Acta Botanica Sinica
植 物 学 报
2004, 46 (3): 363－370
http://www.chineseplantscience.com
Analysis of Seed-expressed Sequence Tags in Triticum aestivum
LI Jia-Rui, WANG Fang, ZHAO Xiang-Yu, DONG Yu-Xiu, ZHANG Li-Yuan, AN Bao-Yan, ZHANG Xian-Sheng*
(College of Life Sciences, Shandong Agricultural University, Taian 271018, China)
Abstract: To isolate seed-expressed sequences, a cDNA library was constructed using wheat (Triticum
aestivum L.) seed tissues at 12 d after pollination. Plasmid DNAs of 10 000 clones randomly picked out from
the library were prepared. The preparation of high density filters were made with the Biomek 2000 HDRT
system, and then hybridized separately with three probes prepared by reverse transcription of RNA of
unpollinated ovary, embryo and endosperm. Based on the hybridization results, 800 clones expressed in
embryo and/or endosperm were chosen for further analysis of expressed sequence tags (ESTs). Finally,
216 different genes were identified preliminarily. Of them, 24 (11.5%) were considered identical to known
wheat genes, 122 (56%) were identified as putative new plant genes which may be involved in seed storage
proteins, biochemical metabolisms, development, and other biological processes of seeds, while 70 (32.5%)
sequence identities could not be determined.
Key words: wheat seeds; cDNA array; differential screening; expressed sequence tag
Wheat is one of the important crops. Its seed is composed of the embryo, the endosperm, and the seed coat
(including pericarp). The embryo developed from a fertilized egg possesses the shoot and root meristems which
form vegetative organs of plant, and the cotyledon or
scutellum. The endosperm is a stored reserve, and it accumulates nutritive materials for germination and seedling
development. As a protective tissue, the seed coat wraps
the embryo and endosperm (Buchanan et al., 2000).
Although a lot of data have been accumulated on developmental events and biochemical metabolism during
seed development, little is known on their molecular mechanisms (Lopes and Larkins, 1993; Buchanan et al., 2000). In
recent years, new molecular tools have been used to identify the genes of plants, such as cDNA array and expressed
sequence tag techniques which have led to the rapid isolation of genes in many organisms, and accelerated studying
the expression profiles of genes in given organs, or tissues
under physiological and environmental conditions (Hu et
al., 1999; Girke et al., 2000; Voibelt et al., 2001; Bao and Li,
2002; Che et al., 2002; Endo et al., 2002; Seki et al., 2002; Yu
and Setter, 2003). Combining the appropriate biochemical
knowledge with gene expression data can provide direct or
indirect evidence for the elucidation of gene function.
In this study, we constructed the cDNA library of wheat
seeds at 12 d after pollination. At that time, the organs of
the embryo have been formed, and synthesis of organic
materials is actively progressing in the endosperm (Yu and
Wan, 1995). Using cDNA array method, a number of clones
have been screened from the library by differential
hybridization. Then, we chose some of them for further
analysis by expressed sequence tag technique. Our data
provide important information to understand the gene function that may be involved in biochemical metabolisms, developmental events and other biological processes during
seed development.
1 Materials and Methods
1.1 Plant materials
Plants of wheat (Triticum aestivum L. cv. PH82-2-2) were
grown in the soil at the campus of Shandong Agricultural
University, China. The seed tissues at 12 d after pollination
were harvested into liquid N2 and stored at –80 ℃　for later
cDNA library construction. For RNA isolation, the roots,
leaves, ovaries, embryos and endosperms at different stages
were collected separately in liquid N2 and also stored at
–80 ℃　until used.
1.2 Construction of cDNA library
Total RNA of wheat seeds at 12 d after pollination was
extracted from 5 g seed tissues by using the total RNA
isolation system (Gibco, BRL), and mRNA was subsequently isolated by mRNA isolation system (TaKaRa, Dalian,
China). mRNA was reversely transcripted into first strand
cDNA, and second strand cDNA was synthesized by DNA
polymerase Ⅰ according to the manufacturer’s protocols
(TaKaRa, Dalian, China). The reaction product was
Received 30 May 2003 Accepted 15 Sept. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (2001AA212211) and the State Key Basic Research and
Development Plan of China (G19990116).
