全 文 ：Journal of Genetics and Genomics
(Formerly Acta Genetica Sinica)
June 2007, 34(6): 527-535


Received: 2006−09−17; Accepted: 2006−11−22
This work was supported by the National Natural Science Foundation of China (No. 30070485) and Southwest University Initial
Research Foundation Grant to Doctor (No. D200404).
① Corresponding author. E-mail: limy@swu.edu.cn
§ Present address. College of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huai’an, Jiangsu Province.

www.jgenetgenomics.org
Research Article
Cloning of a MADS Box Gene (GhMADS3) from Cotton and
Analysis of Its Homeotic Role in Transgenic Tobacco
Yulong Guo1,2 , Qinlong Zhu1 , Shangyong Zheng2, § , Mingyang Li1,①
1. College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China;
2. Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture, Chongqing 400716, China
Abstract: A MADS box gene (GhMADS3) was cloned from cotton (Gossypium hirsutum L.) based on EST sequences. The pre-
dicted protein sequence of GhMADS3 showed 85%, 73%, and 62% identity with Theobroma cacao TcAG, Antirrhinum majus FAR,
and Arabidopsis thaliana AG, respectively, and was grouped with AG homologues when the full length sequences excluding
N-extensions were compared. GhMADS3 expressed in the wild type cotton flower primarily in stamens and carpels, which was
comparable to AG in Arabidopsis. However, it was not expressed in floral buds of a homeotic cotton variant chv1. Ectopic expres-
sion of GhMADS3 in tobacco (Nicotiana tabacum L.) resulted in flowers with sepal-to-carpel and petal-to-stamen transformation.
The carpelloid first whorl organs, with stigmatic tissue on their upper edges, had a white appearance when compared with the dark
green color of the wild type sepals. At times, long filaments were observed at the fusion site of the first carpelloid oranges. The
second whorl organs in staminoid were usually smaller than the wild type and the color was changed from pink to white. These
results suggest that GhMADS3 has a homeotic role in flower development.
Keywords: MADS-box; AG subfamily; homeotic role; cotton; flower development


MADS box genes are defined by the highly con-
served 56-amino-acids-long motif known as the
MADS (MCM1-AGAMOUS-DEFICIENS-SRF) box
and are present in animals, fungi, and plants [1]. Plant
MADS box genes form a large family for transcrip-
tion factors and are involved in various aspects of
developmental processes, including flowering time
control, floral meristem identity, floral organogenesis,
fruit formation, seed pigmentation and endothelium
development [2], control of root structure [3], and fruit
ripening [4]. During flower development, MADS box
genes exert pivotal roles: all Arabidopsis floral organ
identity genes, except AP2, are MADS box genes.
Mutations of these genes usually result in homeotic
transformation of floral organs. Extensive researches
on MADS box genes revealed that the angiosperms
employ homologous genes in floral organ identity but
the regulation of the floral organ identity is complex.
Cotton (Gossypium hirsutum L.) is the world’s
most important fiber crop, and its flower development
is quite different from the model plant Arabidopsis
thaliana. Cotton plants simultaneously develop both
vegetative and reproductive organs. The branches on
a cotton plant can be classified as either vegetative
528 Journal of Genetics and Genomics 遗传学报 Vol.34 No.6 2007
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branches or fruiting branches. Flowers arise from
fruiting branches, and once a flower forms, the initial
growth of a fruiting branch is terminated[5]. The ar-
rangement of a wild cotton flower is generally similar
to Arabidopsis, comprising of sepals, petals, stamens,
and carpels. However, at the outmost of a cotton
flower, there is an extra ‘bract whorl’, which does not
exist in Arabidopsis. It is certain that the study on
cotton flower development is important from either
theoretical or practical point of view. However, ho-
meotic abnormalities and floral MADS box genes
have rarely been reported in cotton. A stable homeotic
variant, chv1, has been identified from somatic em-
bryo regenerated cotton plants. Morphological analy-
ses suggest that all floral organs of chv1 plants are
converted into bract-leaf-like organs[6]. For the pur-
pose of studying the expression of homeotic genes in
the chv1 flowers, a number of floral MADS-box
genes were cloned. The isolation and expression pat-
tern of the GhMADS1 have been reported previ-
ously[7]. In this article, the characteristics of the
GhMADS3 are reported.
