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RESEARCH PAPER
Abstract: To advance the understanding of gibberellins (GA) functions in cotton (Gossypium hisutum L.) development, especially fiber
development, 6 cotton GID1 homologous genes, GhGID1-1 to GhGID1-6 (GenBank accession numbers FJ790125 to FJ790130), were
cloned by searching expressed sequence tag (EST) sequences, extending the flanking sequences by Y-shaped adaptor dependent extension
(YADE) method, and amplifying the full-length cDNA by 3 rapid amplification of cDNA ends (RACE). Multiple sequence alignment
indicated that the deduced GhGID1-1 to GhGID1-6 proteins shared high sequence identity with GID1s from other species and contained
multiple binding sites with GA and DELLA protein, as well as HGG and GXSXG domains conserved in hormone-sensitive lipase (HSL)
family. Three amino acid residuals (G169, G196, and R251) related to rice (Oryza sativa L.) GID1 were also conserved in cotton GID1s. In
the phylogenetic tree of GhGID1s and GID1 proteins with known functions, GhGID1-1 to GhGID1-6 were clustered together with 3
Arabidopsis GID1 proteins, and distantly related to GID1s from rice and Selaginella moellendorffii. According to the results by
quantitative reverse transcription polymerase chain reaction (RT-PCR), the transcripts of GhGID1-1 to GhGID1-6 were detected in roots,
hypocotyls, leaves, petals, anthers, ovules, and fibers. GhGID1-1 and GhGID1-2 were expressed mainly in floral organs, and GhGID1-4
was expressed preferentially in fiber and root. Exogenous GA in medium resulted in alteration of gene expression level in the ovules
cultivated in vitro, and the expression of GhGID1-1 and GhGID1-2 was obviously inhibited by GA. These results suggested that the 6
GhGID1 homologous genes might encode biologically functional GID1 homologues involved in GA signaling in cotton. GhGID1-1 was
predominantly expressed in ovules; its expression level reached the climax at 10 d post anthesis (DPA) and maintained the high level at
14 DPA and 18 DPA. In fibers, GhGID1-4 was the main GID1 homologue expressed, and its highest-level expression occurred at 6 DPA
and declined to a very low level at 14 DPA and 18 DPA. This result strongly suggested that these are relatively independent GA signaling
systems in ovules and attached fibers.
Keywords: GID1; cotton; fiber development; expression specificity
Cotton (Gossypium hisutum L.) fibers are extremelyelongated single-celled fibers originating from the outermost
epidermis of ovule, and the mature fiber is composed of over
95% cellulose. The unique cell structure and chemical
composition make cotton fiber to an excellent model system
for studying certain physiological processes including cell
elongation and carbon partitioning [8]. Previous studies have
demonstrated that GA plays an important role in regulating
the growth and development of cotton fiber [915]. Addition of
GA3 in the medium might promote fiber initiation and
elongation on in vitro cultured ovules [913], whereas GA3
applied to floral bud may increase fiber number per ovule [14].
Chen et al. [13] showed that IAA and GA3 could enhance fiber
elongation in the fiber-less mutant, and had synergistic effect
on promoting fiber elongation. Du et al. [15] reported that
endogenous GA1+3 could increase ovule weight and fiber
length after fertilization. In recent years, transcriptomic
studies have revealed up-regulation of some GA synthetic and
responsive genes during fiber initiation and elongation [1618],
which implies that both GA biosynthesis and response are
involved in the regulation of cotton fiber.
To reveal the GA responsive mechanism in cotton, Aleman
et al. [19] cloned each 2 genes for GID1 and DELLA proteins,
and elucidated the functions and interactions of these proteins
by prokaryotic expression, yeast two-hybridization, and
transgenic complementation in Arabidopsis. However, these
GID1 genes were preferentially expressed in floral organs,
implying that these genes were mainly related to the
development of floral organs. To elucidate the functions of
GA response in fiber development, we cloned 6 GID1
homologous genes from cotton on the basis of expressed
sequence tags (ESTs). Real-time reverse transcription
polymerase chain reaction (RT-PCR) analysis indicated that
all these GID1 homologous genes displayed tissue- or
organ-specific expression, of which GhGID1-4 was expressed
preferentially in elongating fiber.
