|
Short Communication |
Molecular
Cloning and Characterization of Rice Pollen Profilin
YE
Qiu, XU Yin, YAN Fang, TANG Lin, CHEN Fang*
( The College of Life Science, Sichuan
University, Chengdu 610064, China )
Abstract Profilin is a low molecular weight protein
and is identified as an actin-binding protein in higher plants. Two
pollen-specific cDNA clones of rice, RproA and RproB, were
isolated from a cDNA library of rice mature pollen. The two cDNA clones was 821
bp and 805 bp long and contained an open reading frame of 131 amino acids, 5′
and 3′ untranslated regions and a long poly(A) tail. The comparison of deduced
amino acid sequences from RproA and RproB cDNA and other plant
profilin cDNA indicated that the two rice profilin are high homologous to each
other, showed that 89% similarity to maize (ZmPRO3), 87% to C.
dactylon, 83% to H. brasiliensis and 89% to P. pratense.
Genomic southern blot analysis suggested the existence of two genes at least.
Northern blot and reverse transcriptase polymerase chain reaction analysis
demonstrated that the two rice profilin cDNA are anther or pollen specific.
Key
words actin cytoskeleton;
profilin; cloning and expression; pollen
Profilin
is a low molecular weight protein that was originally identified as an
actin-binding protein in calf spleen[1]. Profilin is known to
regulate the organization of the actin cytoskeleton in a complex fashion; It
can promote or prevent actin nucleation or polymerization, depending on the
profilin-to-actin ratio[2-4]. Profilin forms a one to one complex
with the actin monomer and can thereby affect the rate and extent of actin
polymerization[5]. It also binds to protein with proline-rich motifs
and may be linked to signal transduction cascades through binding to
polyphosphoinositides[4-6]. Profilin has been found in organisms
from all eukaryotic kingdoms and plays a significant role in regulating the
dynamics of the actin cytoskeleton[3,4,7].
In
plants, profilin was originally discovered as a major birch pollen allergen[8]
and has subsequently been isolated or cloned from many plants[4].
Such as maize[9], timothy grass[10], wheat[11],
tobacco[12], common bean[13], tomato[14] and Arabidopsis[15].
Plant profilin are members of large multigene families with unique expression
patterns and can be grouped into two classes: those that are constitutively
expressed in vegetative tissues and those expressed primarily in reproductive
tissues. Multiple profilin isoforms can be expressed in individual tissues and cells,
particularly in pollen[15].
To
investigate the function of profilin gene in rice, we have identified and
characterized two members of the profilin multigene family from the plant rice
(Oryza sativa L.c v. IR 36). Two cDNAs was isolated from a rice pollen
library (designated RproA and RproB) and their transcripts were
expressed in mature pollen and anther.
1 Materials
and Methods
1.1 Materials
Rice
plants (Oryza sativa L.c v.IR36) were grown under greenhouse conditions.
Mature pollen grains and anthers were collected and immediately frozen in
liquid nitrogen. Leaves, stems and roots were collected from two-week-old
seedlings. Frozen tissues were stored at -70 ℃
until use.
1.2 Construction
of rice pollen cDNA library
The
poly(A) RNA was purified from mature pollen using poly Attract System 1000 kit
(Promega) and the rice pollen cDNA library was constructed using SMARTTM
cDNA library construction kit(Clontech). The first-strand cDNA was primed with the
CDSIII /3′ primer and the SMARTTMIII Oligo serves as a short,
extended template at the 5′ end of the mRNA. After first-strand cDNA was
synthesized, the primer-extension step generates full-length dscDNA. The dscDNA
was digested with sfi1 and size fractionation using CHROMA SPINTM
columns, 1.5 ml
cDNA was ligated into the sfi1-digested lTrip1Ex2TM
vector and packaged using Gigapach Gold system(Stratagene).
1.3 Isolation
and characterization of two cDNA from rice pollen cDNA library
Prior
to the screening, an approximately 400 bp cDNA fragment was obtained using
RT-PCR with a pair of primers, P1(5′-GTNGACGAGCACCTNAT-3′) and P2(5′-RTARTCNCCNAGNCGYTC-3′)
which were synthesized according to the homogeneity. This amplified product was
subcloned into the PMD18-T vector (TaKaRa). The nucleotide sequence of this
fragment was determined and it was recognized as a partial fragment of profilin
cDNA by the comparison with other known plant profilin cDNA. Using this PCR
fragment, we screened a cDNA library, which was made from pollen of rice by
standard method[16].