* Author for correspondence. E-mail: <[email protected]>.
３６４
fractionated by Sizesep 400 Spun Column, and the fractions containing cDNA larger than 500 bp were collected
and ligated into pBluescript Ⅱ SK(+) at the NotⅠ and
EcoRⅠ sites (Stratagene). Then, the ligation products were
transformed into Escherichia coli JM109 (TaKaRa, Dalian,
China).
1.3 Differential screening
Basically, cDNA array was carried out as described by
Hu et al. (2001). The high density filters were prepared
using the Biomek 2000 HDRT system. For plasmid DNA
preparation, ten thousand clones were picked out randomly.
Twenty µL plasmid DNA for each clone was transferred
into the wells of 384-well plates, and denatured with equal
volume of 0.4 mol/L NaOH. After that, the denatured DNA
was spotted onto nylon membranes using the Biomek 2000
HDRT system. The membranes were briefly washed with 2
× SSC three times. When the membranes were dry, they
were baked at 80 ℃ for 2 h. Each filter was made in triplicate.
Prehybridization was carried out at 65 ℃ for 4 h, in 6×
SSC, 5×Denhart’s, 6% SDS and 20 µg/mL salmon sperm
DNA. Three probes were prepared by reverse transcription
of 10 µg total RNA of ovaries at the anthesis, embryos and
endosperms at 12 d after pollination with RAV-2 reverse
transcriptase. Overnight hybridization was made with three
different probes, respectively.
The membranes were washed in 2×SSC and 0.1% SDS
at 55 ℃ twice, and then autoradiography was performed at
–70 ℃.
Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．３　２００４
1.4 Sequence analysis
Based on hybridization results, eight hundred clones
were chosen and sequenced with ABI 377 DNA sequencer
(Perkin Elmer, USA). Analysis of DNA or protein homology
in GenBank was performed using the program BLAST.
1.5 Northern hybridization
Total RNA isolation and Northern hybridization were
performed as described by Sambrook et al. (1989). Twenty
µg of total RNA was fractionated by gel electrophoresis in
1.2 % formaldehyde agarose gels and transferred from agarose gels to nylon membrane. Prehybridization was performed at 42 ℃ for 12 h. Hybridization was conducted at 42
℃ for 48 h. Filters were washed subsequently in 2×SSC
with 0.2 % SDS and 0.2×SSC with 0.2 % SDS. Autoradiography was performed at –70 ℃.
2
Results
2.1 Identification of seed-specific cDNA clones
A cDNA library was constructed using seed tissues at
12 d after pollination. Detection of PCR indicated that the
library contains 5.0 × 105 clones, the average size of inserts was 1.0 kb and 84% clones had inserts. Three copies,
each with nine high density filters containing triplicate
10 000 cDNA clones picked out randomly from the library
were prepared with Biomek 2000. Then, they were hybridized separately with the probes by reverse transcription of
RNA of ovaries (negative), embryos or endosperms (positive).
The partial hybridization signals are shown in Fig.1.
Fig.1. Hybridization signals by differential screening with three probes. A. Ovary. B. Embryo. C. Endosperm. a, embryo-specific clone;
b, endosperm-specific clone; c, both embryo- and endosperm-specific clones.
３６５
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum
As indicated in Table 1, the hybridization signals of total
6 826 clones were detected. The number of clones representing seed-expressed sequences is 6 288 (92% of 6 826 clones)
except 538 clones detected only in the ovary and among
them, 1 856 clones (30% of 6 288 clones) corresponding to
sequences were expressed in the embryo, endosperm or
both embryo and endosperm, but not in the ovary. The
hybridization signals of 3 016 clones were not detected.
Table 1 Result of differential hybridization with the probes of
ovary, embryo or endosperm
No.
1
2
3
4
5
6
7
8
Expressive tissue
Ovary
Embryo
Endosperm
Embryo and endosperm
Embryo and ovary
Endosperm and ovary
Embryo, endosperm and ovary
Undetected in three tissues
Clone No.