1
1.1
Cotton DNA was extracted using the modified cetyltrimethylammonium bromide method [20]. Y-shaped adaptor
dependent extension (YADE) method was used to amplify the
flanking sequences of ESTs that lacked the initiation ATG
(AI055546 and Ghi4864) as previously described [20, 21].
The cDNA genes were amplified by 3 rapid amplification
of cDNA ends (RACE) with specific primers designed
according to the sequences upstream to the putative initiation
ATGs. Approximate 5 g of total RNAs from root, flower, or
10-DPA (days post anthesis) ovules (including fibers) were
used in the reverse transcription reaction. Amplification of
cDNA was conducted using the reverse transcription kit
(TaKaRa, Dalian, China) according to the manufacturers
instructions. PCR products were separated in argarose gels,
cloned into pGEM-T (Promega, Shanghai, China), and
sequenced by Invitrogen (Shanghai, China).
1.4
Unigene or EST
GhGID1-1
Ghi9117
AGTCTGGTTTGTTCGGCCAT
ACCATCCAGCTTGTAATCCA
GhGID1-2
GhGID1-3
GhGID1-4
GhGID1-5
GhGID1-6
Histone3
Ghi10228
Ghi2061
GTTACGGTTCAGTGGAGACT
GAGCCATGGCTGGTAGCAA
CGAAAAGTCTGGTAGTCGTG
CTACGCAGGGTGAGTGGTA
CATGGCTGGTAGAAATGAAG
CTGAAGGCGAAGATCGAGA
GTCCGACTTAATAGACCCTT
AI055546
TTCATGGTGGGAGCTTTGCA
Ghi4864
TTCCTCGGCGAATAGTGCTA
GGATTCCACAGCTCGTGC
Ghi13407
Ghi13407
CGCAACATGGTGTGCGATG
GGAATGGCTGGAAGTAATGA
ACCTAACGGTAGAAGTCTTG
AATCACCCCGAGATCGTGAA
GGAAATGGCTAGAAGTAATGA
CTAACGGTAGAAGTCTCGAG
GAATGTCAAGCTCCTGTCGT
CCTAACGGTAGAAGTCTTGAA
TAACAGTATGGAAGTGATTGC
GAAGCTGCAGAGGCATACC
CTACCACTACCATCATGGC
Usage
Results
Accession number
GhGID1-1
FJ790125
GhGID1-2
DNA (bp)
Amino acid
Protein
Identity (%)
1249
1035
344
GhGID1a
99.7
FJ790126
1472
1035
344
GhGID1b
98.5
GhGID1-3
FJ790127
1153
1038
345
GhGID1b
88.7
GhGID1-4
FJ790128
1370
1035
344
AtGID1c
82.3
GhGID1-5
FJ790129
1568
1035
344
AtGID1c
81.7
GhGID1-6
FJ790130
1089
1035
344
AtGID1c
80.8
Fig. 1 Multiple sequence alignment of GhGID1s and GID1 proteins from other plants
Numbers on right of the figure indicate positions of the amino acid sequences of cDNA.
Solid bars indicate the binding sites of GA and DELLA proteins. HGG and GXSXG conserved domains in HSL family are marked with asterisks.
Amino acids related to HSL activity are indicated with black dots. Arrowheads present the amino acid residuals essential to the rice GID1 protein.
AtGID1A to AtGID1C, Arabidopsis GID1 proteins (GenBank accession numbers NP_187163, NP_191860, and NP_198084);
OsGID1: Rice GID1 protein (AB211399); SmGID1a and SmGID1b: S. moellendorffii GID1 proteins (ABX10756 and ABX10757).
0.0
0.0
0.000
0.00
0.000
0.000
Fig. 3 Expression levels of six GID1 homologous genes in different cotton organs and tissues
Ro: Root; Hy: Hypocotyls; Le: leaf; Pe: Petal; An: anther; O0: 0-DPA ovule; F6: 6-DPA fibers; O6: 6-DPA ovule.
Discussion
0.00
0.000
0.000
0.00
0.0000
0.000
Fig. 4 Expression of six GID1 genes at various stages in ovules and fibers of cotton
Conclusions
0.00
0.00
0.000
0.0000
0.0000
0.00000
Acknowledgment
This study was financially supported by the National High
Technology Research and Development Program of China
(2007AA10Z134).
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