Báo cáo khoa học: Identification of rice TUBBY-like genes and their evolution Qingpo Liu docx

Identification of rice TUBBY-like genes and their evolution
Qingpo Liu
School of Agriculture and Food Science, Zhejiang Forestry University, Hangzhou, Lin’an, China
TUBBY-like proteins (TULPs) are present in eukary-
otes from single-celled to multicellular organisms. The
first TUBBY gene was identified in obese mice [1]. A
typical feature of TUBBY proteins is the approxi-
mately 270-amino acid tubby domain at their C-termi-
nal region. Accordingly, a number of such genes have
been successively isolated from other organisms, such
as Homo, Gallus, Xenopus, Zea and Arabidopsis [2],
based on sequence characteristics. Interestingly, these
genes form a small family in mammals, which consists
of TUBBY and four TULPs [1,3]. By contrast, plants
appear to harbour a large number of TULPs [2,4].
Moreover, compared with the high divergence of the
N-terminal sequence of animal TULPs, most plant
TULPs contain a conserved F-box domain at the cor-
responding region [5].
Although the high conservation of the tubby domain
across species suggests that TULPs may carry out cer-
tain fundamental biological functions in common [4],
little is known about their target genes or precise
action mechanisms. In animals, TULPs are important
for normal neuronal development and function [4]. It
has been shown that TULPs play crucial roles in vesic-
ular trafficking [6], mediation of insulin signalling [7]
and gene transcription [8]. In Arabidopsis, Lai et al. [2]
have demonstrated that TULP9 (AtTLP9) interacts
with Skp1-like 1 (ASK1). They speculated that, as well
as acting as transcription regulators, F-box domain-

containing plant TULPs should have cellular function
activities of F-box proteins in signal transduction. Sup-
pression and overexpression analyses of Arabidopsis
TULPs(AtTLPs) have shown that at least one AtTLP
may function in the abscisic acid-regulated pathway
[2].
Rice is one of the most important crops for human
consumption. The final completion of the Oryza sativa
genome [9] has made it possible to identify all the
TUBBY-like family members in this plant species at
the genome-wide level. In order to investigate the
evolution and divergence of TULPs in eukaryotes, a
phylogenetic, evolutionary and functional divergence
analysis of the TUBBY-like gene family has been
Keywords
evolution; functional divergence; phylogeny;
rice; TUBBY-like
Correspondence
Q. Liu, School of Agriculture and Food
Science, Zhejiang Forestry University,
Hangzhou, Lin’an, China
Fax: +86 571 86971117
Tel: +86 571 86971611
E-mail:
(Received 28 September 2007, revised
7 November 2007, accepted 12 November
2007)
doi:10.1111/j.1742-4658.2007.06186.x
The identification of TUBBY-like genes in organisms ranging from single-
celled to multicellular eukaryotes has allowed the phylogenetic history of

this gene family to be traced back to the early evolutionary stages of
eukaryote development. Rice TUBBY-like genes were located on chromo-
somes 1, 2, 3, 4, 5, 7, 8, 11 and 12 without any obvious clustering. On a
genomic scale, it was revealed that the rice TUBBY-like gene family proba-
bly evolved mainly through segmental duplication produced by polyploidy.
The altered selective constraints (or site-specific rate changes), related to
functional divergence during protein evolution between plant and animal
TUBBY-like genes, were statistically significant. Based on posterior proba-
bility analysis, five amino acid sites (103, 312, 315, 317 and 319) are
thought to be responsible for functional divergence.
Abbreviations
EST, expressed sequence tag; GEO, gene expression omnibus; NCBI, National Center for Biotechnology Information; RED, Rice Expression
Database; RGP, Rice Genome Project; TIGR, Institute for Genomic Research; TULP, TUBBY-like protein.
FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS 163
performed. This first description of the whole rice
TUBBY-like gene family will aid in our understanding
of the function of TULPs in plants.
Results and Discussion
Identification and sequence analysis
of rice TULPs
After carefully surveying the rice genome, 14 genes
were defined as rice TULPs(OsTLPs; Table 1).
Domain analysis showed that, with one exception
(OsTLP13), a conserved F-box domain (PF00646) was
found at the N-terminal region of OsTLPs. In addition
to the tubby (PF01167) and F-box domains, most
OsTLPs had two PROSITE signature patterns, termed
TUB1 (PS01200) and TUB2 (PS01201), at their C-ter-
minal region, evidence that strongly supports their
reliability as members of the rice TUBBY-like family.