538
274
1 107
478
464
522
3 443
3 016
2.2 Expressed sequence tags (ESTs) analysis and database comparison
Based on the hybridization results, 800 of 1 856 clones
and a few clones undetected in three tissues were chosen
for sequencing. By the analysis of sequences, they represent 216 unique clones after removing redundant ESTs,
and the uni-ESTs were registered in GenBank.
The homologous analysis of 216 ESTs was carried out
by comparing with the sequences against public databases.
Sequences were translated in the three open reading frames
and compared with protein sequence databases using the
program BLASTx (Alschul et al., 1990). We also compared
these sequences with the genes in the unigene database of
wheat in GenBank.
Among them (Tables 2, 3), 24 (11.5%) were considered
identical to known wheat genes, 122 (56%) were identified
as the putative new plant genes, while 70 (32.5%) sequence
identities could not be determined.
2.3 ESTs identical to known genes
Among the sequences showing significant similarity to
them in the databases, 24 ESTs were considered identical
to known wheat genes (Table 2). We defined it identical if
sequences showed more than or equal to 95% identity
over a length of 40 amino acids or 100% identity over a
length of 24 amino acids. As Table 2 shows, some genes (8)
encode the storage proteins including low molecular
Table 2 Expressed sequence tags (ESTs) identical to wheat genes
Accession No.
BU607223
BU607210
BU607213
BU607216
BU607192
BU607195
BU607224
BU607170
BU607168
BU607165
BU607165
BU607168
BU607188
BU607169
AF 479046
BU607199
BU607183
BU607176
BU607211
BU607172
BU607250
BU607177
BU607205
AY290720
Putative product
Chloroform/methanol soluble
(CM16) protein
CM1 protein of alpha-amylase
tetrameric inhibitor
CM2 protein of alpha-amylase inhibitor
CM3 protein of alpha-amylase inhibitor
Starch branching enzyme 2
Small subunit ADP glucose pyrophosphorylase
LMW glutenin U86029
LMW glutenin U86030
Gamma-gliadin class B-III
Gamma-gliadin class mrna
LMW glutenin U86027
LMW glutenin U86029
LMW glutenin U86028
LMW glutenin U86029
Elongation factor 1
Ras related GTP binding protein
Histone H2B
Serpin
Beta purothionin precursor
Puroindoline-a
Sec 61 protein
Cytosolic glyceraldehydes-3-phosphatee
hydrogenase
Inducible phenylalanine ammonia-lyse
Carboxypeptidase D
Identity (aa)
139/139 (100%)
Tissue
Endosperm
Clone No.
2
144/145 (99%)
Endosperm
2
72/72 (100%)
137/138 (99%)
53/54 (98%)
173/173 (100%)
40/41 (97%)
39/41 (95%)
45/45 (100%)
36/36 (100%)
36/36 (100%)
45/45 (100%)
24/24 (100%)
60/62 (97%)
127/131 (96%)
185/185 (100%)
92/92 (100%)
217/220 (98%)
115/116 (99%)
148/148 (100%)
33/33 (100%)
215/217 (99%)
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Embryo
Embryo
Embryo
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
2
4
1
1
7
7
4
1
1
5
1
2
2
1
1
3
3
1
1
1
178/179 (99%)
119/119 (100%)
Undetected
Endosperm embryo
embryo
embryo
embryo
embryo
embryo
embryo
1
1
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Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．３　２００４
Table 3 ESTs highly homologous to known genes in GenBank
Accession No.
