, stress damage
control and repair, and growth regulation [1]. Plant
棉 花 学 报 24 卷
exposure to saline environments leads to many
physiological, biochemical, and molecular alter-
ations that involve massive changes in gene expres-
sion profiles. Transcript analysis allows simultane-
ous detection of multiple gene expression changes
and enables the elucidation of salt tolerance mecha-
nisms, with the ultimate goal of improving crop
productivity in saline soil.
Cotton, the world’s leading fiber source, is a
very important global cash crop. Domestic cotton is
an ideal plant for research on the molecular basis of
plant responses to water deficit and salinity, as it is
derived from wild perennials adapted to semi-arid
sub-tropical environments associated with highly
saline soils and experiencing periodic drought and
temperature extremes[2]. The geographical distribu-
tions of wild amphidiploids are on or near the path
of prevailing currents in both the Pacific and At-
lantic oceans. They have floated to their current lo-
cations or been carried there, intentionally or acci-
dentally, by human beings. This long-distance dis-
persal via oceanic drift has played an important role
not only in diversification of major evolutionary
lines, but also in speciation within Gossypium lin-
eages. Salt water tolerance of wild species seeds
enabled this long-distance dispersal [3]. In contrast,
modern cotton cultivars are the result of intensive
selection often carried out under unstressed condi-
tions to produce large amounts of specific fiber
types. Selection has thus unintentionally narrowed
the genetic variability for salt tolerance. In one
study, more than 3000 upland cotton lines were
screened for salt tolerance, but only 3 lines tolerant
to 0.4% NaCl stress during the seedling stage were
identified [4]. To improve salt tolerance in upland
cotton, exploitation of valuable alleles from wild
Gossypium species is therefore needed.
Gossypium aridum is a D genome diploid na-
tive to the Pacific coast of Mexico. Although many
wild Gossypium species are becoming extremely
rare due to the disappearance of in situ area, a re-
cent expedition report from American scientists in-
dicates that G. aridum does not appear to be threat
ened [5]. This favorable situation is most probably
due to its high genetic diversity, which arises from
its resistance to abiotic stresses, including drought
and salinity. Research on seed viability of coastal
and inland forms of Gossypium has shown that the
seeds of most coastal ecotypes are remarkably toler
ant to salt water immersion [6]. In seed germination
experiments in our own lab, we have observed seed
germination rates as high as 92% for G. aridum in
1.2% NaCl solution(data not shown). All this infor-
mation suggests that G. aridum is a valuable species
for understanding salt tolerance mechanisms in
Gossypium and improving salinity resistance in up-
land cotton.
Although several techniques, such as suppres-
sive subtractive hybridization(SSH), cDNA microar-
rays, and RNA sequencing, are currently available
for transcriptome analysis, the PCR-based technique
of cDNA-amplified fragment length polymorphism
(cDNA-AFLP) is still a valuable mRNA fingerprint
method for isolation of differentially-expressed
genes due to its high sensitivity and low cost[7-8]. The
objective of this study was to develop transcripts
that were differentially expressed in G. aridum
leaves in response to salinity stress using cD-
NA-AFLP, and to validate changes in their expres-
sion patterns at different stress stages using real
time PCR(qRT-PCR).
1 Materials and methods
1.1 Plant materials and salinity treatments
The Gossypium wild species G. aridum (D4)
and the upland cotton cultivar Sumian 12 (G. hirsu-
tum) were used for this study. In addition, G. david-
sonii(D3-k)，which belongs to the same subgenome
as G. aridum and has salt tolerance potential[9], was
used as a positive control for cDNA-AFLP analysis.
Young uniform plants that were 20 cm tall were se-
lected and treated with 300 mmol·L-1 NaCl for 24 h.
436
5 期
For qRT-PCR analysis, young G. aridum plants
were treated with 200 mmol·L-1 NaCl. Leaves were
collected at different intervals(unstressed, 1, 3, 6, 12,
24, and 72 h after stress). All tissues were frozen
immediately in liquid nitrogen and stored at -70℃.
