全 文 ：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: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 363-370
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 com-
posed of the embryo, the endosperm, and the seed coat
(including pericarp). The embryo developed from a fertil-
ized 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 accu-
mulates 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 de-
velopmental events and biochemical metabolism during
seed development, little is known on their molecular mecha-
nisms (Lopes and Larkins, 1993; Buchanan et al., 2000). In
recent years, new molecular tools have been used to iden-
tify the genes of plants, such as cDNA array and expressed
sequence tag techniques which have led to the rapid isola-
tion 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 func-
tion that may be involved in biochemical metabolisms, de-
velopmental 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 subse-
quently 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
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004364
fractionated by Sizesep 400 Spun Column, and the frac-
tions 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 mL 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 mg/mL salmon sperm
DNA. Three probes were prepared by reverse transcription
of 10 mg 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 ℃.
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
mg of total RNA was fractionated by gel electrophoresis in
1.2 % formaldehyde agarose gels and transferred from aga-
rose gels to nylon membrane. Prehybridization was per-
formed 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. Autorad-
iography 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 in-
serts 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 hybrid-
ized 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.
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum 365
As indicated in Table 1, the hybridization signals of total
6 826 clones were detected. The number of clones represent-
ing 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.
and a few clones undetected in three tissues were chosen
for sequencing. By the analysis of sequences, they repre-
sent 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 1 Result of differential hybridization with the probes of
ovary, embryo or endosperm
No. Expressive tissue Clone No.
1 Ovary 538
2 Embryo 274
3 Endosperm 1 107
4 Embryo and endosperm 478
5 Embryo and ovary 464
6 Endosperm and ovary 522
7 Embryo, endosperm and ovary 3 443
8 Undetected in three tissues 3 016
2.2 Expressed sequence tags (ESTs) analysis and data-
base comparison
Based on the hybridization results, 800 of 1 856 clones
Table 2 Expressed sequence tags (ESTs) identical to wheat genes
Accession No. Putative product Identity (aa) Tissue Clone No.
BU607223 Chloroform/methanol soluble 139/139 (100%) Endosperm 2
(CM16) protein
BU607210 CM1 protein of alpha-amylase 144/145 (99%) Endosperm 2
tetrameric inhibitor
BU607213 CM2 protein of alpha-amylase inhibitor 72/72 (100%) Endosperm 2
BU607216 CM3 protein of alpha-amylase inhibitor 137/138 (99%) Endosperm 4
BU607192 Starch branching enzyme 2 53/54 (98%) Endosperm 1
BU607195 Small subunit ADP glucose pyrophosphorylase 173/173 (100%) Endosperm 1
BU607224 LMW glutenin U86029 40/41 (97%) Endosperm 7
BU607170 LMW glutenin U86030 39/41 (95%) Endosperm embryo 7
BU607168 Gamma-gliadin class B-III 45/45 (100%) Endosperm 4
BU607165 Gamma-gliadin class mrna 36/36 (100%) Endosperm 1
BU607165 LMW glutenin U86027 36/36 (100%) Endosperm 1
BU607168 LMW glutenin U86029 45/45 (100%) Endosperm embryo 5
BU607188 LMW glutenin U86028 24/24 (100%) Endosperm embryo 1
BU607169 LMW glutenin U86029 60/62 (97%) Endosperm embryo 2
AF 479046 Elongation factor 1 127/131 (96%) Embryo 2
BU607199 Ras related GTP binding protein 185/185 (100%) Embryo 1
BU607183 Histone H2B 92/92 (100%) Embryo 1
BU607176 Serpin 217/220 (98%) Endosperm embryo 3
BU607211 Beta purothionin precursor 115/116 (99%) Endosperm 3
BU607172 Puroindoline-a 148/148 (100%) Endosperm 1
BU607250 Sec 61 protein 33/33 (100%) Endosperm embryo 1
BU607177 Cytosolic glyceraldehydes-3-phosphatee 215/217 (99%) Endosperm 1
hydrogenase
BU607205 Inducible phenylalanine ammonia-lyse 178/179 (99%) Undetected 1
AY290720 Carboxypeptidase D 119/119 (100%) Endosperm embryo 1
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004366
Table 3 ESTs highly homologous to known genes in GenBank
Accession No. Putative products Organism Identity (aa) Tissue Clone No.