AY290731
BU607232
AY290721
BU607225
BU607229
AF470353
BU607197
BU607198
BU607212
BU607215
BU607209
BU607202
BU607171
BU607201
BU607167
BU607218
BU607180
BU607217
BU607230
BU607194
BU607174
BU607221
BU607166
AF475121
AF469489
BU607262
BU607255
Putative products
ADP glucose pyrophosphorylase
0.19 Alpha-amylase inhibitor
Alpha-amylase inhibitor
CM17 protein of alpha-amylase inhibitor
Trypsin inhibitor CMx precursor
Beta-amylase
High molecular weight (HMW) glutenin subunit
HMW glutenin subunit 1Ax1
Low molecular weight (LMW) glutenin U86026
LMW glutenin subunit group 11 type VI
LMW glutenin subunit group 3 type II
LMW glutenin X84960
LMW glutenin protein 1Agi
LMW glutenin M11335
LMW glutenin subunit group 10 type V
LMW glutenin subunit group 7 type IV
Alpha-gliadin storage protein
Gamma-gliadin clone 10d11
Gamma-gliadin clone G2656
Alpha-/beta-gliadin
Alpha-/beta-gliadin precursor protein
Gamma-gliadin class B-I
Alpha-gliadin
Seed globulin
Avenin
Avenin fast component N9
Avenin-3 precursor
Organism
Triticum aestivum
T. aestivum
Hordeum vulgare
T. aestivum
T. aestivum
H. vulgare
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
T. aestivum
Aegilops tauschii
Avena sativa
A. sativa
A. sativa
Identity (aa)
162/221 (73%)
98/124 (79%)
130/131 (99%)
122/139 (87%)
119/146 (81%)
220/260 (84%)
105/167 (62%)
265/387 (68%)
115/171 (67%)
102/166 (61%)
91/161 (56%)
101/161(63%)
41/63 (65%)
41/63 (65%)
85/173 (49%)
69/145 (47%)
83/161 (51%)
78/131 (59%)
95/145 (65%)
81/120 (67%)
89/144 (61%)
106/115 (92%)
113/141 (80%)
162/222 (72%)
38/66 (57%)
49/86 (57%)
31/62 (50%)
Tissue
Embryo
Embryo
Embryo
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Clone No.
1
19
1
2
1
embryo
4
embryo
5
embryo
2
5
embryo 1 1
embryo
4
embryo
6
embryo
1
embryo
3
1
1
embryo 1 8
2
1
2
embryo
1
3
2
embryo
3
embryo
8
embryo
1
embryo
1
AF470352
BU607139
BU607220
BU607175
BU607187
AY290722
AF475098
AF475122
AF475123
AF475114
AF475116
AF479033
AY290723
AF 479045
AF 479049
AF 479048
AF475108
AF542969
AF475099
AY290724
AY290725
BU607196
AF470354
AF475129
AF470357
BU607219
AF479046
AF479057
Alcohol-soluble avenin-3=23.2 kD protein
Thaumatin-like protein
12s Globulin
Hordoindoline-b
Puroindoline-b
Ribosomal protein L39
Ribosomal protein S4
Ribosomal protein
Acidic ribosomal protein P2a-2
60S Ribosomal protein L21
Ribosomal protein L30
40S Ribosomal protein S3
Acidic ribosomal protein P1a
Ribosomal protein S28
60S Ribosomal protein L2
Ribosomal protein L17
Acidic Ribosomal protein P2b
Ribosomal protein L19
60S Ribosomal protein L15
Ribosomal protein S7
Ribosomal protein S3a
Tap-nuclear mRNA export protein
Putative zinc finger protein
Elongation factor 2
Transcription factor TFIIB
RNA-binding protein
Elongation factor 1 elongation beta
Ocs-binding factor 1
A. sativa
Oryza sativa
A. sativa
H. vulgare
T. aestivum
O. sativa
Nicotiana tabacum
O. sativa
Z. mays
O. sativa
Z. mays
T. aestivum
Z. mays
H. vulgare
Arabidopsis thaliana
Castanea sativa
Z. mays
O. sativa
Homo sapiens
H. vulgare
O. sativa
T. aestivum
O. sativa
Beta vulgaris
O. sativa
T. aestivum
T. aestivum
Z. mays
68/130 (52%)
65/150 (43%)
63/91 (69%)
34/79 (43%)
113/143 (79%)
44/46 (96%)
47/56 (83%)
93/113 (82%)
61/112 (54%)
149/164 (90%)
36/46 (78%)
76/78 (97%)
59/68 (87%)
50/51 (98%)
77/77 (100%)
139/140 (99%)
35/113 (30%)
133/159 (83%)
145/193 (75%)
54/55 (98%)
162/211 (76%)
55/78 (70%)
82/110 (74%)
153/166 (92%)
45/49 (91%)
24/60 (40%)
127/131 (96%)
108/133 (81%)
Endosperm
Endosperm
Endosperm
Undetected
Endosperm
Embryo
Embryo
Endosperm embryo
Endosperm embryo
Embryo
Embryo
Endosperm
Embryo
Embryo
Embryo
Embryo
Endosperm
Embryo
Undetected
Embryo
Undetected
Endosperm
Embryo
Endosperm embryo
Embryo
Endosperm
Embryo
Endosperm
1
1
1
1
1
1
1
2
2
2
1
1
1
2
1
2
2
1
1
1
1
1
1
1
1
1
2
1
３６７
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum
Table 3
(continued)
Accession No.