1.2 cDNA synthesis and cDNA-AFLP analysis
Total RNA was isolated from frozen tissues us-
ing a cold-acidic phenol method with a modified
extraction buffer (10 mmol·L-1 Tris-HCl, pH 8.0;
25 mmol·L-1, EDTA pH 8.0; 2% CTAB; 2% PVP).
The isolated RNA was precipitated with ethanol,
dissolved in DEPC water, and stored at -70 ℃. Us-
ing a Takara M-MLV RTase cDNA synthesis kit
(Dalian, China), first strand synthesis was carried
out with 20 μg of total RNA, which was then fol-
lowed by second strand synthesis. Second strand
cDNAs were purified by chloroform extraction and
dissolved in 30 μL TE solution to give an approxi-
mate concentration of 100 ng·μL-1. The dou-
ble-stranded cDNA was digested with restriction
enzymes MseI/PstI for 3 h at 37 ℃, and then ligated
to MseI and PstI adapters using T4 DNA ligase
(Takara) for 12 h at 16 ℃ . The adaptor sequences
were as follows: PstI adapter, 5-CTCGTAGACT-
GCGTACATGCA-3, 3-CATCTGACTGT-5; MseI
adapter, 5-GACGATGAGTCCTGAG-3, 3-TAC-
TCAGGACTCAT-5. Ligated products were pream-
plified with the appropriate preamplification primers
(PstI: 5-GACTGCGTACATGCAG-3, MseI: 5-G-
ATGAGTCCTGAGTAAT-3). The pre-amplified
products were then diluted 20-fold and selectively
amplified with 200 primer combinations. About 8
μL of 95 ℃ heat-denatured AFLP product was re-
solved on a 6% denaturing PAGE gel containing 7
mol·L-1 urea, and the resulting bands were visual-
ized with silver staining.
1.3 Isolation of transcript -derived fragment
(TDF)
Polymorphic TDFs, which were verified based
on presence and absence of bands in the two toler-
ant wild species(G. aridum and G. davidsonii) com-
pared against the sensitive upland cotton cultivar
Sumian 12, were cut from the gel with a sharp razor
blade, avoiding any contaminating fragment (s).
The excised bands were eluted in 50 μL of sterile
double distilled water at 95 ℃ for 15 min and hy-
drated overnight at 4 ℃ . About 5 μL of the eluent
was used as a template for re-amplification in a total
volume of 25 μL PCR reaction mixture using the
same set of corresponding selective primers and
PCR conditions as follows: 95 ℃ denaturation for
3 min, followed by 36 cycles at 94 ℃ for 30 s, 56 ℃
for 30 s, and 72 ℃ for 1 min. After the last cycle,
the amplification was extended for 10 min at 72 ℃.
PCR products were resolved on a 2% TAE-agarose
gel; each single band was isolated and eluted using
an AxyGEN gel extraction kit (Axygen Scientific,
USA).
1.4 Cloning, sequencing and sequence anal-
ysis of TDFs
Eluted TDFs were cloned into a plasmid
pTG19-T vector(GENEray, Shanghai) following the
manufacturers protocol. Monoclones were se-
quenced byBeijing Genomics Institute(BGI) (Shang-
hai, China). TDF sequences were analyzed using
NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
For some smaller TDFs ranging from 50 bp to 500
bp long，BLAST results were unsuccesful. We
therefore assembled these sequences from related
Gossypium ESTs using an in silico cloning strategy
and CAP3 software; The assembled cDNA se-
quences were then analyzed and sequence-verified
with G. aridum cDNA. The verified sequences were
re-submitted for BLAST analysis.
1.5 Quantitative reverse transcriptase PCR
(qRT-PCR) analysis
Total RNA was isolated from leaves at various
time intervals up to 72 h of salt stress using the
method described above. Gene-specific primers
were designed using PRIMER3(http://frodo.wi.mit.
edu/primer3/input.htm)(Table 1). For qRT-PCR, as-
says were performed in triplicate on 1 μL aliquots
刘章伟等：盐胁迫下棉属野生种旱地棉（Gossypiumaridum）差异表达基因的 cDNA-AFLP分析 437
棉 花 学 报 24 卷
2 Results
2.1 Identification of salinity-regulated TDFs
A total of 200 primer combinations were used
for cDNA-AFLP analysis. Expression profiles were
compared among the two salt-tolerant wild species
and a sensitive variety under both control conditions
and after 24 h of salt stress based on presence or ab-
sence of bands. Fragments greater than 50 bp long
were considered for analysis. On average, about 40
clear bands(TDFs) were generated with each primer
combination, yielding a total of about 8000 bands.