1 Materials and Methods
1. 1 Plant materials
Cotton and tobacco (Nicotiana tabacum L.)
plants were grown under natural conditions during the
growing seasons in Chongqing, China, and were
transferred to a green house in winter. Two cotton
cultivars, Xuzhou 142 and Chuanmian 239, and a
cotton homeotic variant, chv1, were used for mRNA
isolation preparation. Variant chv1 was regenerated
from the cell culture of Chuanmian 239, and all floral
organs of this variant were transferred into bract-
leaf-like organs [6]. The tobacco cultivar K326 was
used for transgenic research.
1. 2 Cloning and sequence analysis of the coding
region of GhMADS3
The MADS-box region of Arabidopsis AG was
used as the probe sequence to search the GenBank
cotton EST database (database before January 2002)
using the tBLASTn procedure. All the hit sequences
were used for contig analysis by the Seqman II pro-
cedure (DNASTAR Inc., Madison, WI, USA). The
3′-end common sequence excluding MADS-box of
each contig were used for further search with the
purpose of extending 3′-end sequence. PCR was car-
ried out for the interested contig sequences to amplify
the complete ORF. For the GhMADS3, PCR primers
were designed as: GME-up 5′-TCAAGTTAG-
GAAGCATGGTG-3′ and GME-dn 5′-CCCATAAC
ATTAGACTAGTGA-3′. The floral buds cDNA of
Xuzhou 142 was used as the template. PCR products
were cloned into the pUCm-T vector and the
determination of DNA sequences was performed on
the Abi Prism 3700 sequencer. Sequences analyses
were carried out using the LASERGENE sequence
analysis software (DNASTAR Inc., Madison, WI,
USA).
1. 3 Phylogenetic analysis
Full-length amino acid alignment of 21 pub-
lished AG homologues and GhMADS3 was performed
using the ClustalX version 1.83 package [8]. ClustalX
multiple alignment parameters were gap opening 8
and gap extension 2, using the Gonnet series protein
weight matrix. The N-extensions present in several
AG subfamily genes were excluded from the align-
ments. The phylogenetic tree was obtained by the
Protdist (Dayhoff PAM model) and the Neighbor
(neighbor joining method) programs of the PHYLIP
package (version 3.65, provided by Felsenstein J,
Department of Genome Sciences, University of
Washington, Seattle. WA, USA). The tree was visual-
ized using the TreeView package [9]. The protein se-
quences used in this study were retrieved from the
GenBank, and their accession numbers are listed be-
low: AG (CAA37642), CUM1 (AAC08528), CUM10
(AAC08529), FAR (CAB42988), FBP6 (CAA48635),
FBP7 (CAA57311), FBP11 (CAA57445), GGM3
(CAB44449), GhMADS3 (AAL92522), MASAKO
C1 (BAA90744), MASAKO D1 (BAA90743), NAG1
(AAA17033), PLE (AAB25101), PMADS3 (CAA51
417), PTAG1 (AAC06237), PATG2 (AAC06238),
Yulong Guo et al.: Cloning of a MADS Box Gene (GhMADS3) from Cotton and Analysis of Its Homeotic Role in Transgenic Tobacco 529
www.jgenetgenomics.org
SHP1/AGL1 (AAA32730), SHP2/ AGL5 (AAA3273
5), STK/AGL11 (AAC49080), TAG1 (AAA34197),
TcAG (ABA39727), and VvMADS1 (AAK58564).