AY290726
BU607190
AF475102
BU607238
BU607240
BU607242
BU607236
AF479034
AF479053
AF470356
AF469490
BU607235
AF475105
AY290732
AF542972
AF469492
AF479043
BU607249
Putative products
Translation initiation factor
Eukaryotic translation initiation factor 4B
26S Proteasome AAA-ATPase subunit RPT5a
Similarity to guanylate binding protein
Putative DEAD/DEAH box RNA helicase protein
bZIP protein
Ara4-interacting protein
ADP-ribosylation factor
S-locus protein 5
QM protein (tumor suppressor)
Gigantea-like protein (controlling flowering time)
Putative embryogenesis-abundant protein
Possible apospory-associated protein
Putative senescence-associated protein
Putative tumor suppressor
MCM protein-like protein
Cyc 07
Histone H3
AY290727
AF470355
AF479051
Putative cytochrome P450
Metallothionein type 2
Predicted RNA methylase like
BU607252
BU607162
AF475128
AF475124
110 kDa 4SNc-Tudor domain protein
Putative UV-damaged DNA binding factor
Abscisic acid-induced protein
Glutathione peroxidase-like protein
AF479039
AF542185
AF475100
BU607200
AY290733
BU607154
Mosaic virus helicase domain binding protein
Glutaredoxin
Catalase
Wali7 induced by aluminum
Cyclophilin
Similarity to gb|AF181686 membrane
protein TMS1d
Mitochondrial inner membrane translocating
protein-like
Protein disulfide isomerase 2 precursor
Putative 2,3-bisphosphoglycerate-independent
phosphoglycerate mutase
Formate dehydrogenase
Putative 1-acyl-glycerol-3-phosphate
acyltransferase
Cytosolic 3-phosphoglycerate kinase
Phosphoethanolamine methyl-transferase
Triosephosphat isomerase
Pectinesterase-like protein
NADPH-dependent mannose 6-phosphate
reductase
Pin1-type peptidyl-prolyl cis/trans isomerase
Enoyl-Acp reductase
Succinate dehydrogenase subunit 3
Holocarboxylase synthetase
Histone acetyltransferase
Ubiquinol reductase Cytochrome C reductase
Similar to methylenetetrahydrofolate dehydrogenase
BU607141
BU607206
AF475111
AF479036
AF479037
BU607214
BU607233
BU607189
BU607184
AF475103
BU607185
AF475125
AF475119
AF479038
AF475113
AF479054
BU607138
Organism
Pisum sativum
T. aestivum
A. thaliana
A. thaliana
O. sativa
A. thaliana
A. thaliana
O. sativa
Brassica rapa
O. sativa
H. vulgare
O. sativa
Pennisetum ciliare
P. sativum
O. sativa
N. tabacum
O. sativa
Onobrychis
viciaefolia
O. sativa
Poa secunda
Caenorhabditis
elegans
P. sativum
A. thaliana
H. vulgare
H. vulgare
Identity (aa)
158/228 (69%)
70/230 (30%)
179/184 (97%)
81/188 (43%)
164/189 (86%)
58/152 (38%)
88/266 (33%)
178/189 (94%)
115/175 (65%)
151/160 (94%)
170/186 (91%)
94/150 (62%)
113/123 (91%)
173/259 (67%)
196/217 (90%)
154/199 (77%)
139/186 (74%)
127/127 (100%)
Tissue
Endosperm
Endosperm
Endosperm
Endosperm
Endosperm
Embryo
Embryo
Endosperm
Embryo
Embryo
Embryo
Embryo
Endosperm
Embryo
Embryo
Embryo
Embryo
Embryo
Clone No.