AY290731 ADP glucose pyrophosphorylase Triticum aestivum 162/221 (73%) Embryo 1
BU607232 0.19 Alpha-amylase inhibitor T. aestivum 98/124 (79%) Embryo 19
AY290721 Alpha-amylase inhibitor Hordeum vulgare 130/131 (99%) Embryo 1
BU607225 CM17 protein of alpha-amylase inhibitor T. aestivum 122/139 (87%) Endosperm 2
BU607229 Trypsin inhibitor CMx precursor T. aestivum 119/146 (81%) Endosperm 1
AF470353 Beta-amylase H. vulgare 220/260 (84%) Endosperm embryo 4
BU607197 High molecular weight (HMW) glutenin subunit T. aestivum 105/167 (62%) Endosperm embryo 5
BU607198 HMW glutenin subunit 1Ax1 T. aestivum 265/387 (68%) Endosperm embryo 2
BU607212 Low molecular weight (LMW) glutenin U86026 T. aestivum 115/171 (67%) Endosperm 5
BU607215 LMW glutenin subunit group 11 type VI T. aestivum 102/166 (61%) Endosperm embryo 11
BU607209 LMW glutenin subunit group 3 type II T. aestivum 91/161 (56%) Endosperm embryo 4
BU607202 LMW glutenin X84960 T. aestivum 101/161(63%) Endosperm embryo 6
BU607171 LMW glutenin protein 1Agi T. aestivum 41/63 (65%) Endosperm embryo 1
BU607201 LMW glutenin M11335 T. aestivum 41/63 (65%) Endosperm embryo 3
BU607167 LMW glutenin subunit group 10 type V T. aestivum 85/173 (49%) Endosperm 1
BU607218 LMW glutenin subunit group 7 type IV T. aestivum 69/145 (47%) Endosperm 1
BU607180 Alpha-gliadin storage protein T. aestivum 83/161 (51%) Endosperm embryo 18
BU607217 Gamma-gliadin clone 10d11 T. aestivum 78/131 (59%) Endosperm 2
BU607230 Gamma-gliadin clone G2656 T. aestivum 95/145 (65%) Endosperm 1
BU607194 Alpha-/beta-gliadin T. aestivum 81/120 (67%) Endosperm 2
BU607174 Alpha-/beta-gliadin precursor protein T. aestivum 89/144 (61%) Endosperm embryo 1
BU607221 Gamma-gliadin class B-I T. aestivum 106/115 (92%) Endosperm 3
BU607166 Alpha-gliadin T. aestivum 113/141 (80%) Endosperm 2
AF475121 Seed globulin Aegilops tauschii 162/222 (72%) Endosperm embryo 3
AF469489 Avenin Avena sativa 38/66 (57%) Endosperm embryo 8
BU607262 Avenin fast component N9 A. sativa 49/86 (57%) Endosperm embryo 1
BU607255 Avenin-3 precursor A. sativa 31/62 (50%) Endosperm embryo 1
AF470352 Alcohol-soluble avenin-3=23.2 kD protein A. sativa 68/130 (52%) Endosperm 1
BU607139 Thaumatin-like protein Oryza sativa 65/150 (43%) Endosperm 1
BU607220 12s Globulin A. sativa 63/91 (69%) Endosperm 1
BU607175 Hordoindoline-b H. vulgare 34/79 (43%) Undetected 1
BU607187 Puroindoline-b T. aestivum 113/143 (79%) Endosperm 1
AY290722 Ribosomal protein L39 O. sativa 44/46 (96%) Embryo 1
AF475098 Ribosomal protein S4 Nicotiana tabacum 47/56 (83%) Embryo 1
AF475122 Ribosomal protein O. sativa 93/113 (82%) Endosperm embryo 2
AF475123 Acidic ribosomal protein P2a-2 Z. mays 61/112 (54%) Endosperm embryo 2
AF475114 60S Ribosomal protein L21 O. sativa 149/164 (90%) Embryo 2
AF475116 Ribosomal protein L30 Z. mays 36/46 (78%) Embryo 1
AF479033 40S Ribosomal protein S3 T. aestivum 76/78 (97%) Endosperm 1
AY290723 Acidic ribosomal protein P1a Z. mays 59/68 (87%) Embryo 1