A total of 25 TDFs were isolated from the sil-
ver-stained gels based on their presence in G.
aridum and G. davidsonii under salt stressed condi-
tions and absence under unstressed conditions, or,
for G. hirstrum Sumian 12, their absence under both
stressed and unstressed conditions(Fig. 1). The ex-
periment for these 25 TDFs was repeated twice for
reproducibility and verification of the data.
2.2 Sequence extension and analysis of TDF
clones
All 25 identified TDFs were sequenced. The
BLASTN program failed to locate homologous EST
sequences or unigenes of some of the small TDFs in
GenBank. An in silico strategy was thus used to as-
semble original TDFs with homologous Gossypium
ESTs. Most were greatly extended, and some
full-length genes were identified (TDF3 , TDF4 ,
TDF18, TDF22, TDF23, TDF24, and TDF25).
To verify consistency between assembled se-
quence s and known G. aridum sequences, primers
of each cDNA dilution using SYBR Green Master
Mix(Applied Biosystems, Dalian) on an ABI 7500
sequencedetection system(AppliedBiosystems). PCR
was performed for each sample in triplicate. UBQ, a
constitutive protein, was used as an internal control
to normalize all data (Forward primer: 5-GAAG-
GCATTCCACCTGACCAAC-3; Reverse primer:
5-CTTGACCTTCTTCTTCTTGTGCTTG-3). The
relative quantification method (ΔΔCT) was used for
quantitative evaluation of variation between repli-
cates.
Table 1 Primers used for qRT-PCR
TDF Forward primer (5’-3’) Reverse Primer (5’-3’) Size of amplicon /bp
TDF2 CGCTTACAAGACCAGTGGTGAGGAT CGGGACTAGATGTTTCTGCAGCATT 296
TDF3 GCAGACTCGGTCTCGGTATGCTTAT CCACTGCAGCAACATTGACTCTTAG 208
TDF8 GATTGTTCGAGCATCCGAGGACGAT ACTGCTGCTGCAGCTGAATGTTACG 257
TDF10 CCAGACCAGCAGAGGCTTATCTTTG AGCTGGTTGGTGTGTCCACACTTCT 245
TDF11 CCTTGCTGAGCTTGGCCTTATGCAA CCAGGGCATTACGATCTGGACTTGA 263
TDF13 GGATCTCAGTACCTATACTAGATCG ATCGAGTGGGAGACCATCCATGTAT 167
TDF18 GCTGTTGTGTCCATGCCTGCAGTAA TGTTTCCATTGGAGGAGGAGGCTTT 223
TDF21 GAAGACAATGGAGACCGCAGAAGCA GGCATCCGCCATTCCAAAGGTATGT 251
TDF24 GCTTAGCAATAGAATCGGCCTCACT GCTGCACGGTTCAACATGCCAGAAA 243
438
5 期
from TDFs 2, 3, 8, 10, 11, 13, 18, 21, and 24 were
designed based on assembled sequences and used to
amplify corresponding sequences with G. aridum
cDNA. Amplified products were sequenced as de-
scribed above. The resulting match rates between
assembled and verified sequences ranged from
90.3% to 100% (TDF2 95.58% , TDF3 99.26% ,
TDF8 97.16% , TDF10 95.24% , TDF11 97.74% ,
TDF13 90.3%, TDF18 98.37%, TDF21 100%, and
TDF24 100%), indicating the in silico cloning strat-
egy was an effective way to extend target sequences
in Gossypium.
An NCBI database search revealed that 23 cD-
NA fragments had homology to known proteins,
with their corresponding genes involved in respons-
es to various stresses, such as drought, cold, and
salinity(Table 2). Most of the TDFs and assembled
fragments of the homologous genes were found to
be involved in functions such as ion transport
(TDF2), ROS scavenging (TDF23), cell signaling
(TDF1, 7, 15, 25), cell division(TDF11, 17), tran-
scription regulation (TDF19), protein metabolism
Table 2 Nucleotide-homology of TDFs with known gene sequences in GenBank using the BLASTN algorithm
Note: All data are BLASTN scores except those marked with * which are tBLASTx scores, full-length ORF obtained
through assembling marked by #.