The results of expressed sequence tag (EST) and
cDNA blast searches showed that 13 of the 14
OsTLPs matched at least one significant EST sequence,
and 11 of the 14 OsTLPs exactly matched a corre-
sponding full-length cDNA sequence in the GenBank
or KOME database (Table 1). These results indicated
that most TULPs are expressed in the rice genome.
The comparison of gene structure showed a con-
served exon number pattern in OsTLPs, although the
length of introns was different (Table 1). With three
exceptions (OsTLP7, OsTLP13 and OsTLP14), all of
the OsTLPs had four exons. Further analysis found
that the F-box domain, except for OsTLP13, was
encoded by the first exon, whereas the tubby domain
was encoded by the following three exons.
Phylogenetic analysis
blast search against the GenBank database showed
that both single-celled (Ostreococcus tauri, Tetrahy-
mena thermophila, etc.) and multicellular (mammals,
plants, etc.) organisms possessed tubby domain-con-
taining proteins, indicating their functional importance
for eukaryotes. In this study, in order to avoid biased
analysis, the C-terminal tubby domain, rather than the
highly divergent N-terminus, was used to perform phy-
logenetic analysis. Supplementary Fig. S1 shows the
multiple sequence alignment of tubby domains. The
phylogenetic trees reconstructed using the neighbour-
joining and minimal evolution methods in mega v3.1
(trees not shown) and the neighbour-joining method in
phylip (Fig. 1) revealed similar topologies.

At first glance, three clades (plant, animal and mixed
clades) were evident in the tree (Fig. 1). In animals,
the T. thermophila TULP (TtTLP) was evolutionarily
distant from the other animal TULPs, suggesting long
periods of divergence from this metazoan. Further
analysis showed that invertebrate (insects, nematodes
and Echinodermata) and vertebrate metazoans were
not clustered into distinct clades, indicating that the
divergence of animal TULPs probably occurred before
the invertebrate–vertebrate split. In addition, mamma-
lian TULPs were clustered into four subclades, three
of which were evolutionarily clustered together with
Actinopterygii TULPs; this indicates that divergence
may have predated the Mammalia–Actinopterygii split.
With five exceptions (AtTLP8, OsTLP13, PtaTLP10,
CrTLP1 and CrTLP2), plant TULPs were tightly clus-
tered together, supported by highly significant boot-
strap values (996; Fig. 1). In the plant-specific clade,
Table 1. List of rice TUBBY-like family members.
OsTLP
Accession
number Chromosome
Amino
acid length Gene structure cDNA
OsTLP1 AAT07611 5 445
AK103583
OsTLP2 BAD33172 8 451
AK120706
OsTLP3 CAE01783 4 462
AK104333

OsTLP4 ABA95864 12 445
AK102298
OsTLP5 BAC01219 1 448
AK102221
OsTLP6 AAX95106 11 440
OsTLP7 BAC20077 7 406 AK071159
OsTLP8 BAD08037 2 428
AK064855
OsTLP9 BAD73373 1 356
AK070401
OsTLP10 AAU03104 5 372
AK061747
OsTLP11 AAU10642 5 352
OsTLP12 BAB90233 1 368 AK106853
OsTLP13 BAD28008 2 427
OsTLP14 ABF95936 3 403 AK102370
TUBBY-like genes in rice Q. Liu
164 FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS
monocotyledon and dicotyledon TULPs were not
found in distinct groups, but, instead, were inter-
spersed, suggesting that the significant expansion of
plant TULPs should be no younger than the diver-
gence time between monocots and dicots (approxi-
mately 200 million years ago [10]).
The mixed clade is very special, and consists of one
fungus, one euglenozoan and five plant TUBBY-like
proteins. In addition, one tubby domain-containing
protein was identified in both Plasmodium yoelii yoelii
and Plasmodium falciparum. These two proteins were
excluded from phylogenetic analysis because of their