AF 479045 Ribosomal protein S28 H. vulgare 50/51 (98%) Embryo 2
AF 479049 60S Ribosomal protein L2 Arabidopsis thaliana 77/77 (100%) Embryo 1
AF 479048 Ribosomal protein L17 Castanea sativa 139/140 (99%) Embryo 2
AF475108 Acidic Ribosomal protein P2b Z. mays 35/113 (30%) Endosperm 2
AF542969 Ribosomal protein L19 O. sativa 133/159 (83%) Embryo 1
AF475099 60S Ribosomal protein L15 Homo sapiens 145/193 (75%) Undetected 1
AY290724 Ribosomal protein S7 H. vulgare 54/55 (98%) Embryo 1
AY290725 Ribosomal protein S3a O. sativa 162/211 (76%) Undetected 1
BU607196 Tap-nuclear mRNA export protein T. aestivum 55/78 (70%) Endosperm 1
AF470354 Putative zinc finger protein O. sativa 82/110 (74%) Embryo 1
AF475129 Elongation factor 2 Beta vulgaris 153/166 (92%) Endosperm embryo 1
AF470357 Transcription factor TFIIB O. sativa 45/49 (91%) Embryo 1
BU607219 RNA-binding protein T. aestivum 24/60 (40%) Endosperm 1
AF479046 Elongation factor 1 elongation beta T. aestivum 127/131 (96%) Embryo 2
AF479057 Ocs-binding factor 1 Z. mays 108/133 (81%) Endosperm 1
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum 367
Table 3 (continued)
Accession No. Putative products Organism Identity (aa) Tissue Clone No.
AY290726 Translation initiation factor Pisum sativum 158/228 (69%) Endosperm 1
BU607190 Eukaryotic translation initiation factor 4B T. aestivum 70/230 (30%) Endosperm 1
AF475102 26S Proteasome AAA-ATPase subunit RPT5a A. thaliana 179/184 (97%) Endosperm embryo 1
BU607238 Similarity to guanylate binding protein A. thaliana 81/188 (43%) Endosperm embryo 1
BU607240 Putative DEAD/DEAH box RNA helicase protein O. sativa 164/189 (86%) Endosperm embryo 1
BU607242 bZIP protein A. thaliana 58/152 (38%) Embryo 1
BU607236 Ara4-interacting protein A. thaliana 88/266 (33%) Embryo 1
AF479034 ADP-ribosylation factor O. sativa 178/189 (94%) Endosperm 1
AF479053 S-locus protein 5 Brassica rapa 115/175 (65%) Embryo 1
AF470356 QM protein (tumor suppressor) O. sativa 151/160 (94%) Embryo 1
AF469490 Gigantea-like protein (controlling flowering time) H. vulgare 170/186 (91%) Embryo 1
BU607235 Putative embryogenesis-abundant protein O. sativa 94/150 (62%) Embryo 1
AF475105 Possible apospory-associated protein Pennisetum ciliare 113/123 (91%) Endosperm embryo 1
AY290732 Putative senescence-associated protein P. sativum 173/259 (67%) Embryo 1
AF542972 Putative tumor suppressor O. sativa 196/217 (90%) Embryo 1
AF469492 MCM protein-like protein N. tabacum 154/199 (77%) Embryo 1
AF479043 Cyc 07 O. sativa 139/186 (74%) Embryo 2
BU607249 Histone H3 Onobrychis 127/127 (100%) Embryo 2
viciaefolia
AY290727 Putative cytochrome P450 O. sativa 54/90 (60%) Endosperm 2
AF470355 Metallothionein type 2 Poa secunda 44/56 (78%) Endosperm embryo 2
AF479051 Predicted RNA methylase like Caenorhabditis 65/154 (42%), Endosperm embryo 1
elegans
BU607252 110 kDa 4SNc-Tudor domain protein P. sativum 112/266 (42%) Embryo 1
BU607162 Putative UV-damaged DNA binding factor A. thaliana 16/26 (61%) Endosperm 1