TDF Originalsize/bp
Assembled
size/bp Homologous gene
GenBank Accession
Number E-value Similarity
TDF1* 158 164 Phospholipase D (PLD) (Gossypium hirsutum) GU569955.1 9E-14 100%
TDF2 133 1475 MscS 2 like protein (Arabidopsis thaliana ) XP_002533682 1.7E-151 69.5%
TDF3 # 153 1311 Threonine aldolase (Populus trichocarpa) XP_002325334 2.7E-161 88.1%
TDF6 95 95 No significant similarity
TDF7* 175 660 WD-repeat protein GhTTG3 gene (Gossypium hirsutum) AF530911.1 3E-05 41%
TDF8 131 1035 Beta-fructofuranosidase/hydrolase (Citrus sinensis) BAF34362 7.8E-147 85.75%
TDF9 79 900 No significant similarity
TDF10 111 258 Ubiquitin fusion protein (Populus trichocarpa) ABK93568 1.0E-40 95.6%
TDF11 110 931 Cyclin B1 (Populus trichocarpa) XP_002314016 3.6EE-81 80.75%
TDF14 131 992 Carbonic anhydrase (Gossypium hirsutum) AAM22683 8.0E-154 82.4%
TDF15 64 1423 Stress-induced receptor-like kinase (Vitis vinifera) XP_002268775 9.2E-133 79.05%
TDF16 89 204 Early fruit mRNA (Populus trichocarpa) XP_002324260 1.8E-5 77.75%
TDF18# 272 1757 Cytochrome P450 (Populus trichocarpa) XP_002308860 0 92.15%
TDF19 202 1860 Transcription factor LHY(Populus nigra) BAH09382.1 3E-134 65%
TDF20 301 613 Alcohol oxidase (Populus trichocarpa) XP_002314488 6.0E-81 80.35%
TDF22# 125 684 Copper binding protein 6 (Gossypium hirsutum) ADV57641 1E-89 100%
TDF23# 169 1159 Beta carotene hydroxylase (Vitis vinifera) XP_002273581 2.8E-87 80.65%
TDF4 # 131 1863 3-chloroallyl aldehyde dehydrogenase/
oxidoreductase (Corylus heterophylla)
ADW80331 0 88.8%
TDF5 91 1869 4-hydroxy-3-methylbut-2-en-1-yl diphosphate
synthase (Vitis vinifera)
XP_002285130 0 95.15%
TDF12 106 1708 Serine-type endopeptidase/ serine-type peptidase
(Ricinus communis)
XP_002515283 0 86.4%
TDF13 160 1050 ABC transporter B family member 26 (TAP1)
(Arabidopsis thaliana)
NM_105729.5 1E-96 73%
TDF17 54 192 Transitional endoplasmic reticulum ATPase
(Vitis vinifera)
XP_002282146 2.6E-42 90.25%
TDF21 139 411 Late embryogenesis abundant protein D-7
(Gossypium hirsutum)
CAA31589 2.4E-15 74.22%
TDF24# 121 765 RNA recognition motif-containing protein
（Populus trichocarpa）
XP_002323849 1.3E-78 71.8%
TDF25# 139 1950 Armadillo/beta-catenin-like repeat-containing
protein (Arabidopsis thaliana)
NP_001190236.1 1E-122 77.5%
刘章伟等：盐胁迫下棉属野生种旱地棉（Gossypiumaridum）差异表达基因的 cDNA-AFLP分析 439
棉 花 学 报 24 卷
TDF2 was homologous to Arabidopsis MscS-
like genes AtMSL2 and AtMSL3. MscS-like genes
are responsible for perceiving mechanical stimuli
such as touch and osmotic pressure. In one study,
Arabidopsis insertion mutants MSL3 and MSL2
showed abnormalities in plastid size and shape, and
MSL2-GFP and MSL3-GFP, along with the plastid
division protein AtMinE, were found to be localized
to discrete foci on the plastid envelope[10]. Our qRT-
PCR results showed that TDF2 expression increased
significantly, peaking at 12-24 h before declining.