failure in the chi-squared test for homogeneity of
amino acid composition. However, if these sequences
were arbitrarily included in the phylogenetic analysis,
they were clearly classified into this clade (bootstrap
value 920; tree not shown). Although tubby domain-
containing proteins were found in several protozoans,
this type of protein was only identified in one fungus
after an exhaustive database search. It should be noted
that O. tauri belongs to the Prasinophyceae, that
diverged at the base of the monophyletic green lineage,
which includes green algae and land plants [11]. Thus,
the identification of one TULP in O. tauri and three
TULPs in Chlamydomonas reinhardtii (CrTLPs)
allowed the origin of this gene family to be traced
back to the early evolutionary stages of eukaryote
development.
Genomic organization of OsTLPs
Gene families can arise through tandem amplification,
resulting in a clustered occurrence, or segmental dupli-
cations of chromosomal regions, resulting in a scat-
tered occurrence of family members [12]. It was
observed that OsTLPs were located on chromosomes
1, 2, 3, 4, 5, 7, 8, 11 and 12. By contrast with only one
OsTLP found on chromosomes 3, 4, 7, 8, 11 and 12,
three (OsTLP5, OsTLP9 and OsTLP12), two (OsTLP8
and OsTLP13) and three (OsTLP1, OsTLP10 and
OsTLP11) genes were located on chromosomes 1, 2
and 5, respectively (Fig. 2A). Although the distribution
of OsTLPs on rice chromosomes was obviously
uneven, no two OsTLPs were found to be located

Fig. 1. Phylogenetic tree of eukaryote TUBBY domains. The
neighbour-joining method wrapped in
PHYLIP [28] was used to
reconstruct the phylogenetic tree based on the multiple sequence
alignment of tubby domains (supplementary Fig. S1). The numbers
beside the branches represent bootstrap values (‡ 600) based on
1000 replications. To identify the species of origin for each tubby
domain, a species acronym is included before the protein name:
Aa, Aedes aegypti; Ag, Anopheles gambiae; Am, Apis mellifera;
At, Arabidopsis thaliana; Bt, Bos taurus; Ca, Cicer arietinum; Cb,
Caenorhabditis briggsae; Ce, Caenorhabditis elegans; Cf, Canis
familiaris; Cr, Chlamydomonas reinhardtii; Dm, Drosophila mela-
nogaster; Dp, Drosophila pseudoobscura; Dr, Danio rerio; Ec,
Encephalitozoon cuniculi; Gg, Gallus gallus; Hs, Homo sapiens;
Lm, Leishmania major; Lp, Lemna paucicostata; Mam, Macaca
mulatta; Mm, Mus musculus; Mt, Medicago truncatula; Os, Oryza
sativa; Ot, Ostreococcus tauri; Pc, Pyrus communis; Pt, Pan trog-
lodytes; Pta, Populus trichocarpa; Pxa, Platanus · acerifolia; Rn,
Rattus norvegicus; Sp, Strongylocentrotus purpuratus; Tc, Triboli-
um castaneum; Tn, Tetraodon nigroviridis; Tt, Tetrahymena ther-
mophila; Xl, Xenopus laevis; Xt, Xenopus tropicalis.
Q. Liu TUBBY-like genes in rice
FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS 165
close to each other, for example on the same scaffolds
or bacterial artificial chromosomes. Thus, segmental
duplications probably contributed to the expansion of
the rice TUBBY-like gene family. To test this hypothe-
sis, the method of Schauser et al. [12] was performed
to investigate the evolutionary relationships between
duplicated segments. In this way, five pairs of dupli-

cated segments, including OsTLP1 ⁄ 5, OsTLP4 ⁄ 6,
OsTLP7 ⁄ 14, OsTLP9 ⁄ 11 and OsTLP10 ⁄ 12, were iden-
tified (Fig. 2B). Further examination of the rice dupli-
cation blocks identified by Yu et al. [13] and Wang
et al. [14] revealed that the five pairs of OsTLPs con-
stituted three duplication blocks corresponding to part
of the long arm of chromosome 1 (OsTLP9, OsTLP12
and OsTLP5) and part of the long arm of chromo-
some 5 (OsTLP11, OsTLP10 and OsTLP1), part of
the short arm of chromosome 3 (OsTLP14) and part
of the long arm of chromosome 7 (OsTLP7), and part
Fig. 2. Genomic organization of OsTLPs. (A) Localization of OsTLPs on rice chromosomes. Black boxes indicate the three duplication blocks
between chromosomes 1 and 5, 3 and 7, and 11 and 12. The relative sizes of chromosomes are derived from RGP. (B) Detection of seg-
mental duplications in regions of the rice genome encompassing OsTLPs. The sequences of 10 proteins surrounding each OsTLP (five on
each side) were concatenated to form one block. A vertical black bar indicates the concatenation of two protein sequences. This was per-
formed for all 14 OsTLPs, resulting in 14 blocks, which were then searched against each other using a reciprocal best-hit
BLAST strategy.
The five pairs of OsTLPs identified resulting from segmental duplications are shown here.
TUBBY-like genes in rice Q. Liu
166 FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS
of the short arm of chromosome 11 (OsTLP6) and
part of the short arm of chromosome 12 (OsTLP4).
The three pairs (OsTLP9 ⁄ 11, OsTLP12 ⁄ 10 and
OsTLP5 ⁄ 1) located on chromosomes 1 and 5 may
originate from an ancient whole genome duplication,
followed by a segmental inversion. Another two pairs
(OsTLP6 ⁄ 4 and OsTLP7 ⁄ 14) may be the result of
recent segmental duplication of chromosomes 11 and
12 and chromosomes 3 and 7, respectively [13,14].
With regard to OsTLP8 and OsTLP13, they may have

