Download cDNA Sequences of Three Kinds of /3

DNA RESEARCH 2, 21-26 (1995)
cDNA Sequences of Three Kinds of /3-tubulins from Rice
Yasunori KOGA-BAN,*-* Tomoya NIKI,^ Yoshiaki NAGAMURA, Takuji SASAKI, and Yuzo MiNOBEtt
Rice Genome Research Program, National Institute of Agrobiological Resources, 1-2
Kannondai 2-chome, Tsukuba, Ibaraki 305, Japan/Institute of Society for Techno-Innovation of
Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305, Japan
(Received 31 January 1995)
Abstract
Complete nucleotide sequences of three kinds of rice /3-tubulin cDNA clones (pTUB22, R1623 and
R2242) were determined. Southern hybridization indicated that these /3-tubulins consist of one gene family.
Using RFLP mapping, these three /3-tubulin cDNAs were mapped to different chromosomes indicating at
least three loci for the /3-tubulin gene. The deduced amino acid sequences of these cDNAs showed a high
similarity to other plant /3-tubulins. The asparagine residue located at the 100th amino acid from the Nterminus of plant /3-tubulins was also conserved with these three /3-tubulins. This asparagine is thought to
be responsible for the sensitivity against rhizoxin, the toxin of the pathogen of rice seedling blight, Rhizopus
sp. a soil-borne microorganism. Expression of the three /3-tubulin genes was analyzed by Northern blotting
and all three clones were expressed in root, the possible target tissue of rhizoxin. These results suggest
that these clones are candidates of /3-tubulins targeted by rhizoxin.
Key words: Oryza sativa; rhizoxin sensitivity; seedling blight disease; gene family; RFLP mapping
1.
Introduction
Microtubules are involved in several basic processes of
eukaryotic cells, including cell division, cell motility, intracellular transport and as a component of the cytoskeleton determining cell morphology. Especially in higher
plants, cortical microtubules orient the extracellular deposition of cellulose microfibrils.1"3 The major structural
components of microtubules are the highly conserved aand /3-tubulins. Both a- and /3-tubulins are encoded
by a large gene family in higher organisms, including
plants.4~6 For example, Arabidopsis thaliana contains at
least six expressed a-tubulin genes and at least nine expressed /3-tubulin genes.5'6 Since each tubulin gene shows
a different tissue specific expression pattern, each /3tubulin is suggested to have a different function.6 It is
also assumed that each /3-tubulin has a different role at
different intracellular locations.
Rice seedling blight is one of the most serious diseases
of rice at the seedling stage. This disease is induced by
the rhizoxin, produced by the fungus Rhizopus chinensis,
and causes the inhibition of root growth and finally results in plant death.7'8 This symptom is attributed to the
*
t
tt
Communicated by Mituru Takanami
To whom correspondence should be addressed. Tel. +81-17728-1015, Fax. +81-177-28-1017
Present address: Division of Gene Engineering, Aomori Green
Bio Center, 221-10, Yamaguchi, Nogi, Aomori, 030-01, Japan
Present address: Department of Molecular Biology, National
Institute of Agrobiological Resources, 1-2, Kannondai 2-chome,
Tsukuba, Ibaraki 305, Japan
inhibition of polymerization of /3-tubulin by rhizoxin.8'9
The sensitivity of /3-tubulin to rhizoxin is due to the
100th amino acid residue of /3-tubulin, since isoleucine
or valine instead of asparagine at the 100th residue in
yeast results in a resistant phenotype.9 For the progress
in breeding new types of rice which have resistance to
seedling blight disease using genetic engineering, characterization of /3-tubulin genes is important. Therefore,
we have characterized three /3-tubulin cDNA sequences,
genomic structures and expression patterns in various tissues.
2.
Materials and Methods
2.1. Cloning of (3-tubulin cDNAs
The PCR products from rice (Nipponbare) genomic
DNA were synthesized using two primers: one from
amino acids 158 to 164 of maize /3-tubulin, and the other
from 397 to 403.4 The amplified DNAs were used as a
probe for plaque hybridization. About 5xlO5 plaques in
rice root callus cDNA library in AgtlO (gift from Dr. M.
Wada of Tsukuba University) were screened and about
100 plaques were picked up as first positives. After secondary screening, 17 clones were isolated and 1 of them
was chosen as pTUB22. Two other cDNA clones, R1623
and R2242, were picked up in a large-scale cDNA sequencing of root cDNA library, and the method of cDNA
cloning was the same as that used for a rice callus cDNA
library.10
[Vol. 2.