AF475128 Abscisic acid-induced protein H. vulgare 67/75 (89%) Endosperm 1
AF475124 Glutathione peroxidase-like protein H. vulgare 107/115 (93%) Endosperm 1
AF479039 Mosaic virus helicase domain binding protein N. tabacum 96/127 (75%) Endosperm 1
AF542185 Glutaredoxin O. sativa 83/113 (73%) Endosperm embryo 1
AF475100 Catalase H. vulgare 236/246 (95%) Undetected 1
BU607200 Wali7 induced by aluminum T. aestivum 162/224 (72%) Endosperm embryo 1
AY290733 Cyclophilin A. thaliana 145/178 (81%) Embryo 1
BU607154 Similarity to gb|AF181686 membrane Drosophila 131/174 (75%) Embryo 1
protein TMS1d melanogaster
BU607141 Mitochondrial inner membrane translocating O. sativa 71/90 (78%) Endosperm 1
protein-like
BU607206 Protein disulfide isomerase 2 precursor T. aestivum 123/190 (65%) Endosperm embryo 3
AF475111 Putative 2,3-bisphosphoglycerate-independent O. sativa 232/261 (88%) Endosperm 1
phosphoglycerate mutase
AF479036 Formate dehydrogenase H. vulgare 215/240 (89%) Endosperm 1
AF479037 Putative 1-acyl-glycerol-3-phosphate Z. mays 204/266 (77%) Endosperm 1
acyltransferase
BU607214 Cytosolic 3-phosphoglycerate kinase T. aestivum 221/258 (85%) Endosperm 1
BU607233 Phosphoethanolamine methyl-transferase T. aestivum 113/132 (86%) Endosperm embryo 1
BU607189 Triosephosphat isomerase T. aestivum 208/219 (94%) Embryo 2
BU607184 Pectinesterase-like protein O. sativa 68/109 (62%) Embryo 1
AF475103 NADPH-dependent mannose 6-phosphate Orobanche ramosa 163/231 (70%) Endosperm embryo 8
reductase
BU607185 Pin1-type peptidyl-prolyl cis/trans isomerase Malusx domestica 76/112 (67%) Embryo 1
AF475125 Enoyl-Acp reductase O. sativa 94/135 (69%) Endosperm 1
AF475119 Succinate dehydrogenase subunit 3 O. sativa 108/129 (83%) Endosperm 1
AF479038 Holocarboxylase synthetase H. sapiens 166/192 (86%) Endosperm 1
AF475113 Histone acetyltransferase Z. mays 69/110 (62%) Embryo 1
AF479054 Ubiquinol reductase Cytochrome C reductase O. sativa 62/68 (91%) Embryo 1
BU607138 Similar to methylenetetrahydrofolate dehydrogenase O. sativa 188/256 (73%) Endosperm 1
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004368
glutenins, gliadins and other storage proteins. Some are
involved in starch metabolism (6), such as small subunit
of ADP glucose pyrophosphorylase and starch branch-
ing 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, elon-
gation factors, and some transcriptional factors; second,
encoding seed storage proteins (24), such as low and high
molecular weight glutenins, gliadins and other seed stor-
age proteins. Most of sequences for storage proteins are
more than one, and even 18 clones, such as the gene en-
coding alpha-gliadin storage protein (Table 3). Results of
Table 2 and Table 3 indicate that the genes for the synthe-
sis of storage proteins were actively transcribed and
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 flower-
ing 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 homolo-
gies with the sequences in GenBank (data not shown).