arisen as a result of duplication events, and lost their
counterparts over the long period of evolution, because
there was only one copy of each in the duplicated
regions on chromosome 2. This explanation is reason-
able, as it has been shown that the rice genome was
probably generated through two rounds of ancient
polyploidy events that were followed by massive gene
losses and numerous chromosome rearrangements [15].
Functional divergence (altered functional
constraint) analysis
A maximum likelihood test of functional divergence
was performed on the basis of the Gu method [16],
using the program diverge [17], which evaluates the
shifted evolutionary rate after gene duplication or spe-
ciation [18]. The advantage of the Gu method [16] is
that it is not sensitive to saturation of synonymous
sites. The estimation was based on the phylip neigh-
bour-joining tubby domain tree (Fig. 1). The result
showed that the coefficient of type I functional diver-
gence between the plant and animal clades was statisti-
cally significant (h = 0.387 ± 0.149; likelihood ratio
test statistic, 6.669; P < 0.05), indicating that signifi-
cantly different site-specific shifts of evolutionary rate
may take place at certain amino acid sites [18] between
plant and animal tubby domains. In order to identify
these variant amino acid sites, the posterior probability
of divergence was determined for each site. The results
showed that the functional divergence between plant
and animal TUBBY-like proteins could be partially
attributed to variation on at least five amino acid sites

(103, 312, 315, 317 and 319, counting from PtaTLP10;
supplementary Fig. S1 and Fig. 3). Boggon et al. [8]
performed X-ray crystallographic analysis of the tubby
domain of mouse TUBBY and found a unique protein
structure: a 12-stranded b-barrel conformation filled
with a central hydrophobic core that traversed the
entire barrel. It was observed that these five amino
acids fall within the fifth (E5, site 103) and ninth
(E9A, site 312; E9B, site 319) b-strands, and the loop
between E9A and E9B (sites 315 and 317) (supplemen-
tary Fig. S1).
Santagata et al. [19] demonstrated experimentally
that the amino acids that interact with l-a-glycero-
phospho-d-myo-inositol 4,5-bisphosphate (GPMI-P
2
)
are mostly in b-strands 4, 5 and 6 and helix 6A. More
importantly, three positively charged amino acid resi-
dues, R332, R363 and K330 (corresponding to
sites 103, 182 and 101 in this study), were found to be
crucial for the tubby domain of mouse TUBBY
protein to bind phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P
2
] [19]. Figure 3 shows that site 103
(Q
k
= 0.877) is invariant arginine in animal tubby
domains, a result supporting its importance for animal
TULPs specifically binding a number of phosphory-

lated phosphoinositides [20]. By contrast, the same
position in plant tubby domains contains several
amino acids (arginine, serine or lysine) with different
chemical properties, such as the uncharged polar
amino acid serine (S). This variation may be explained
as a relaxed selective constraint at site 103 in plant
tubby domains. In addition, it was observed that site
182 was not an arginine (R) and showed a plant-spe-
cific deletion (supplementary Fig. S1). These results
suggest that at least some of the plants may have lost
their ability to bind phosphatidylinositol phosphates.
Boggon et al. [8] observed a groove of highly posi-
tive charge that was bordered at the top by helix H8
and at the bottom by the large 7–8 loop and the three-
stranded ‘extra’ 9ABC sheet. The result showed that
the amino acid sites 312, 315, 317 and 319 were func-
tional divergence related, in which one site was located
at E9A (site 312) and E9B (site 319) respectively, and
two sites (315 and 317) were positioned in the E9A–
E9B loop (supplementary Fig. S1). It was observed
that the four amino acid sites were significantly con-
served in plants (Fig. 3), indicating that strong func-
tional constraints were imposed on these sites. In
comparison with plants, great variation was observed
in these sites in animal tubby domains. Of these sites,
site 317 was predicted to be highly functional diver-
gence related (Q
k
= 0.954). It was observed that the
chemical properties of the amino acid at site 317 were