Rice /3-tubulins cDNA Sequences
32
pTUB22
1854 bp
R1623
1567 bp
R2242
1668 bp
ATG"
5'UTS = 120bp
ORF = 1335bp (444 amino acids)
AAAAAA
ORF = 1344bp (447 amino acids)
3'UTS = 202bp
FTTAAH
DATG"
5'UTS = 58bp
3'UTS = 399bp
"TAAI
lATG"
5'UTS = 21 bp
1AAAAAA
"TAGf
ORF = 1344bp (447 amino acids)
IAAAAAA
3'UTS = 266bp
Figure 1. The outline of the three /3-tubulin cDNA clones of rice. Each clone contains 5'-untranslated sequences (5' UTS), one open
reading frame (ORF) and 3'-untranslated sequences (3' UTS).
library.10
2.2. DNA sequencing
pTUB22 was subcloned into pBluescriptll SK+. Two
/3-tubulin cDNA clones, R1623 and R2242. were cloned
as plasmids in pBluescriptll SK+. Both strands of
these three clones were then sequenced. DNA fragments either produced by restriction enzyme or sonication were inserted into M13mpl8 or M13mpl9. Singlestranded template DNAs were prepared 11 and sequenced
by the dideoxynucleotide chain termination method. 12
The DDBJ accession numbers are D13224 for pTUB22,
D30716 for R1623 and D30717 for R2242.
2.3. Southern and Northern hybridization
Rice genomic DNA was isolated from Oryza sativa
japonica c.v. Nipponbare and Oryza sativa indica c.v.
Kasalath using green leaves.13 DNA (5 /xg) was digested
with each of four kinds of restriction enzymes and transferred onto a nylon membrane. RNA was prepared from
root, callus, green and etiolated shoot according to Hattori et al. 14 Poly(A) + RNA was isolated with latex particles of Oligotex™-dT30. 15 Both Southern and Northern
hybridizations were performed by ECL labeling and detection system (Amersham, Buckinghamshire, UK).
2.4- Linkage analysis
The map positions of /3-tubulin cDNAs were determined using MAPMAKER 16 by placing them onto a
map of approximately 1,000 restriction fragment length
polymorphism (RFLP) markers reported by N. Kurata
et al. 17
3.
Results and Discussion
3.1. Structure and features of rice 0-tubulins
Figure 1 shows the structure of three completely sequenced /3-tubulin cDNA clones. Similarity in the nucleotide sequence among the three clones was high in
their open reading frames. For example, similarities between pTUB22 and R2242, pTUB22 and R1623. and
R1623 and R2242 are 86.2%, 82.4%, and 81.8%, respectively. Rice /3-tubulin coding sequences showed high similarity to maize /3-tubulin. For example, there is 83.3% homology between pTUB22 and maize 0-2 tubulin (accession no. X52879).4 However, there are no significant similarities in the 5'- and 3'-untranslated sequences among
these clones. Even the length of the 3'-untranslated
regions is different (Fig. 1). The nucleotide composition is different in the open reading frame and the 3'untranslated region. GC contents of the coding regions
are 56.2%: (pTUB22), 52.5% (R1623) and 59.3% (R2242).
indicating relatively high GC contents. On the other
hand, GC contents of 3'-untranslated regions are 45.6%,
46.5% and 40.6% for pTUB22, R1623 and R2242. respectively.
Deduced amino acid sequences of rice /3-tubulins were
aligned with those of maize 0-2, Arabidopsis TUB1 (accession no. M20405) TUB2 (accession no. M84700) and
TUB9 (accession no. M84706) (Fig. 2). The gene family
of Arabidopsis /3-tubulin has been characterized extensively, and nine /3-tubulin genes were grouped into three
sub-families.6 The three Arabidopsis /3-tubulins aligned
are representative of each sub-group. The similarity of
the amino acid sequence in the /3-tubulin family is quite
high. More than 90% of amino acid residues are identical to each ./3-tubulin, and the sites of different amino
acids are specific. Some 10 amino acid residues at the
carboxyl terminus vary considerably. This characteristic
in the C-terminal region of /3-tubulin is quite common
among other plant ./3-tubulins. The region from amino
No. 1]
Y. Koga-Ban et al.
pTUB22
R1623
R2242
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QCGNQIGAKF
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WEVICDEHGV
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DATGRYAGDS DLQLERINVY
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51
pTUB22
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>». thaliana
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LISKIREEYP DRMMLTFSVF PSPKVSDTW EPYNATLSVH QLVENADECM
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R2242
********** ********** ********** **********
**********
Z.maysf>2
********** ****M***** ********** **********
**********
A. thaliana
TUB1
********** ********** ********** **********
**********
4 . thaliana
TUB2
********** ********** ********** **********
**********
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TUB9
********** * * * * j j * * * » *
********** **********
**********
pTUB22
R1623
R2242
Z.maystti
A. thaliana TUB1
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201
pTUB22
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A. thaliana TUB2
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VLDNEALYDI CFRTLKLATP TFGDLNHLIS
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RKLAVNLIPF PRLHFFMVGF APLTSRGSQQ YRALTVPELT QQMWDAKNMM
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CAADPRHGRY LTASAMFRGK MSTKEVDEQM LNVQNKNSSY FVEWIPNNVK
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Figure 2. Amino acid sequence comparisons of /3-tubulin polypeptides from rice (pTUB22, R1623 and R2242), maize (/32)4 and
Arabidopsis (TUB1, TUB2 and TUB9).6 Asterisks indicate amino acids identical to those of pTUB22. Amino acid sequence of the
widely varying carboxy terminal is indicated.