TDF3 showed similarity with threonine al-
dolase, which is an important enzyme splitting thre-
onine into glycine and acetaldehyde. An investiga-
tion in Arabidopsis revealed that N-methyltrans-
ferase genes catalyze the transformation of glycine
to betaine via a three-step methylation process, and
demonstrated that glycine accumulation may play a
role in plant salt stress response [11]. The qRT-PCR
results in our study revealed that TDF3 expression
was greatly induced after 12 h salt stress, with
17-fold overexpression at 24 h salt stress compared
with the untreated control.
TDF8 was identified as soluble invertase,
which can convert sucrose into glucose and fruc-
tose, used not only for plant growth and
metabolism, but also for maintenance of cell osmot-
ic pressure. Under drought stress during early devel
(TDF3, 5, 7, 10, 12), protein and membrane protec-
tion(TDF16, 21, 22, 24), and penetration regulation
(TDF4, 8, 13, 18, 20). Only two TDFs(TDF6 and
TDF9) had no significant similarity with known se-
quences or mapped to unclassified proteins with un-
known function(Table 2).
2.3 TDF expression analysis
Of the 25 sequences, 9 assembled TDFs that
had been confirmed with G. aridum cDNA were
further analyzed for their expression patterns and
their relative abundance in G. aridum leaves at dif-
ferent stress stages(0, 1, 3, 6, 12, 24, 48, and 72 h)
was quantitatively assessed. The quantitative ex-
pression patterns for all 9 TDFs were similar in both
cDNA-AFLP and qRT-PCR experiments (up-regu-
lated in leaves under salt stress). The expression of
most TDFs reached a peak at 12-24 h(Fig. 2).
440
5 期
opment, repression of the soluble invertase gene
leads to ovary abortion in Zea mays[12]. In our study,
this gene was up-regulated 4.4-fold at 12 h of salt
stress.
TDF10 was homologous to the Arabidopsis u-
biquitin extension protein gene. Ubiquitination
takes place in diverse cellular processes in higher
plants, including differentiation, cell division, hor-
monal responses, and biotic and abiotic stress re-
sponses. In one study, pub22 and pub23 knockout
mutants were more drought-tolerant than wild-type
plants, with a pub22 pub23 double mutant display
ing even greater drought tolerance[13]. These results
indicated that PUB22 and PUB23 function as nega-
tive regulators in water stress response. Using dif-
ferential hybridization technology, the Arabidopsis
ubiquitin extension protein was one of three ERD
(early-responsive to dehydration) proteins strongly
induced by dehydration stress, but it was not signifi-
cantly affected by ABA[14]. Plant ubiquitin extension
protein thus appears to play an important role in de-
hydration stress through an ABA-independent path-
way. According to our qRT-PCR results, it was
3-fold overexpressed at 24 h of salt stress.
TDF11 was homologous to the cyclin B gene,
which is involved in cell mitosis. Overexpression of
the OsMYB3R-2 gene, which specifically promotes
the OsCycB1 gene, has been shown to increase pro-
line content and cold tolerance in rice[15]. The CycB
protein is thus seen to be important in plant stress
resistance. We found that TDF11 expression was
rapidly altered in response to salt stress, with 4-fold
overexpression at 6 h.
TDF13 corresponded to the ATP-binding cas
sette transporter protein gene, and, according to
BLAST results, had highest homology with the Ara-
bidopsis thaliana ABC transporter B family member
26 gene. Related research has shown that this type
of plant protein is transcriptionally active and en-
codes forward-orientation half-molecule proteins
that localize to the membranes bounding small in-
tra-cellular compartments. The precise functions of
this type of protein remain unclear[16]. The AtABCB27
(TAP2) gene is involved in plant aluminum toler-
ance, suggesting the protein may be associated with
plant abiotic stress[17]. In our study, TDF-13 expres-
sion increased significantly under salt stress. The
qRT-PCR results revealed an increase after 3 h,
which reached its highest level at 24 h of salt stress.