1. 4 RNA analysis
Total mRNA was isolated from mature floral
buds (bracts, sepals, petals, stamens, and carpels)
and 10-days-seedlings (roots, stems, and leaves) of
Xuzhou 142, and from floral buds of Chuan-
mian239 and chv1. cDNA was synthesized using
MBI kits (MBI Fermentas, Lithuan). The RT-PCR
primers used for amplifying GhMADS3 were the
same as the GhMADS3 cloning primers. Ubiquitin
gene was used as the RNA standard. The RT-PCR
primers used for amplifying the ubiquitin gene were
Ubi-up (5′-CAGATCTTCGTCAAAACCCT-3′) and
Ubi-dn (5′-GACTCCTTCTGGATGTTGTA-3′).
1. 5 Plant transformati
The GhMADS3 coding region was first inserted
into the KpnⅠ-XbaⅠ sites of pBIN AR, and placed
between the 35S promoter and the OCS terminator.
Then, the 35S-GhMADS3-OCS cassette was inserted
into Hind Ⅲ and EcoRⅠsites of pCAMBIA 2301.
This construct was introduced into Agrobacterium
tumefaciens strain LBA4404 and subsequently trans-
ferred into tobacco using the leaf disc method as de-
scribed by Horsh et al [10]. Transgenic plants were
confirmed by GUS staining and RT-PCR analysis of
GhMADS3 using RNA extracted from leaves.



Fig. 1 Alignment of the EST contig sequence, the nucleotide sequence, and deduced amino acid sequence of the GhMADS3 gene
The EST contig sequence is printed in lowercase; k represents G or T; r represents A or G; y represents C or T; s represents C or G;
m represents C or A; and w represents A or T. The sequences used to design the two primers are boxed. A thick line is drawn under
the MADS-box, a thin line under the K-box, and a double line under the N-terminal extension preceding the MADS-box; the aster-
isk indicates the stop codon. The differential bases between the EST contig sequence and the cloned gene are highlighted.
530 Journal of Genetics and Genomics 遗传学报 Vol.34 No.6 2007
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2 Results
2. 1 Isolation and sequence analysis of GhMADS3
Contig analysis of cotton MADS-box EST se-
quences showed that three sequences, BM359874,
BG444491, and BG445265, coalesced into a consen-
sus sequence, and designated GMEC1 (GhMADS
EST Contig 1). PCR primers were designed to amplify
the deduced ORF of GMEC1. The amplified cDNA is
almost the same as the corresponding region of the
GMEC1, except for 26 positions (Fig. 1); it consists of
763 bp and designats GhMADS3 (GenBank accession
No. AY083173), which possesses an uninterrupted
open reading frame of 732 bp encoding a protein of
244 amino acids. The deduced GhMADS3 protein
contains the conserved MADS domain from amino
acid 17−72 and a less conserved K-box from amino
acid 105−171 (Fig. 1), and exhibits the MIKC struc-
ture that is typical for the MADS-domain protein from
higher eudicots. When this deduced amino acid se-
quence was compared with that in the GenBank data-
base, the nearest match was found with the translation
product of TcAG, a MADS-box gene expressed in
flower and fruit of cocoa[11]. The full length of the
GhMADS3 protein showed 85% (208/244), 73%
(179/246), 72% (176/244), and 62% (177/285) identity
with TcAG, FAR, PMADS3, and AG, respectively. In
the 56 amino acid MADS-domain, 96%, 95%, 96%,
and 96% identity were observed for GhMADS3 with
TcAG, FAR, PMADS3, and AG, respectively.
GhMADS3 has two in-frame putative start codons
preceding the MADS-box. If the first start codon is
used in vivo, GhMADS3 contains an N-terminal ex-
tension preceding the MADS-domain, which is com-
monly present in the C lineage of the AG subfamily
MADS-box genes [12].
Phylogenetic analysis indicates that Arabidopsis
AG, together with SHP1/2 and STK, likely represents
a monophyletic clade, which includes members that
appear to control the development of reproductive
organs in both gymnosperms and angiosperms, and
therefore this clade is called the AG subfamily[13].