1.4 Southern
blot analysis
Total
rice DNA was isolated from two-week-old seedlings of rice (Oryza sativa.L.c.v.IR36)
according to the method[17]. For analysis of the profilin gene copy
number in rice, genomic DNA was completely digested with EcoRI, BamHI,
HindIII, PstI and XbaI and then separated in a 1% agarose
gel. DNA was transferred onto Hybond N＋
filter (Biohringer Mannheim) The EcoRI/XbaI RproA insert
labelled with [32P]dCTP by the random priming technique was used as
a probe. Blots were prehybridized at 65 ℃
in prehybridization solution[6×SSC(1×SSC
is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate), 0.5% SDS, 10% denatured
salmon sperm DNA and 5×Denhardt’s
solution [1×Denhardt’s
solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA]) for 3 h
and hybridized at 65 ℃
in prehybridization solution containing [32P]-labeled probes using
primer-a-Gene Labelling System(Promega). Blot were then washed under low
stringency conditions (5 min in 2×SSC
and 0.1% SDS at room temperature and 10 min in 1×SSC
and 0.1% SDS at room temperature) and then exposed on Fuji X-ray film at -70 ℃
overnight.
1.5 Northern
blot analysis
Total
RNA from pollen, anther, shoots, leaves and roots was extracted using a guanadine
thiocyanate extraction method[18]. Fifteen mg
of RNA per lane was separated in an agarose gel containing 10% formaldehyde and
blotted onto a Hybond N+ membrane in 10×SSC(1×SSC
is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate). The labelled EcoRI/XbaI
RproA insert was used as a probe. After baking for 2 h at 80 ℃,
the membrane was hybridized to the profilin cDNA fragment, which has been
labeled with [32P]dCTP using primer-a-Gene Labelling System. The
blot was washed at high stringency (5 min in 2×SSC,
0.01% SDS at 65 ℃,
10 min in 1×SSC
and 0.1% SDS at 65 ℃)
before X-ray film was exposed to them.
1.6 Nucleotide
sequencing analysis
DNA
sequencing was performed by the dideoxy chain-termination method using Bca BEST
dideoxy sequencing kit (TaKaRa).
2 Results
and Discussion
2.1 A
rice pollen-specific cDNA library
To
isolate and characterize the profilin gene in the development of pollen
germination and tube growth, a rice pollen cDNA library was constructed in the
expression vector lTripIEx2TM
(Clontech) using cDNA synthesized from polyadenylated mRNA isolated from rice
mature pollen. The vector lTripIEx2TM,
used in this system allowed conversion from phage l
to plasmid clone by using the cre-lox site-specific recombination system. The
library contained 2×106
independent clones. The efficiency of recombinant clones was about 97%. It was
conform to objective of the cDNA library constructed.
2.2 Isolation
and sequence of cDN
A
clonesA rice cDNA library derived from rice pollen mRNA was screened using a
366 bp nucleotide PCR fragment as a probe(see material and method). Several
positive recombinant phages were isolated after screening 2×105
plaques. These positive phages were then submitted to a second screen.
Thirty-seven positive clones were isolated from a screen of 2×106
clones. Based on the sequence information, we have obtained two full-length
cDNA clones(RproA and RproB). The nucleotide sequence of RproA
and RproB cDNA is 821 bp and 805 bp repectively and exhibits an open reading
frame corresponding to a 333 bp amino acid polypeptide (Fig.1). The presence of
a stop codon (TGA) two codon upstream of the first ATG suggests that this ATG
is the initiation codon and thus that the coding sequence is complete. The cDNA
presents 77 bp of 5′- and 319 bp of 3′-untranslated sequences and a long
poly(A) tail.
Fig.1 Nucleotide and deduced amino
acid sequences of the Rpro cDNA
DNA
sequence corresponding to the Rpro tanscript is shown, with the
predicted translation product shown below. The nucleotide numbering is listed
at the right side.
Comparison
of the nucleotide sequences of RproA and RproB cDNA showed that
the two sequences were homologous (89% identity) but not identical(Fig.2). Some
differences localized in the 3′-untranslated sequence, suggested that the two
cDNAs could derive from two different genes. Nucleotide sequence homology
search in GenBank revealed that the cDNA fragment has a similarity of 89% to
maize ZmPRO3, 87% to C.dactylon, 83% to H.brasiliensis and
89% to P.pratense(Fig.3). A G-actin-binding region, the VERLGDYL is
located at 119―126
amino acid and a phosphatidylinositol 4,5-bisphosphate(PIP2) region
IRGKKGSGGITVKKT is located at 82―97
amino acid, respectively. These results show that the deduced amino acid
sequence of profilin from pollen is high conservation and the profilin is an
actin-binding-protein. It could influence the pollen germination, subsequent
tube growth, and movement of germinal cells down the tube by affecting the
actin cytoskeleton. For example, profilin could both inhibit and promote actin
polymarization because of its role in sequestering actin monomers and in
catalyzing nucleotide exchange associate with monomer actin[19]. In
addition, profilin interacted with phospholipids and probably linked the
remodeling of the actin cytoskeleton to the signal transduction pathway in
response to extracellular stimuli[6,20,21].