Rice /3-tubulins cDNA Sequences
24
1
I 5 I 1
OS
<< NK NK NK NK
[Vol. 2,
j i
00
0Q
NK NK NK NK <<
\
Figure 3. Southern hybridization of rice genomic DNA. Each lane contains 5 fig of genomic DNA digested with the indicated restriction
enzyme. Genomic DNA was extracted from Nipponbare (N) or Kasalath (K). The left panel was hybridized with a coding region
probe, and the right panel was hybridized with 3'-untranslated region probe. Arrowheads indicated the polymorphic bands of
ffindlll-digested DNA.
acids 12 to 20 contains several alterations.
/3-Tubulin is the target protein of rhizoxin, toxin of the
rice seedling blight disease.7'8 The sensitivity to rhizoxin
depends on the species of the 100th amino acid residue
from the N-terminus of /3-tubulin.9 All the three rice /3tubulin clones sequenced had asparagine at the 100th
residue which is always observed in /3-tubulin sensitive
to rhizoxin in yeast18'19 and Neurospora crassa.20
3.2. Gene family and chromosomal loci of rice j3tubulins
To estimate the gene number of rice /3-tubulins, Southern hybridization to rice genomic DNA (c.v. Nipponbare and c.v. Kasalath) was performed. To detect all
the /3-tubulin genes, the sequence in the open reading
frame of pTUB22 was used as a probe. Seven to ten
bands were clearly observed in Nipponbare (Fig. 3, left
panel). For example, ffindlll digestion of Nipponbare
DNA produced positive fragments of 15 kb, 10 kb, 8.2
kb, 4.4 kb, 3.5 kb, 3.0 kb, 2.5 kb, 1.0 kb, 0.76 kb
and 0.57 kb. Some of these bands, such as 15 kb and
1.0 kb, were not detected in Kasalath, thus indicating
polymorphism. We assume that in conjunction with
other data on cDNA sequence analysis (to be published),
that the genes of rice /3-tubulin consist of at least seven
genes in one gene family, as with maize and Arabidopsis
thaliana,4'6 and some of them are polymorphic among
rice varieties. To identify the fragments corresponding to
pTUB22, the 3'-untranslated sequence was probed using
the same filter (Fig. 3, right panel). This hybridization
gave a clone-specific signal, and //irailll-digested DNA
showed polymorphism of 0.76 kb (Nipponbare) and 0.60
kb (Kasalath). Using the 3' untranslated sequence probe,
RFLPs of R1623 and R2242 were also detected. The
three clones were mapped on chromosome 3 (pTUB22),
chromosome 1 (R1623) and chromosome 2 (R2242), respectively (Fig. 4). In animals, gene families are often
clustered at one chromosomal region, such as human
globin genes.21 On the other hand, plant gene families
are often dispersed throughout the genome. From our
rice RFLP map, peroxidase, chitinase, alcohol dehydrogenase and other gene families were also dispersed.17
Y. Koga-Ban et al.
No. 11
Chr. 1
25
Chr. 3
Chr.2
O
pTUB22•*
5(1
•£
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(0
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o>
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o
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pTUB22
R 1 6 2 3 T ••
R1623
R2242
R2242 - -
Figure 5. Northern hybridization of poly(A)+ RNA from several
rice tissues. Each lane contains 1 fig of poly(A)"1" RNA from
green shoot, etiolated shoot, root and callus. The filter was
hybridized with 3'-untranslated region probe.
Figure 4. Location of /3-tubulin cDNA sequenced in this paper
on the RFLP linkage map.
3.3.
Expression profile of rice [3-tubulins
The existence of a gene family suggests the possibility that each /3-tubulin is expressed and functions in a
specific manner within a specific tissue. To study this,
the tissue-specific expression pattern of pTUB22, R1623
and R2242 was analyzed by Northern hybridization using each 3'-untranslated sequence as a probe (Fig. 5).
The three clones showed different expression patterns.
As rice seedling blight disease causes growth defects in
root, /3-tubulin which is expressed in root is a logical target of rhizoxin. Although the gene of R1623 is relatively
highly expressed in root, the other two genes examined
are also expressed in root. This means that at least three
/?-tubulin genes may be involved in rhizoxin sensitivity in
root.
Acknowledgments: We thank Dr. Ichiro Yahara
for kindly providing information on the /3-tubulin gene
and rhizoxin at the start of this research and Dr. Ilkka
Havukkala for critical reading of this manuscript and Dr.
K. Kobayashi for his support and encouragement, and
Mrs. N. Kawaguchi and S. Hoshi for their technical assistance.
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Rice /3-tubulins cDNA Sequences
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