Within the angiosperms, the AG subfamily mem-
bers are divided into two major clades, the C and D
lineages. The core eudicot C lineage is further split
into two separate lineages, the PLE lineage and the
euAG lineage[12]. Phylogenetic tree reconstruction
of GhMADS3 and the representative plant MADS-box
genes known to date revealed that GhMADS3 belongs
to the AG subfamily. To visualize more accurately the
position of GhMADS3 in this subfamily, a phyloge-
netic tree of GhMADS3 and 21 published AG homo-
logues was created. The tree reveals that GhMADS3
belongs to the C subclade, and is closely related to the
euAG lineage and is closest to a cocoa AG homologue
TcAG, whereas it is clearly distinct from the D sub-
clade and the PLE lineage (Fig. 2).



Fig. 2 Phylogenetic tree of GhMADS3 and MADS-box
gene members with C and D functions of the AG subfamily
The numbers next to each node indicate bootstrap support from
1,000 replicate analysis. The taxon of origin is shown in pa-
rentheses after each gene name. The name of the gene
(GhMADS3) isolated in this study is underlined.
2. 2 RT-PCR analysis
The spatial expression pattern of GhMADS3 in
floral organs of cotton was investigated by RT-PCR.
The results showed that the GhMADS3 was expressed
in stamens (whirl 3) and carpels (whirl 4) of the mature
floral buds of Xuzhou 142, and was not expressed in
sepals (whirl 1) and petals (whirl 2). Expression of
GhMADS3 was not detected in vegetative organs, such
as roots, stems, leaves, and bract-leaves (Fig.3). This
Yulong Guo et al.: Cloning of a MADS Box Gene (GhMADS3) from Cotton and Analysis of Its Homeotic Role in Transgenic Tobacco 531
www.jgenetgenomics.org
expression pattern corresponds to the Arabidopsis gene
AG [14] and suggests that this gene was involved in the
development of reproductive floral organs while it re-
mained inert in the perianth and vegetative organs.

Fig. 3 Expression analysis of GhMADS3 by RT-PCR

Total mRNA was also isolated from the floral
buds of chv1 variant and the wild type (Chuanmian
239) cotton plants. RT-PCR analysis showed that
GhMADS3 was expresseed in Chuanmian 239 floral
buds, but not in chv1 variant (Fig. 4). These results
suggest that the chv1 variant does not have normal
expression patterns of floral genes.

Fig. 4 Different expressions of GhMADS3 between the
chv1 and the wild type cotton floral buds
2. 3 Ectopic expression of GhMADS3 in tobacco
Several Kanamycin-resistant plants were regener-
ated. Leaf-disks of 30 independent Kanamycin- resis-
tant plants were subjected to GUS staining. 26 plants
showed detectable GUS activity and were transferred
to soil. Sixteen survived transformants were investi-
gated for GhMADS3 expression and phenotypic
changes. The GhMADS3 expression of all 16 plants
was detected by RT-PCR but only 12 had flowers with
more or less phenotypic changes compared to wild type,
and the remaining (4 lines) showed very weak altera-
tions. The phenotypes of the changed flowers from 4
representative transgenic plants (M3106, M3702,
M3104 and M3101) are shown in Fig. 5.
For comparison of the flower phenotype of the
wild tobacco and transgenic plants, it is necessary to
briefly review the structure of wild type tobacco flowers
(Fig. 5, A and G), which has been described previously
[15]. The first whorl of wild type flower is occupied by
five green sepals that are connately fused for most of
their length. The second whorl is occupied by five pet-
als that are also connately fused to form trumpet-like
corolla with pink limb. Five stamens, each consisting of
a long filament capped by a pollen-bearing anther and
fused to the second whorl petal tube for most of its
length, occupy the third whorl. The fourth whorl is oc-
cupied by a two-carplellate gynoecium.
The growth and leaf phenotypes were not af-
fected by ectopic expression of the GhMADS3 gene,
and the number and position of organs in each whorl
were the same as that in the wild-type flowers in most
cases. However, the transgenic plants usually had
flowers, which were smaller than the wild type, with
carpelloid first and staminoid second whorl organs.
During early flower development, the growth and
the completely fused sepals with stigmatic structure on
the upper edges in some cases impeded the growth of the
interior organs, resulting in distorted morphology (Fig.