1
1
embryo
1
embryo
1
embryo
1
1
1
1
1
1
1
1
embryo
1
1
1
1
2
2
54/90 (60%)
44/56 (78%)
65/154 (42%),
Endosperm
Endosperm embryo
Endosperm embryo
2
2
1
112/266 (42%)
16/26 (61%)
67/75 (89%)
107/115 (93%)
Embryo
Endosperm
Endosperm
Endosperm
1
1
1
1
N. tabacum
O. sativa
H. vulgare
T. aestivum
A. thaliana
Drosophila
melanogaster
O. sativa
96/127 (75%)
83/113 (73%)
236/246 (95%)
162/224 (72%)
145/178 (81%)
131/174 (75%)
Endosperm
Endosperm embryo
Undetected
Endosperm embryo
Embryo
Embryo
1
1
1
1
1
1
71/90 (78%)
Endosperm
1
T. aestivum
O. sativa
123/190 (65%)
232/261 (88%)
Endosperm embryo
Endosperm
3
1
H. vulgare
Z. mays
215/240 (89%)
204/266 (77%)
Endosperm
Endosperm
1
1
T. aestivum
T. aestivum
T. aestivum
O. sativa
Orobanche ramosa
221/258 (85%)
113/132 (86%)
208/219 (94%)
68/109 (62%)
163/231 (70%)
Endosperm
Endosperm embryo
Embryo
Embryo
Endosperm embryo
1
1
2
1
8
Malusx domestica
O. sativa
O. sativa
H. sapiens
Z. mays
O. sativa
O. sativa
76/112 (67%)
94/135 (69%)
108/129 (83%)
166/192 (86%)
69/110 (62%)
62/68 (91%)
188/256 (73%)
Embryo
Endosperm
Endosperm
Endosperm
Embryo
Embryo
Endosperm
1
1
1
1
1
1
1
３６８
Table 3
Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．３　２００４
(continued)
Accession No.
AY290734
AF 479042
AY290735
BU607246
AY290736
AY290729
AF475110
AF475115
AY290730
AF542966
Putative products
Similar to putative lipase
Putative fructose-bisphosphate aldolase
Cytochrome b5 reductase isoform II (NFR II)
Putative carboxyl-terminal proteinase
Isopentenyl pyrophosphate:dimethyllallyl
pyrophosphate isomerase
DTDP-glucose-4-6-dehydratase-like protein
Myo-inositol 1-phosphate synthase
Ethylene-forming-enzyme-like dioxygenaselike protein
MGDG synthase type A
Geranylgeranylated protein ATGP1
Glycolytic glyceraldehydes-3-phosphate
dehydrogenase
Fructan 6-fructosyltransferase
Photosystem II OE17 protein
C13 endopeptidase precursor
Ubiquitin
Lon protease
AF475109
AF479035
AF475127
Ubiquitin fused to ribosomal protein L40
Proteinase inhibitor (Rgpi9)
Hypothetical protein XP-196551
AF542190
AF542968
BU607140
AF542970
AF475112
AY290728
glutenins, gliadins and other storage proteins. Some are
involved in starch metabolism (6), such as small subunit
of ADP glucose pyrophosphorylase and starch branching enzyme 2. Some other genes (10) were identified as
well.
2.4 Putative new genes
To classify these ESTs, the criteria to define sequence
identity was referred to ESTs research in Arabidopsis (Höfte
et al., 1993). The limit values are as follows: more than or
equal to 30% over a length of 50 amino acids, or 50% over
a length of 20 amino acids. Among the ESTs identified as
putative new plant genes (Table 3), thirty-two cDNAs (26%)
represented new wheat gene family members and 90 (74%)
were similar to genes from other plant species.
Most abundant sequences identified were classified into
two categories (Table 3): first, corresponding to the genes
(31) involved in gene/protein expression machinery, such
as ribosomal proteins, translation initiation factors, elongation factors, and some transcriptional factors; second,
encoding seed storage proteins (24), such as low and high
molecular weight glutenins, gliadins and other seed storage proteins. Most of sequences for storage proteins are
more than one, and even 18 clones, such as the gene encoding alpha-gliadin storage protein (Table 3). Results of
Table 2 and Table 3 indicate that the genes for the synthesis of storage proteins were actively transcribed and
Organism
Identity (aa)
O. sativa
110/136 (81%)
O. sativa
173/178 (97%)
Z. mays
56/59 (94%)
Gossypium hirsutum 64/184 (34%)
O. sativa
159/189 (84%)
Tissue
Clone No.
