Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00202.X |
Differentially
Expression of Tua1, a Tubulin-encoding Gene, during Flowering of Tea
Plant Camellia sinensis (L.) O. Kuntze Using cDNA Amplified Fragment
Length Polymorphism Technique
Wan-Ping
FANG1,2, Chang-Jun JIANG1*, Mei YU1, Ai-Hua YE1,
and Zhao-Xia WANG1,3
1 Key Laboratory of Tea Biochemistry and Biotechnology,
Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, China;
2 College Of Horticulture, Nanjing Agriculture University,
Nanjing 210095, China;
3 Auhui Institute of Education, Hefei 230036, China
Received:
April 19, 2006
Accepted:
May 30, 2006
This
work was supported by a grant from the Natural Science Foundation of Anhui
Province (No. 050410102)
*Corresponding
author: Tel, 86-551-5156265; Fax, 86-551-5156265; E-mail, [email protected]
Abstract The complementary DNA (cDNA) amplified
fragment length polymorphism technique was used to isolate transcript-derived
fragments corresponding to genes involved in the flowering period of tea plant.
Comparative sequence analysis of an approximately 300 bp differential fragment
amplified by primer combination E11M11
revealed 80%–84%
similarity to the corresponding part of an a-tubulin gene of other
species. The complete cDNA sequence of this a-tubulin was cloned by
the rapid amplification of cDNA ends technique; its full length is 1537 bp and
contains an open reading frame of 450 amino acid residues with two
N-glycosylation sites and four protein kinase C phosphorylation sites. The
deduced amino acid sequences did show significant homology to the a-tubulin from
other plants that has been reported to be a pollen-specific protein and could
be correlated with plant cytoplasm-nucleus-interacted male sterility. We named
this complete cDNA Tua1. The nucleotide and amino acid sequence data of Tua1
have been recorded in the GenBank sequence database under the accession No.
DQ340766. This Tua1 gene was cloned into the pET-32a expression system and
expressed in Escherichia coli BL21trxB(DE3). The molecular weight of
expressed protein was deduced to be approximately 49 kDa. Western blot analysis
was used to identify the temporal expression of Tua1 in tea plant. The further
study of the effect of Tua1 protein on pollen tube growth indicated the Tua1
solution obviously promoted the growth of tea pollen tube.
Key words cDNA-AFLP; Camellia sinensis;
flower bud; a-tubulin;
Western blot; pollen
Tea plant (Camellia
sinensis) is an important economic crop in China. It is evergreen,
perennial and cross-pollinated. Under cultivated conditions, a bush height of
60–100 cm is maintained for harvesting the
tender leaves, a process that can continue for more than 100 years. It bears
flowers and fruit 2–3 years after being planted.
Under natural pollination conditions, the flowering rate of most tea plant
varieties is usually very high, but there is a big difference in the fruiting rate among different tea
varieties. The fruiting rate of some varieties is very low (e.g., approximately
1% in Wulong and Maoxie) or absolutely blank (e.g., 0% in Zhenghe big white tea
and Foshou), but in other varieties the rate is very high (e.g., 10% in
Longjing43 and Wuniuzao) [1]. The flower bud differentiation of tea plant usually
begins in early summer and lasts until late fall. It takes approximately 4
months from bud differentiation to bloom (Fig. 1). The reproductive growth of
tea plant, including flower bud differentiation, flowering, blossoming and
fruiting, requires large amounts of energy and nutrients, which restrains
vegetative growth and severely influences immunity function. As a result,
reproductive growth is not only reducing the production of tea leaves, but also
influencing the quality and resistance character of tea, which directly relates
to the income of tea farmers. Additionally, tea is propagated either
through seed or cutting. The segregation character of tea plant filial
generation is often unavoidable due to the limitation of its cross-fertility.
At the beginning of the 20th century, some tea plant breeding
researchers attempted to obtain a pure-breeding variety by self-fertilization,
but failed. Tea vegetative propagation is an effective method for maintaining
the excellent character of the maternal plant. Until now, it has been a key
issue to control the reproductive growth of tea plant due to increasing product
demand, the need for quality improvement, and for tea plant breeding.
Flowering of tea plant is a continuous process, meaning different developmental
stages, from flower bud differentiation to full flower period, can occur in one
plant simultaneously. All of these stages are asynchronous processes in nature
and difficult to analyze individually. At present, research on specific
characteristic products related to the quality of tea is developing quickly [2–4], but there is
little research on the flowering period of tea plant. In 1987, Jiang and Wang
published their research on the growth of pollen, the formation of pollen, the
growth of ovule, the formation of embryo and the process of endosperm growth
[5]. Dong classified tea plant into three types: high fruitage rate type; low
fruitage rate type; and sterile type [6]. However, until now, no reports have
been published on the flower developmental genes of tea plant.