TDF18 was homologous to the CYP98A3 sub-
class of cytochrome P450. Plant P450s are involved
in a variety of metabolic pathways, including plant
hormone and defensive secondary metabolite (e.g.,
phytoalexin) biosynthesis and detoxification of ex-
ogenous chemicals, such as herbicides. Although
functional information during stress is unavailable
for this protein, a previous study indicated that cy-
tochrome P450 mono-oxygenases were up-regulat-
ed under salt stress in Arabidopsis, rice, and Mesem
bryanthemum(ice plant) [18]. Our qRT-PCR results
showed that TDF18 expression in G. aridum was
significantly increased at 12-24 h of salt stress.
TDF21 was related to LEA(late embryogenesis
abundant) protein, which is well known for helping
plants resist salt stress. Genetically-engineered rice
plants constitutively overexpressing the barley LEA
gene(HVA1) driven by a rice actin-1 promoter gen-
erally showed better salt tolerance (at 200 mmol·L-1
NaCl) compared with wild-type plants[19]. Four LEA
genes, Td 29b, Td11, Td16, and Td 25a , were
strongly induced (1000- to 9000-fold) under salt
stress[20]. In our study, TDF21 was strongly expressed
at 12 h of salt stress, but dramatically declined
thereafter.
TDF24 was homologous to a nuclear acid
binding protein. Its counterpart in Arabidopsis had
the highest coexpression potential with the SOS1
gene （http://atted.jp/data/locus/At1g14340.shtml),
an important gene in Arabidopsis salt tolerance [21].
We observed that TDF24 expression increased
steadily after stress, but declined at 48 h of salt
stress.
刘章伟等：盐胁迫下棉属野生种旱地棉（Gossypiumaridum）差异表达基因的 cDNA-AFLP分析 441
棉 花 学 报 24 卷
3 Discussion
Unlike animals, which can move to avoid dif-
ferent stresses, plants must respond and adapt to
stresses in place to survive severe environmental
conditions. During the long course of evolution,
plants have developed a number of mechanisms to
resist these stresses. Cotton is a moderately salt tol-
erant species. Previous studies have revealed some
mechanisms of salt tolerance in cotton. For exam-
ple, the cotton DRE-binding transcription factor
gene (GhDREB) conferred enhanced tolerance to
drought, high salt, and freezing in transgenic wheat[22].
Three ethylene-responsive factors (GhERF2, Gh-
ERF3, and GhERF6) were induced by ethylene, ab
scisic acid, salt, cold, and drought [23]. A stress re-
sponsive group C MAPK gene(GhMPK2) in cotton
(G. hirsutum) was induced by abscisic acid (ABA)
and abiotic stresses such as NaCl, PEG, and dehy-
dration [24]. Expression of an Arabidopsis vacuolar
H+-pyrophosphatase gene(AVP1) in cotton improved
drought and salt tolerance and increased fiber yield[25].
A cDNA clone designated Gossypium hirsutum zinc
finger protein 1(GhZFP1), which was isolated from
a salt-induced G. hirsutum cDNA library using dif-
ferential hybridization screening, encodes a novel
CCCH-type zinc finger protein. GhZFP1-overex-
pressing transgenic tobacco plants showed en-
hanced salt stress tolerance and resistance to Rhi
zoctonia solani [26]. GhCyp1, an immunophilin pro-
tein cloned from Gossypium hirsutum using rapid
amplification of cDNA ends, conferred higher toler-
ance to salt stress and Pseudomonas syringae pv.
tabaci infection in overexpressed transgenic tobacco
compared with control plants[27]. All of these results
indicate that many kinds of cotton salt stress genes
have important regulatory roles in responses to both
abiotic and biotic stress.
Gossypium wild species that often experienced
periodic drought and extreme temperature and
dispersed their seeds by seawater flow have evolved
a unique stress tolerance system. Exploiting
differentially expressed genes derived from
salt-tolerant Gossypium species should further
enhance our understanding of salt tolerance
mechanisms in cotton. In this study, we investigated
the salt and drought tolerant wild species G. aridum.