2.5 Tissue-specific expression
To confirm the seed-specific cDNAs, Northern hybrid-
izations 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 dif-
ferential 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 differ-
ential screening.
Table 3 (continued)
Accession No. Putative products Organism Identity (aa) Tissue Clone No.
AY290734 Similar to putative lipase O. sativa 110/136 (81%) Endosperm 1
AF 479042 Putative fructose-bisphosphate aldolase O. sativa 173/178 (97%) Embryo 1
AY290735 Cytochrome b5 reductase isoform II (NFR II) Z. mays 56/59 (94%) Endosperm embryo 1
BU607246 Putative carboxyl-terminal proteinase Gossypium hirsutum 64/184 (34%) Endosperm embryo 1
AY290736 Isopentenyl pyrophosphate:dimethyllallyl O. sativa 159/189 (84%) Endosperm embryo 1
pyrophosphate isomerase
AF542190 DTDP-glucose-4-6-dehydratase-like protein A. thaliana 221/258 (85%) Endosperm embryo 1
AF542968 Myo-inositol 1-phosphate synthase H. vulgare 241/243 (99%) Embryo 1
BU607140 Ethylene-forming-enzyme-like dioxygenase- O. sativa 36/82 (43%) Endosperm 1
like protein
AF542970 MGDG synthase type A Glycine max 151/178 (84%) Embryo 1
AF475112 Geranylgeranylated protein ATGP1 O. sativa 133/195 (68%) Undetected 1
AY290728 Glycolytic glyceraldehydes-3-phosphate H. vulgare 84/84 (100%) Undetected 1
dehydrogenase
AY290729 Fructan 6-fructosyltransferase Agropyron cristatum 16/52 (30%) Undetected 1
AF475110 Photosystem II OE17 protein Z. mays 81/182 (44%) Undetected 1
AF475115 C13 endopeptidase precursor H. vulgare 59/76 (77%) Endosperm 1
AY290730 Ubiquitin H. vulgare 80/114 (70%) Endosperm 1
AF542966 Lon protease Dichanthelium 142/175 (81%) Endosperm embryo 1
lanuginosum
AF475109 Ubiquitin fused to ribosomal protein L40 O. sativa 67/73 (92%) Undetected 1
AF479035 Proteinase inhibitor (Rgpi9) O. sativa 41/69 (59%) Endosperm 1
AF475127 Hypothetical protein XP-196551 Mus musculus 24/28 (85%) Endosperm 2
LI Jia-Rui et al.: Analysis of Seed-expressed Sequence Tags in Triticum aestivum 369
3 Discussion
cDNA array screening and sequence analysis have led
to the identification of a large number of new expressed
genes in Arabidopsis, rice and wheat (Höfte et al., 1993;
Kawasaki et al., 2001; Luo et al., 2002; Rao et al., 2002). In
this study, we have isolated 1 856 clones corresponding to
sequences expressed in the embryo, endosperm or both
embryo and endosperm, but not in the ovary. It suggests
the expression of these genes is directly or indirectly regu-
lated 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
sequences in the seeds. If more seed-expressed unique
sequences are to be isolated, in particular, less abundant
cDNAs, the normalized cDNA library of seed is required to
be constructed.
As shown in Table 1, a number of clones (3 016) could
not be detected by differential hybridization, and
theoretically, all of clones should have hybridization sig-
nals with at least one probe. There might be two reasons:
one is the transcripts of some clones representing sequences
which are less abundant in the seeds. In fact, X-ray films
had been in the dark only for 1 d in this experiment and
there is the possibility that the time of X-ray films for the
exposure in the dark was too short to detect the signals;
the other is that some genes from seed coats (including
pericarp) in the cDNA library were not involved in the syn-
thesis of probes.
The sequence analysis demonstrated that those se-
quences 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 im-
prove the starch quality of flour and identify their biologi-
cal 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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(Managing editor: ZHAO Li-Hui)