In this study, we exploited the complementary DNA amplified fragment
length polymorphism (cDNA-AFLP) technique to isolate transcript-derived
fragments (TDFs) corresponding to genes involved in the flowering period of tea
plant and aimed at finding some clues to the mechanism of the plant’s
reproductive growth. Research on the flowering period of tea plant from a
molecular biology angle can provide insight into the theory of the molecular
mechanism of flowering of tea. Thus, it might be possible to control the
reproductive growth of tea plant using biotechnology, according to product
demand. It could also give us a chance to use crop hybridity, so as to lay a
good theoretical foundation for the development of tea’s output, quality and
resistance characters. We will attempt to provide leads for further research on
the flowering mechanism, as well as the fertility mechanism, of tea plant
through our study. To our knowledge, this is the first study of the flowering
character of tea plant from the point of view of molecular biology.
Materials and
Methods
Plant materials and RNA
isolation
In this study, we selected two tea plant cultivars planted in the
tea variety orchard of Anhui Agriculture University (Hefei, China), Longjin43
and Wulong. The flowering rates of both are as high as 60%, but the fruiting
rate of Longjing43 (10%) is considerably higher than that of Wulong (1%). Other
characters of both are basically similar. We harvested the small flower buds
[Diameter (Dm)=3 mm)] of both lines and the big flower buds (Dm=6 mm) of both
cultivars in the same stock plant 1 month later. Flower buds were collected and
immediately frozen in liquid nitrogen (Fig.
2). For each sample, total RNA was extracted from approximately 100 mg of
flower buds using Trizol plant RNA purification reagent (Gibco BRL,
Gaithersburg, USA). The total RNA quantity was measured using a
spectrophotometer at a wavelength of 260 nm. The purity of the RNA was
evaluated by the ratio of absorbency at 260 and 280 nm (A260/A280). Quality of the RNA was checked by
formaldehyde denaturing gel electrophoresis.
cDNA-AFLP procedure
Double-stranded cDNA was synthesized using the SMART polymerase chain
reaction (PCR) cDNA synthesis kit (Clontech, Palo Alto, USA). The cDNA-AFLP
procedure described by Bachem et al. [7] was used with a few
modifications. The EcoRI/MseI enzyme system was used. The cDNA
was then digested using EcoRI and MseI. The following restriction
digests were then mixed: 20 ml cDNA (approximately 100 ng), 1 ml EcoRI (10
U), 4 ml 10´PCR buffer, and 15 ml H2O. The mixture was incubated at a temperature appropriate for the EcoRI
(65 ºC) for 2 h. To the first digest mixture, the following agents were
added: 1 ml 10´PCR buffer, 1 ml MseI (10 U), and 8
ml H2O. This mixture was incubated at 37 ºC for 2 h. For ligation of the
adaptors (Table 1) to the digest mixture, the following agents were
added: 1 ml EcoRI adaptors (5 pM), 1 ml MseI adaptors (5
pM), 0.5 ml of 10 mM ATP, 0.5 ml 10´PCR buffer, 0.2 ml T4 DNA ligase,
and 2 ml double-distilled H2O. The mixture was incubated
for 3 h at 37 ºC. The ligation product was termed the primary template and its
20-fold dilution was used directly for pre-amplification.
For selective amplification, a total of 16 primer pairs were used (Table
1) with the following PCR system: 2 ml secondary template, 2 ml each of primer
E11–E18 and primer M11–M18, 2 ml of 10´PCR buffer, 2.5 ml Mg2+
(25 mM), 1 ml dNTPs (25 mM), 0.3 ml Taq polymerase (10 U), and 10.5 ml
double-distilled H2O. Amplification was carried out for 12 cycles
with 30 s denaturation at 94 ºC, 30 s annealing at 65 ºC (the annealing
temperature touching down to 1 ºC for every cycle) and 1 min extension at 72
ºC; then 23 cycles with 30 s denaturation at 94 ºC, 30 s annealing at 56 ºC,
and a 1 min extension at 72 ºC. After the last cycle, the amplification was
extended for 10 min at 72 ºC.
The selective amplification products were separated on a 6%
polyacrylamide gel containing 8 M urea at 110 W until the bromophenol blue
reached the bottom. The cDNA bands were stained with silver nitrate, following
the protocol described in the DNA sequencing system kit (Promega, Madison,
USA).