Using cDNA-AFLP, we obtained an expression
profile of early response transcripts following
salinity stress. Up-regulated genes were mainly
involved in ion transport, ROS scavenging, cell
signaling, cell division, transcription regulation,
protein metabolism, protein and membrane
protection, and penetration regulation. These
newly-identified TDFs should be useful resources
for future research on saline stress in cotton.
This work resulted in the identification of
different cDNA fragments induced by salt stress in
G. aridum leaves. Some of the cDNAs showed
homology with known proteins involved in various
stress responses, including LEA(TDF-21), β-OHASE
(TDF-23), and BADH (TDF-4). For homologous
genes of some of the other TDFs, however, no such
salt tolerance function has been discovered in other
plants. In Arabidopsis thaliana, for example, the
TDF2 homologous gene regulates perception of
physical force, response to osmotic pressure
changes, and control of plastid size and shape. The
induction of the gene at an early stage of salt stress,
however, suggests a possible regulatory function in
salinity adaptation, probably by preventing cellular
damage and establishing a homeostatic environment.
TDF13 corresponded to the ATP-binding cassette
transporter protein TAP gene. The AtABCB27
(TAP2) gene is involved in plant aluminum toler-
ance, suggesting this type of protein may play a role
in plant abiotic stress response[17]. Further research is
necessary to reveal the function of these genes in
cotton.
References:
[1] ZHU J K. Salt and drought stress signal transduction in plants[J].
Annu Rev Plant Biol，2002，53: 247-273.
442
5 期
[2] KOHEL R J. Influence of certain morphological characters on
yield[J]. Cotton Grow Rev，1974，51:281-292.
[3] WENDEL J F，Brubaker C L，Seelanan T. The origin and
evolution of Gossypium [M]//Stewart J McD，Oosterhuis D，
Heitholt J J，et al. Physiology of Cotton.New York: Springer，
2010，1-182010: 1-17.
[4]刘国强，鲁黎明，刘金定．棉花品种资源耐盐性鉴定研究[J]．
作物品种资源，1993(2)：21-22．
LIU Guo-qiang，Lu Li-ming，Liu Jin-ding. Study on salt tolerance
for cotton germplasm[J]. China Seeds，1993(2): 21-22.
[5] ULLOA M，Stewart J M，Garcia-C E A，et al. Cotton genetic re-
sources in the western states of Mexico: in situ conservation sta-
tus and germplasm collection for ex situ preservation[J]. Genetic
Resources and Crop Evolution，2006，53: 653-668.
[6] STEPHENS S G. Salt water tolerance of seeds of Gossypium
species as a possible factor in seed dispersal[J]. The American
Naturalist，1958，92 (863): 83-92.
[7] BAISAKH N，Subudhi P K，Parami N P. cDNA-AFLP analysis
reveals differential gene expression in response to salt stress in a
halophyte Spartina alterniflora Loisel[J]. Plant Science，2006，
170: 1141-1149.
[8] JAYARAMAN A，Puranik S，Rai N K，et al. cDNA-AFLP Anal-
ysis reveals differential gene expression in response to salt stress
in foxtail millet (Setaria italica L.)[J]. Mol Biotechnol，2008，40:
241-251.
[9] SUN Y Q，Zhang X L，Huang C，et al. Somatic embryogenesis
and plant regeneration from different wild diploid cotton
(Gossypium) species[J]. Plant Cell Rep，2006，25: 289-296..
[10] HASWELL E S，Meyerowitz E M. MscS-like proteins control
plastid size and shape in Arabidopsis thaliana[J]. Current Biolo-
gy，2006，16: 1-11.
[11] WADITEE R，Bhuiyan M N H，Rai V，et al. Genes for direct
methylation of glycine provide high levels of glycinebetaine
and abiotic-stress tolerance in Synechococcus and Arabidopsis
[J]. Proc Natl Acad Sci USA，2005，102: 5，1318-1323.
[12] ANDERSEN M N，Asch F，Wu Y，et al. Soluble invertase ex-
pression is an early target of drought stress during the critical，
abortion sensitive phase of young ovary development in maize
[J]. Plant Physiol，2002，130: 591-604.