Endosperm
1
Embryo
1
Endosperm embryo
1
Endosperm embryo
1
Endosperm embryo
1
A. thaliana
H. vulgare
O. sativa
221/258 (85%)
241/243 (99%)
36/82 (43%)
Endosperm embryo
Embryo
Endosperm
1
1
1
Glycine max
O. sativa
H. vulgare
151/178 (84%)
133/195 (68%)
84/84 (100%)
Embryo
Undetected
Undetected
1
1
1
Agropyron cristatum
Z. mays
H. vulgare
H. vulgare
Dichanthelium
lanuginosum
O. sativa
O. sativa
Mus musculus
16/52 (30%)
81/182 (44%)
59/76 (77%)
80/114 (70%)
142/175 (81%)
Undetected
Undetected
Endosperm
Endosperm
Endosperm embryo
1
1
1
1
1
67/73 (92%)
41/69 (59%)
24/28 (85%)
Undetected
Endosperm
Endosperm
1
1
2
translated during seed development.
Another category of interesting sequences is those
homologous to the genes that may play important roles
during plant development (Table 3). Those genes include
S-locus protein 5, gigantea-like protein (controlling flowering time), putative embryogenesis-abundant protein, etc.
We also found a number of genes encoding enzymes
involved in protein structure formation, protein degradation,
starch metabolism and other biochemical metabolisms. In
addition, there were 70 sequences whose identities were
not determined because they have no or very low homologies with the sequences in GenBank (data not shown).
2.5 Tissue-specific expression
To confirm the seed-specific cDNAs, Northern hybridizations were carried out with three genes, respectively. As
shown in Fig.2, the hybridization signals of both genes
corresponding to gigantea-like protein (AF469490) and
putative embryogenesis-abundant protein (BU607235)
whose expression was detected only in the embryo by differential screening were detected in the young seed and
embryo, and the transcripts of the gene for avenin which
was screened out only in the endosperm by differential
screening (AF469489) were detectable in the endosperm.
These results indicate that the three clones are expressed
in the same tissue-specific manner as that shown by differential screening.
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum
Fig.2. Northern hybridization showing the expression patterns
of three genes. A. A gene encoding gigantea-like protein
(AF469490). B. A gene encoding putative embryogenesis-abundant protein (BU607235). C. A gene encoding avenin (AF469489).
D. 18S rRNA as a control. Lanes 1, root; 2, leaf; 3, ovary at
anthesis; 4, seed at 5 d after pollination (DAP) ; 5, embryo at 10
DAP; 6, embryo at 15 DAP; 7, endosperm at 10 DAP; 8, endosperm at 15 DAP; 9, endosperm at 20 DAP.
３６９
the other is that some genes from seed coats (including
pericarp) in the cDNA library were not involved in the synthesis of probes.
The sequence analysis demonstrated that those sequences correspond to the genes encoding seed storage
proteins or involved in biochemical metabolisms,
development, and other biological processes during seed
development. The data provide important information to
improve flour quality and understand the functions of
genes. Right now, a few genes have been chosen to improve the starch quality of flour and identify their biological functions by genetic transformation .
Acknowledgements: The authors would like to thank Dr.
LI Jia-Yang for his generosity to provide the Biomek 2000
HDRT system, Dr. BAO Fang for her technical assistance
in making the high density filters and Dr. LI Xing-Guo for
making figures.
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3 Discussion
cDNA array screening and sequence analysis have led
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genes in Arabidopsis, rice and wheat (Höfte et al., 1993;
Kawasaki et al., 2001; Luo et al., 2002; Rao et al., 2002). In
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sequences expressed in the embryo, endosperm or both
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the expression of these genes is directly or indirectly regulated by pollination. Among them, 122 putative new seed
expressed genes are identified with above techniques, and
these genes represent new wheat gene family members and
are similar to the ones from other plant species.
From 800 clones, 216 unique sequences have been
identified, indicating that there is higher redundancy of
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(Managing editor: ZHAO Li-Hui)