significantly different between plant and animal tubby
domains. In plants, the amino acid at site 317 was the
invariant nonpolar leucine (L), whereas it was changed
to uncharged polar serine (S), threonine (T) or aspara-
gine (N) in animals (Fig. 3).
Molecular structural and genetic analyses have sug-
gested a common function for animal tubby domains
that can bind to double-stranded DNA and phospha-
tidylinositol phosphates [4,8]. With regard to the
N-terminus, although this region shows a lack of con-
servation in animals, it is able to activate transcription
[8]. Unlike animals, the N-terminal region of most
Q. Liu TUBBY-like genes in rice
FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS 167
plant TUBBY-like family members often contains a
well-conserved F-box domain. Experimental evidence
has shown that AtTLP1, AtTLP2, AtTLP3, AtTLP6,
AtTLP7, AtTLP9, AtTLP10 and AtTLP11 are
expressed in all tested organs, whereas AtTLP5 and
AtTLP8 are tissue specifically expressed in Arabidopsis
[2]. After querying the rice dbEST database at the
National Center for Biotechnology Information
(NCBI), it was found that, with three exceptions
(OsTLP11, OsTLP12 and OsTLP13), OsTLP genes
showed a tissue-specific expression pattern, although
most were expressed in nearly all the examined tissues
(Table 2). However, the expression of several phylo-
genetically closely related OsTLP genes showed similar
or overlapping tissue specificity, for example OsTLP1
and OsTLP5 (Table 2). Interestingly, TULPs can be

expressed with cell-type specificity and can be regu-
lated by their subcellular localization [4,21]. He et al.
[21] found that, in hypothalamic neurones, TUB was
localized in the cytoplasm and nucleus, whereas, in
photoreceptor cells, it appeared to be found only in
the cytoplasm. The reason for the localization of
TULPs in different cell populations still remains
unknown. In addition, Lai et al. [2] demonstrated that
AtTLP9 might participate in the abscisic acid signal-
ling pathway. The Rice Expression Database (RED)
[22] and gene expression omnibus (GEO) in NCBI
were also queried; it was found that OsTLP4, OsTLP5,
OsTLP7, OsTLP9, OsTLP10 and OsTLP12 were prob-
ably involved in the abscisic acid and gibberellin sig-
nalling processes. Nevertheless, more in-depth studies
are needed to establish their distinctive activities and
biological roles.
Experimental procedures
Collection of rice TULPs
The consensus sequence of the tubby domain (PF01167)
was obtained from the Pfam database. The Arabidopsis
TUBBY-like proteins (accession numbers: AtTLP1,
AF487267; AtTLP2, AY045773; AtTLP3, AY045774;
AtTLP5, AY046921; AtTLP6, AF487268; AtTLP7,
Fig. 3. Functional divergence related amino acid site candidates
(Q
k
> 0.6). A site-specific profile based on the posterior probability
(Q
k

) was used to identify critical amino acid sites that were respon-
sible for functional divergence [18] between the animal and plant
tubby domains. According to the definition, a large Q
k
value indi-
cates a high possibility that the functional constraint (or the evolu-
tionary rate) of a site is different between two clusters. (A)
Animals; (B) Plants; and (C) Posterior probability values (Q
k
) of five
amino acid sites.
TUBBY-like genes in rice Q. Liu
168 FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS
AY092403; AtTLP8–AtTLP10, AF487269–AF487271; AtT-
LP11, AY046922) were downloaded from the GenBank
database. In an attempt to obtain all the TULP members,
the rice protein sequences collected in the Rice Genome
Project (RGP) [9], Institute for Genomic Research (TIGR)
and NCBI were downloaded to construct a local rice pro-
tein database. With the tubby domain consensus and the
Arabidopsis TUBBY-like proteins (AtTLPs) as queries, psi-
blast was seeded to search the local and Oryza sativa pro-
tein database in NCBI with an e-value of 10. Moreover, a
psi-blast search against the nonredundant GenBank data-
base was performed to collect tubby domain-containing
proteins in other species using the tubby domain consensus
sequence as query. The collected Arabidopsis and other
published TULPs were used to construct a hidden Markov
model (HMM) profile; this was followed by an hmmer
(version 2.3.2) [23] search of the rice proteome. In addition,