Characterization of AFLP
fragments
Selected fragments were excised from the polyacrylamide gel,
suspended in H2O and eluted DNA was reamplified using the
same PCR conditions and the same primer combination for selective
amplification. Reamplified products were checked on a 1% agarose gel [8]. The
cloned DNA fragments were sequenced by Shanghai Sangon Company (Shanghai,
China). The sequences obtained were compared to those in the GenBank database
using BLAST sequence alignments.
Rapid amplification of cDNA
ends (3‘/5‘ RACE) of Tua1 gene
Comparison of cDNA-AFLP patterns revealed different cDNA fragments
among the four samples. These bands were excised from gels, cloned into the
plasmid, and sequenced. We picked out and further analyzed a 300 bp
differential fragment, ChaH-1, amplified by primer combination E11M11, and it revealed 80%–84% to a-tubulin of other species.
It was particular expressed in the big flower buds of both lines. The RACE
procedure was carried out using the BD SMART RACE cDNA amplification kit
(Clontech). We designed primers and nested primers for RACE on the basis of the
sequence of ChaH-1. We named the complete cDNA sequence Tua1.
The secondary structure of tea Tua1 gene was predicted
using the Garnier method (GOR) biological tool in PROSITE (http://www.expasy.org/prosite/)
and Swiss-Prot (http://www.expasy.org/sprot/).
Sequence analysis was carried out using BLAST-W and DNAStar software in GenBank
(http://www.ncbi.nih.gov/).
Reverse transcription (RT)-PCR
identification
RT-PCR was carried out using 1.0 ml of first-strand cDNA
prepared as described above. Primers specific for the 5‘ RACE of ChaH-1 were
used to standardize the gene transcript levels in different samples (leaves,
small flower buds and big flower buds). PCR reactions of 25.0 ml were subjected
to 30 cycles of 30 s denaturing at 94 ºC, 30 s annealing at 58 ºC and 1 min extension
at 72 ºC. Five microliters of the product was electrophoresed on an agarose gel
alongside a DNA quantification ladder, and the levels of template added to the
reaction were altered according to the amount of expected product.
Prokaryotic expression of tea Tua1
gene
According to the complete cloned cDNA sequence, two primers were
designed using the Primer Select Program in DNAStar: 5‘-CTCGGATCCATGAGAGAGTTCATTTCGATC-3‘
(forward) and 5‘-CTCAAGCTTATACTCATCACCTTCATCATC-3‘ (reverse). PCR
products were cut with HindIII and BamHI, then ligated with the
pET-32a vector, and transformed into Escherichia coli BL21trx (DE3).
Induced expression was carried out according to the manufacturer’s instructions
(Novagen, California, USA). Target protein analysis was also carried out
according to the user manual of the pET-32a vector (Novagen).
Western blot analysis
Total soluble protein was extracted from leaves, small flower buds and
big flower buds of two tea plant cultivars (approximately 250 mg per sample) by
homogenizing in 3 ml extraction buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 500
mM NaCl, 100 mg/ml phenylmethylsulfonyl fluoride) and centrifuging at 15,000 g for
15 min at 4 ºC. Protein concentrations were determined using the Bradford
method with some modification [9]. The supernatant was mixed with 0.1 volume of
Tris saline azide buffer kept on ice for 10 min and centrifuged at 5000 rpm for
5 min. The supernatant was boiled for 5 min in 5´sodium dodecylsulfate (SDS) sample buffer and separated using 10%
SDS-polyacrylamide gel electrophoresis (1 h, 120 V), then transferred to a
nitrocellulose membrane using a semi-dry blotter (Mini Gel Transfer apparatus;
Bio-Rad, California, USA). The membrane was incubated for 2 h in Tris-buffered
saline with 5% non-fat milk power, in Tris-buffered saline/Tween-20 for 1 h at
room temperature, then probed in succession with anti-a-tubulin antibody made in
rabbit at a dilution of 1:1000 in 0.5% bovine gelatin in phosphate-buffered
saline. Bound antibodies were detected with alkalinephosphatase-conjugated goat
anti-rabbit IgG antibodies (Pierce, Rockford, USA), diluted at 1:5000 to
1:10,000 in blocking buffer (both antibodies were purchased from Beijing
Biosynthesis Biotechnology Company, Beijing, China). The membranes were
developed with 0.33 mg/ml 1-nitroblue tetrazolium and 0.165 mg/ml
5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer (100 mM
Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl2).