Fig.2 Nucleotide sequences of two rice
profilin cDNAs(RproA and RproB), isolated from a rice pollen
library
The
RproA is the longest cDNA.
Fig.3 Comparison of the rice pollen profilin
amino acid sequences with other plant profilin sequences
The deduced amino acid sequence of rice
profilin was compared with profilin sequences from Z.mays, C.Dactylon,
P.pratense. H.Brasiliensis, N.Tabacuum. Sequences were
aligned using the DNAStar program. Identical amino acids are highlighted on a
black background.
2.3 Southern blot analysis of the profilin gene family
Unlike
most other organisms, in higher plants there are large multigene families for
profilin and actin[4,22]. Maize was predicted to have at least five
profilin genes[9] and six to eight actin isoforms[23, 24].
In order to determine the size of the profilin multigene family in rice genomic
DNA was digested with EcoRI, BamHI, HindIII, PstI
and XbaI and probed with a cDNA fragment of the RproA and RproB,
respectively (see materials and methods). Each probe hybridized to two or three
bands. These results indicate that the profilin multigene family exist in the
rice genome and have at least two or three profilin gene (Fig.4).
Fig.4 Southern blot analysis of rice
genomic DNA digested with EcoRI, BamHI, HindIII, PstI
and XbaI
Gene-specific
probes made from digested EcoRI/XbaI RproA insert.
2.4 Analysis of the expression of the profilin gene in different
organs of rice plants
The
RNA gel blot analysis showed that a different expression pattern among various
organs. The transcript was abundant in the pollen and anther, no transcripts
present in roots, stems, leaves of rice plants. Identical results were also
obtained through RT-PCR analysis. An abundance of Rpro transcript was
only detected in pollen and anther, but not in other organs. These results
indicated that the Rpro was a cDNA clone which was pollen
specific(Fig.5). Identical results were also obtained in RT-PCR (Fig.6). An
abundance of Rpro transcripts was only detected in pollen grains and
anthers. Huang et al analyzed some plant profilin genes and had
suggested two profilin gene classes(constitutive and pollen-specific) in plant.
The pollen-specific gene in the gene class included profilin genes from the Arabidopsis
PRF4, monocot maize (Zma1-3), wheat(Tac), and timothy
grass(Ppr), and the dicots white birch(Bve) and tobacco(Nta).
Similar results were also obtained in rice, in which genes could only be
detected in mature and germinated pollen grains[15]. Recent data
indicated that profilin interacted with a soluble pollen component(s),
resulting in dramatic alterations in the phosphorylation of a number of
phosphoproteins. This implies a signaling role for profilin in angiosperm
pollen[25], suggests that profilin may act in a signaling capacity
to regulate pollen tube growth through its modulation of protein kinase
activity.
Fig.5 Northern blot analysis of
profilin transcripts in various rice (IR 36) tissures and organs
(A) Northern blot analysis of total RNA
(15 m
in each lane) isolated from different tissue-specific
expression of the profilin gene. Rice tissues: anthers(A), pollen grains(P),
leaves(L), stems(S), root(R). The RNA was blotted onto a Hybond N＋
membrane (Boehringer Mannheim) and probed with [32P]-labeled RproA
cDNA. A strong hybrydization signal was detected in both pollen grains and
anthers, but not in other organs. (B) Fifteen micrograms of total RNA were
loaded in each lane and stained the gel with ethidium bromide.
Fig.6 RT-PCR products amplified from
rice RNA using a 366 bp long PCR fragment as primer
0.5 mg RNA was used to synthesize the cDNA,
the cDNA was amplified by PCR. The lanes are: F×174-HincII
digest DNA marker(M), root(R), stems(S), leaves(L), pollen grains(P),
anthers(A). The RT-PCR products were shown in the pollen or anther.
Acknowledgements We thank LI Jia-Yang
for providing laboratory facilities and MOU Zhong-Lin for technical assistance and
comments on the manuscript.
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Received: March 9, 2001 Accepted: April
3, 2001
This work was supported by the Doctoral
Foundation of the Ministry and the Project for Key Teacher in Higher School of
the Ministry of Education, China
The nucleotide sequence (RproA)
reported in this paper has been submitted to the GenBank under the accession
number AF310253
*Corresponding author: Tel,
86-28-5417281; Fax, 86-28-5417281; e-mail, [email protected]