5C). When the flowers opened fully, the phenotype of
the first whorl organs in transgenic plants changed most
obviously. The carpelloid first whorl organs had a white
appearance, which was in contrast to the dark green
color of wild type sepals (Fig. 5, B, C, D, E, and G).
Three kinds of tissue were observed on the upper edge
of the first whorl organs of transgenic flowers: one
(M3104) was green stigmatic tissue, which was the same
as that found at the tips of wild type styles (Fig. 5, J and
K); the second (M3106) was glassy stigmatic tissue with
long filaments formed at the fusion site of the first whorl
organs (Fig. 5, G, H and I); the third (M3702 and M3101)
was stigmatic tissue without long filaments (Fig. 5, C
and E). The corolla of the transgenic plants was usually
smaller than the wild type, with changes of color from
pink to white. It appeared that the petal tubes were not
altered significantly but the limbs were changed into
staminoid tissue (Fig. 5, D, F, and K) in some cases. The
third whorl organs were not influenced significantly by
the introduction of the GhMADS3. However, the fusion
of filaments to the second whorl was clearly shorter. The
carpels were usually smaller than the wild type and had
a very pale surface.
532 Journal of Genetics and Genomics 遗传学报 Vol.34 No.6 2007
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Fig. 5 Photographs of wild-type and 35S::GhMADS3 transgenic tobacco flowers
A: An inflorescence from a wild-type plant; B to E: Inflorescences from transgenic plant M3106 (B), M3702 (C), M3104 (D), and
M3101 (E); F: An M3101 transgenic flower, top view, showing antheroid tissue developing on the limb; G: Phenotypes of a
wild-type (left) and a transgenic (M3106, right) flower. Note the color of the first and the second whorl organs; H to I: Close views
of the first whorl carpelloid organ top from an M3106 transgenic flower. Note the long filaments (I) and the stigmatic tissue on the
upper edge (H); J: Two pieces of the first whorl carpelloid organs from a M3104 transgenic flower. Note the green stigmatic tissue
at their tips; K: An M3104 transgenic flower with carpelloid first and stamenoid second whorl organs.


3 Discussion
The GhMADS3, a cotton MADS-box gene, was
identified based on EST sequences. Sequence analysis
and phylogenetic tree reconstruction indicated that
GhMADS3 belongs to the C lineage of the AG sub-
family of plant MIKC MADS-box genes. RT-PCR
analysis revealed that the expression pattern of
GhMADS3 is the same as the Arabidopsis AG gene
and is different from the Arabidopsis SHPs and STK
genes. The transgenic tobacco flowers which ectopi-
cly expressed GhMADS3 displayed homeotic conver-
sions of perianth organs into reproductive organs.
Similar alterations in flower development were also
Yulong Guo et al.: Cloning of a MADS Box Gene (GhMADS3) from Cotton and Analysis of Its Homeotic Role in Transgenic Tobacco 533
www.jgenetgenomics.org
observed in transgenic plants that constitutively ex-
pressed the Arabidopsis AG gene[16], or other homo-
logues of AG, such as BAG[15], TcAG[11], MASAKO C1
and D1[17], LLAG1[18], BpMADS6[19], and VvMA-
DS1[20]. The transgenic phenotypes, the sequence
analysis, and the RNA expression data together sup-
port the conclusion that GhMADS3 probably repre-
sents a cotton function orthologue of the Arabidopsis
AG gene.
However, it was found that there were some mi-
nor differences between the phenotype changes
caused by the ectopic expression of GhMADS3 and
AG subfamily genes from other plants. Firstly, long
filaments capped with stigmatic tissue were observed
at the fusion site of the first whorl carpelloid organs
(Fig. 5I) from GhMADS3 transgenic tobacco flowers.