Functional study of tea Tua1
To study the effect of Tua1 protein on pollen tube growth, we first
optimized the isolated culture medium for tea plant pollen germination, then
collected the pollen, and cultivated it for 12 h in two kinds of pollen germination
medium (total volume 5 ml per medium): medium I, 10.0% (W/V)
sucrose, 0.01% (W/V) boric acid, 1.0% agar; medium II, 10.0% (W/V)
sucrose, 0.01% (W/V) boric acid, 1.0% agar, 0.1 mg/ml Tua1 protein
expressed in E. coli BL21trxB(DE3).
Results
cDNA-AFLP analysis of gene
differentially expressed during flower development
The gene differential expression of small flower buds and big flower
buds was compared using cDNA-AFLP technology. In total, 256 primer set
combinations were used per cDNA sample for selective amplification. A typical
example of the obtained cDNA-AFLP gel is shown in Fig. 3(A). The
cDNA-AFLP produced an average 50 amplification products per PCR and therefore a
total estimated number of 12,800 fragments. Distinct transcriptional changes
between the small flower buds and the big flower buds of both varieties were
observed for approximately 90 cDNA fragments, of which 37 were only expressed
in small flower buds of both varieties, and the other 53 appeared in big
flower buds of both varieties. This indicated these fragments, which specially
expressed at different times, were potentially related with the flower
development of tea plant.
Characterization of AFLP
fragments
We selected a part of these special expression fragments, which
appeared simultaneously at different developmental stages of flower buds of
both varieties, for sequencing. Twelve TDFs were successfully re-amplified by
PCR using the selective primers that were used to obtain the fragments in the
cDNA-AFLP analysis. Amplified fragments were sent for DNA sequencing. Those
fragments were compared with those in the GenBank database using the BLAST
search tool (Table 2). One sequence, named ChaH-1 [Fig. 3(B)],
revealed high (80%–84%) similarity to the a-tubulin gene. Another fragment showed 80%
homology to Arabidopsis thaliana threonine kinase AT1G72760. Other
fragments showed no homology to known sequences in GenBank. They might
represent previously uncharacterized genes, or the cDNA fragments might be too
short to reveal significant homology.
Tua1 gene cDNA full-length
sequence
The complete cDNA sequence of this a-tubulin was cloned by the
RACE technique. It has 1537 bp and shares 80%–84% similarity to a-tubulin of
other plants. Its complete cDNA sequence has been submitted to GenBank
(accession No. DQ340766). Analysis of this cloned complete cDNA showed that it
encompassed an open reading frame with 1350 bp encoding 450 amino acid
residues. Homology searches with the deduced 450 amino acid residues revealed
tea Tua1 shares 98% similarity with Nicotiana tabacum a-tubulin, 97%
with A. thaliana Tua6, and 84% with Oikopleura dioica
putative a-tubulin. The predicted protein contains two N-glycosylation
sites, four protein kinase C phosphorylation sites, seven casein kinase II
phosphorylation sites, and one tubulin nucleotide-binding domain-like fragment
(Fig. 4). Homology searches and domain query indicate that tea Tua1 is a
member of the potential conserved tubulin family. The predicted secondary
structure composition for this protein has 35.56% helix, 18.82% sheet, and
46.22% loop.
RT-PCR identification
To quantify the differential expression of the Tua1 gene at
different developmental stages of tea plant, RT-PCR experiments were carried
out on leaves, small flower buds and big flower buds of Longjing43. The results
from these RT-PCR experiments (Fig. 5) clearly show that the expression
of the Tua1 gene only exists in the big flower buds. There was no expression
in leaves or small flower buds of tea plant.
Prokaryotic expression of Tua1
The recombinant plasmid pET-32a-Tua1 was transformed and expressed
in E. coli BL21trxB(DE3), producing a target fusion protein (approximately
70 kDa) not produced by the control (untransformed host pET-32a). The results
of protein electrophoresis are illustrated in Fig. 6(A). The size of the
major protein expressed by the control was 20.67 kDa (containing TrxA, which
consists of a six histidinol tag and 109 amino acids). Therefore, the molecular
weight of the induced mature Tua1 was deduced to be approximately 49 kDa,
consistent with the theoretical value. Fig. 6(A) also shows that the
yield of the expressed fusion protein increased with time.