[13] CHO S K，Ryu M Y，Song C，et al. Arabidopsis PUB22 and
PUB23 are homologous U-box E3 ubiquitin ligases that play
combinatory roles in response to drought stress[J]. Plant Cell，
2008，20(7): 1899-1914．
[14] KIYOSUE T，Yamaguchi-Shinozaki K，Shinozaki K. Cloning
of cDNAs for genes that are early-responsive to dehydration
stress(ERDs) in Arabidopsis thaliana L.: identification of three
ERDs as HSP cognate genes[J]. Plant Mol Biol，1994，25(5): 791-
798.
[15] MA Q B，Dai X Y，Xu Y Y，et al. Enhanced tolerance to chill-
ing stress in OsMYB3R-2 transgenic rice is mediated by alter-
ation in cell cycle and ectopic expression of stress genes [J].
Plant Physiol，2009，150: 244-256.
[16] REA P A. Plant ATP-binding cassette transporters[J]. Annu Rev
Plant Biol，2007，58，347-375.
[17] LARSEN P B. Arabidopsis ALS1 encodes a root tip and stele
localized half type ABC transporter required for root growth in
analuminiumtoxicenvironment[J].Planta，2006，225:1447-1458.
[18] ABDULRAZZAK N，Pollet B，Ehlting J，et al. A coumaroyl-es-
ter-3-hydroxylase insertion mutant reveals the existence of
nonredundant meta-hydroxylation pathways and essential roles
for phenolic precursors in cell expansion and plant growth [J].
Plant Physiol，2006，140: 30-48.
[19] XU D，Duan X，Wang B，et al. Expression of a late embryogen-
esis abundant protein gene，HVA1，from barley confers toleran-
ce to water deficit and salt stress in transgenic rice[J]. Plant Phys-
iol，1996，110: 249-257.
[20] ALI-BENALI M A，Alary R，Joudrier P，et al. Comparative ex-
pression of five LEA genes during wheat seed development and
in response to abiotic stresses by real-time quantitative RT-PCR
[J]. Biochim Biophy Acta，2005，1730: 56-65.
[21] KATIYAR-AGARWAL S，Zhu J H，Kim K，et al. The plasma
membrane Na+/H+ antiporter SOS1 interacts with RCD1 and
functions in oxidative stress tolerance in Arabidopsis[J]. Proc
Natl Acad Sci USA，2006，103 (49): 18816-18821.
[22] GAO S Q，Chen M，Xia L Q，et al. A cotton(Gossypium hirsu-
tum) DRE binding transcription factor gene，GhDREB，confers
enhanced tolerance to drought，high salt，and freezing stresses
in transgenic wheat[J]. Plant Cell Rep，2009，28(2): 301-311.
[23] JIN L G，Li H，Liu J Y. Molecular characterization of three
ethylene responsive element binding factor genes from cotton
[J]. Journal of Integrative Plant Biology，2010，52 (5): 485-495.
[24] ZHANG L，Xi D M，Li S W，et al. A cotton group C MAP ki-
nase gene，GhMPK2，positively regulates salt and drought toler-
ance in tobacc[J]. Plant Mol Biol，2011，77(1/2): 17-31
[25] PASAPULA V，Shen G X，Kuppu S，et al. Expression of an
Arabidopsis vacuolar H+-pyrophosphatase gene(AVP1) in cot-
ton improves drought- and salt tolerance and increases fibre
yield in the field conditions[J]. Plant Biotechnology Journal，
2011，9: 88-99.
[26] GUO Y H，Yu Y P，Wang D，et al. GhZFP1，a novel
CCCH-type zinc finger protein from cotton，enhances salt stress
tolerance and fungal disease resistance in transgenic tobacco by
interacting with GZIRD21A and GZIPR5[J]. New Phytol，2009，
183(1): 62-75.
[27] ZHU C，Wang Y，Li Y，et al. Overexpression of a cotton cy-
clophilin gene(GhCyp1) in transgenic tobacco plants confers du-
al tolerance to salt stress and Pseudomonas syringae pv. tabaci
infection [J]. Plant Physiol Biochem，2011，49 (11): 1264-1271.
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刘章伟等：盐胁迫下棉属野生种旱地棉（Gossypiumaridum）差异表达基因的 cDNA-AFLP分析 443