This is the first report on this kind of structure ap-
pearing in the first whorl of plant flowers constitu-
tively expressing AG homologues. Secondly, the car-
pelloid first whirl organs, corolla, and rind (the skin of
fruit) of the GhMADS3 transgenic tobacco had a
white appearance. This is similar to the BpMADS6 [19]
or VvMADS1[20] over-expressed tobacco flowers hav-
ing sepals that are very pale, but the color change is
more pronounced in this report. Color changes also
observed in Arabidopsis flowers ectopicly-expressed
AG; the wild type white petals were changed to yel-
lowish in transgenic plants[16]. These results indicate
that AG subfamily genes probably have regulated
roles in flower pigment biosynthesis. All these differ-
ences indicate that GhMADS3 probably has its spe-
cific functions.
Extensive research of flower development in
Arabidopsis and Antirrhinum resulted in the ‘ABC’
model. Further investigation into the functions of
other floral MADS box genes has led to a revised
‘ABCDE’ model[21]. In this model, the D function was
proposed to be responsible for the establishment of
ovule identity. E class genes, represented in Arabi-
dopsis by SEPALLATA1-3, have redundant functions
that are required for petal, stamen, and carpel devel-
opment. In triple mutants, in which SEP1, SEP2, and
SEP3 had all lost their functions, all flower organs
resembled sepals. The phenotype is similar to that of
double mutants, which are defective in both B- and
C-class floral genes. However, SEP1-3 are still ex-
pressed in B and C loss-of-function mutant, and the
initial expression patterns of B- and C-class genes are
not altered in the sep1: sep2 : sep3 triple mutant[22].
Morphological analysis showed that all flower organs
of the cotton variant chv1 were bract-leaf-like or-
gans[6]. Previously, it was shown that a SEP subfamily
MADS box gene GhMADS1 was not expressed in
chv1 floral buds[7]. Here, it is demonstrated that a
cotton AG homologue gene GhMADS3 is also not
expressed in chv1 floral buds. This provides a new
support to the previous conclusion that all flower or-
gans of chv1 are converted into vegetative organs. It
is possible that loss-of-function of a floral homeotic
gene activator is responsible for the chv1 novel phe-
notype.
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Yulong Guo et al.: Cloning of a MADS Box Gene (GhMADS3) from Cotton and Analysis of Its Homeotic Role in Transgenic Tobacco 535
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棉花 MADS 框基因 GhMADS3 的克隆及其组成型表达对烟草
花器官决定的影响
郭余龙1, 2, 祝钦泷1, 郑尚永2, 李名扬1
1. 西南大学园艺园林学院，重庆 400716；
2. 农业部生物技术与作物品种改良重点实验室，重庆 400716
摘 要：MADS 框基因在植物花器官发育中发挥着关键性作用。为研究棉花花器官发育的机理，以徐州 142 花蕾为材料，
利用 EST 数据库资料，通过 EST 序列整合，克隆出了一个 MADS 域蛋白的编码区段，GenBank 登录号为 AY083173。 该
片段(GhMADS3)包含一个 732 bp 的开放阅读框，推导的氨基酸序列(244 氨基酸)与可可，黄瓜，烟草，矮牵牛，金鱼草等
的 AG 亚家族基因的序列相似性高。进化树重建分析将 GhMADS3 基因归入 MADS 框基因 AG 亚家族 C 功能分支的 euAG
分支。RT-PCR 分析显示，该基因在雄蕊和心皮中表达，在根、茎、叶等营养器官，萼片，花瓣，花器官变异体 chv1(所有
花器官均变为苞叶状器官)的花蕾中不表达。将 GhMADS3 与 35S 启动子融合构建成嵌合基因转化烟草，转基因烟草植株花
朵出现萼片(轮 1)向心皮，花瓣(轮 2)向雄蕊的转变，花器官表现明显的白化倾向。同时，在轮 1 观察到丝状结构的出现，
该结构在此前类似的研究中尚无报道。这些结果说明，实验中克隆了一个有生物学功能的棉花的 AG 亚家族 MADS 框基因，
该基因可能在棉花花器官发育中有重要的功能。
关键词：MADS 框；AG 亚家族；同源异型作用；棉花；花发育
作者简介：郭余龙（1969-），男，重庆人，博士，副研究员，研究方向：植物发育与生物技术。E-mail: yulong@swu.edu.cn