Western blot analysis
The pET-32a-Tua1 fusion expression proteins were subjected to
SDS-PAGE and detected by Western blot analysis with anti-a-tubulin
specific polyclonal antibodies. The results showed that stably accumulated proteins
appeared in the anti-tubulin staining pattern on the blot. A distinct spot of
tubulin protein appears in the Tua1 position, whereas the control sample failed
to produce a protein spot in the corresponding location [Fig. 6(B)].
We further used this antibody to detect the expression pattern of
Tua1 protein in tea plant. The immunoblot analysis of protein obtained from
different tissue is shown in Fig. 6(B). The expected band was detected
in the big flower buds, but there was no protein corresponding to this band in
the leaves or small flower buds. Results demonstrated that Tua1 protein was
especially expressed in big flower buds of tea plant. This result is consistant
with our cDNA-AFLP and RT-PCR analyses.
Effect of the Tua1 protein on the
pollen tube growth of tea plant
The promotive experiments of pollen germination were carried out on
various mediums. The pollen germination rates and the pollen tube growth of tea
plant in various media were measured (Table 3 and Fig. 7). The
Tua1 solution did not promote the germination of pollen, but it clearly
promoted the growth of the pollen tube.
Discussion
The combination of developmentally staged flower bud samples with the
cDNA-AFLP technique allowed us to carry out a large screening for genes showing
differential expression patterns during the flowering period. Mascarenhas
distinguished two patterns of flower gene expression. “Early” genes
are transcribed soon after meiosis and are reduced or undetectable in mature
pollen. Transcripts of the “late” genes are first detected around the
time of microspore mitosis and continue to accumulate as pollen matures [10].
In our study, we chose those TDFs that appeared simultaneously in big flower
buds of both varieties. Thus, we excluded the possibility of discrepancy in
the two breeds themselves and confirmed that these TDFs were exactly relevant
to developmental stages of flower buds. According to the BLAST result of these
fragments, we selected a “late” gene fragment that was deduced to be
an a-tubulin
gene for further research and named it Tua1. The remaining TDFs obtained
by cDNA-AFLP will be cloned and studied in more detail in the future. The a-tubulin gene of
other species has been reported to be a key protein in flower development [11–13]. The
phenomenon that the Tua1 gene was expressed preferentially in the big
flower buds, and no expression was found in the small flower buds or leaves of
either line, indicates that the Tua1 gene might belong to the group of
genes expressed at the late stage of flower development.
Microtubules are components of the filamentous cytoskeleton of
eukaryotic cells and participate in many cell processes, including cell
division, intracellular transport, cell motility, and cell morphogenesis [14–17]. In plants,
microtubules have a number of specialized roles [18–20]. The major structural
component of microtubules is tubulin, a heterodimeric protein composed of two
highly conserved subunits, a and b. A less abundant form, g-tubulin, is also found in higher plants
[21,22]. Both a– and b-tubulins are encoded by multigene families in eukaryotes [23,24].
Tissue-specific preferences in accumulation of tubulin transcripts have been
reported in both Arabidopsis and maize [11,25–27]. Up to the present,
developmentally regulated patterns of a-tubulin transcription in
pollen have been mainly studied in Arabidopsis. The Arabidopsis a-tubulin gene, Tua1,
is differentially expressed in flower organ of Arabidopsis, but no
expression was detected in root or leaf, and the peak transcription level of
Tua1 was noted at the flowering period. It can be concluded that the Tua1 gene
plays a key role in the development of pollen of Arabidopsis [11,12].
The research on maize also indicates that the a-tubulin gene has a close
relationship with cytoplasm-nucleus-interacted male sterility [13,28–30].
In our present study, the transcription and characterization
analysis showed that Tua1 was expressed differentially during the
flowering period and might be related to fertility of tea plant. The further
functional study indicated the Tua1 solution did not promote the germination of
pollen, but clearly promoted the growth of the pollen tube. It will be useful
to verify the gene function more deeply and elucidate the biological roles and
their relationship with the flower development of tea plant. Further work will
be focused on identifying the functional mechanism of Tua1 in tea plant. A
long-term goal of our research is to identify expressed transcripts during
flower development, and to manipulate these genes or their promoter elements
to regulate flower development and fertility mechanisms in tea plant. This
work constitutes the first report of genes activated during the flowering
period of tea plant and has significance for tea plant breeding.
Acknowledgements
We wish to thank Prof. Zheng-Zhu ZHANG (Key Laboratory of Tea
Biochemistry and Biotechnology, Anhui Agriculture University) for helpful
suggestions to our research, and Prof. Yu-Bao LI (Shanghai Institutes for
Biological Sciences, Chinese Academy Sciences) for his critical reading of this
manuscript.
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