a tblastn search against the rice genomic sequences depos-
ited in RGP, NCBI and TIGR was also conducted. Signifi-
cant hits were collected and redundant hits were removed
by manual inspection. The domain architecture of eukary-
ote TUBBY-like proteins was analysed using the domain
analysis program interproscan [24] with the default
parameters. The accession numbers of the collected TULPs
are listed in supplementary Table S1.
Analysis of rice TULP evolution
TULPs were found to show a scattered distribution pattern
on rice chromosomes. Consequently, segmental duplication
was assumed to have contributed to the expansion of this
gene family. Schauser et al. [12] demonstrated that the
effective way to detect this type of duplication event was to
identify additional paralogous protein pairs in the neigh-
bourhood of each of the family members. Accordingly, the
present study focused on 10 proteins encoded by genes
flanking each of the 14 rice TULPs (five on each side).
Multiple sequence alignment and construction
of the phylogenetic tree
Alignment of the tubby domains was performed using the
clustalw program [25] with the default parameters. The
multiple aligned sequences were initially subjected to a
chi-squared analysis for homogeneity of amino acid com-
position, implemented in tree-puzzle v5.2 [26]. Sequences
that failed in this test were excluded. To investigate the
evolutionary relationships amongst tubby proteins, a phy-
logenetic tree was reconstructed by employing the neigh-
bour-joining method and the minimal evolution method
wrapped in mega v3.1 [27]. For both methods, the param-

eters p-distance model and pairwise deletion of gaps ⁄ miss-
ing data were selected. In addition, phylip [28] was
employed to reconstruct a neighbour-joining tree from the
same data. A bootstrap test of phylogeny was performed
with 1000 replications for each method. The programs
njplot and mega v3.1 [27] were used to display the phy-
logenetic trees.
Estimation of functional divergence
diverge, a program developed by Gu and Velden [17], was
used to detect functional divergence between members of a
protein family [29]. In the TUBBY-like gene family, two
gene clusters of interest, including plant and animal
TULPs, were selected. The coefficient of type I functional
divergence h and the likelihood ratio test statistic between
Table 2. EST-derived expression profile for rice TUBBY-like genes.
OsTLP
Tissue
Root Leaf Shoot Panicle Flower Callus
OsTLP1 ++ + +++ +++ + ++++++++
OsTLP2 ++++++ +++ +++++++++ +++ + +
OsTLP3 ++ +++ ++++++++++++ ++++++++ ++++ +++++++++++++
OsTLP4 ++ ++++++++++ ++++++++ ++++++++ + +++++++
OsTLP5 ++++++ + ++++++ ++++++ ++++ ++++++++
OsTLP6 ++ +++ ++++ ++ ++
OsTLP7 ++ ++ +++ ++ +++
OsTLP8 ++++ +++ ++++ +++++++++++++++++++ ++
OsTLP9 + +++++++ ++++++++ ++++ +++ ++
OsTLP10 +++++++++++++++ ++++++++++++ ++++++ +++++
OsTLP11
a

OsTLP12 +
OsTLP13 + + ++
OsTLP14 ++ ++++ ++ +++ ++
+, Presence of gene sequences in EST collection derived from the indicated tissues.
a
No significant EST hit was found for OsTLP11 in the present EST database, indicating that this gene might be weakly expressed in rice.
Q. Liu TUBBY-like genes in rice
FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS 169
the two clusters were quickly calculated. A h value signifi-
cantly greater than zero indicates altered selective con-
straints of amino acid sites after gene duplication [18].
Acknowledgement
This project was supported by the China Postdoctoral
Science Foundation (No. 20060390348).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Multiple sequence alignment of eukaryote
tubby domains.
Table S1. Accession numbers of collected TULPs in 33
species.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Q. Liu TUBBY-like genes in rice
FEBS Journal 275 (2008) 163–171 ª 2007 The Author Journal compilation ª 2007 FEBS 171