Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

Abstract

Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

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Construction of cDNA library and preliminary analysis
of expressed sequence tags from green microalga
Ankistrodesmus convolutus Corda
Tran Thanh•Vu Thi Quynh Chi•Mohd Puad Abdullah•
Hishamuddin Omar•Mostafa Noroozi•Huynh Ky•
Suhaimi Napis
Received: 15 October 2009/Accepted: 15 March 2010/Published online: 31 March 2010
? Springer Science+Business Media B.V. 2010
Abstract
Corda is a fast growing alga which produces appreciable
amount of carotenoids and polyunsaturated fatty acids. To
our knowledge, this is the first report on the construction of
cDNA library and preliminary analysis of ESTs for this
species. The titers of the primary and amplified cDNA
libraries were 1.1 9 106and 6.0 9 109pfu/ml respec-
tively. The percentage of recombinants was 97% in the
primary library and a total of 337 out of 415 original cDNA
clones selected randomly contained inserts ranging from
600 to 1,500 bps. A total of 201 individual ESTs with sizes
ranging from 390 to 1,038 bps were then analyzed and the
BLASTX score revealed that 35.8% of the sequences were
classified as strong match, 38.3% as nominal and 25.9% as
weak match. Among the ESTs with known putative func-
tion, 21.4% of them were found to be related to gene
expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to
metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress
Green microalga Ankistrodesmus convolutus
response, and the remaining 45.8% were classified as novel
genes. Analysis of ESTs described in this paper can be an
effective approach to isolate and characterize new genes
from A. convolutus and thus the sequences obtained rep-
resented a significant contribution to the extensive database
of sequences from green microalgae.
Keywords
Ankistrodesmus convolutus ? cDNA library construction ?
Expressed sequence tags
Green microalgae ?
Introduction
Algae are accountable for around 50% of the total organic
carbon produced on earth each year [1], and they form the
basis of the food chain for over 70% of the world’s biomass
[2]. Presently, about 107tons of algae are harvested each
year by algae biotechnology industries for different pur-
poses [3]. Green microalga Ankistrodesmus convolutus is a
fast growing alga which produces appreciable amount of
carotenoids and polyunsaturated fatty acids [4]. The ability
of A. convolutus to form floating aggregates during its
normal growth to facilitate harvesting as well as other
beneficiary attributes make it an interesting candidate for
many biotechnological applications such as production of
natural products or expression of therapeutic proteins.
There were two reports involving studies on the green
microalga species A. convolutus. The first report mentioned
about the promising microalgae for production of useful
chemicals [4], and the second described the influence of
carbon source on growth, biochemical composition and
pigmentation of A. convolutus [5]. At the time the present
study was initiated, there was no report of any molecular
biology studies on A. convolutus.
Electronic Supplementary Material
article (doi: 10.1007/s11033-010-0092-4contain supplementary
material, which is available to authorized users.
The online version of this
T. Thanh ? V. T. Q. Chi ? M. P. Abdullah ? H. Ky ?
S. Napis (&)
Department of Cell and Molecular Biology, Faculty of
Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, 43400 UPM-Serdang, Selangor Darul Ehsan, Malaysia
e-mail: suhaimi@putra.upm.edu.my; s.napis@gmail.com
H. Omar ? M. Noroozi
Department of Biology, Faculty of Science, Universiti Putra
Malaysia, 43400 UPM-Serdang, Selangor Darul Ehsan, Malaysia
T. Thanh ? V. T. Q. Chi
Rubber Research Institute of Vietnam, 301 Nguyen Van Troi
Street, Tan Binh District, Ho Chi Minh City, Vietnam
123
Mol Biol Rep (2011) 38:177–182
DOI 10.1007/s11033-010-0092-4
211

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A cDNA library containing the information from the
mRNA of a particular tissue or organism is an effective
tool for research on gene structure, function and manipu-
lation [6]. A standard cDNA library, once constructed from
a specific tissue or organism, is a faithful and direct rep-
resentation of the ratio of mRNAs presence under exact
conditions at the time of sampling. Therefore, poorly
expressed genes will be poorly represented within libraries,
and genes that are not expressed will be absent [7]. The
production of ESTs starts with the construction of cDNA
libraries. Since the first description of ESTs from humans
was reported in 1991 [8], EST analysis has become the
approach of choice to investigate the transcribed genome in
various living model systems. A large number of ESTs
have been accumulated for a wide variety of organisms
including several important species of green microalgae.
ESTs can also provide a robust sequence resource that can
be exploited for gene discovery, genome annotation and
comparative genomics [7]. Additionally, analysis of ESTs
can provide an overall picture of transcripts involved in
organ or tissue development [9]. Analysis of ESTs on green
microalgae species has been reported in Chlamydomonas
reinhardtii [10], Dunaliella salina [11], Ostreococcus tauri
and Acetabularia acetabulum [12]. To date, there are no
reports on the construction of cDNA library as well as the
generation and submission of ESTs in public databases for
green microalga A. convolutus. A study on the cDNA
library construction and preliminary analysis of ESTs from
A. convolutus cells conducted in our laboratory is hereby
described.
Materials and methods
Culture condition
Green microalga A. convolutus Corda was collected from
axenic freshwater and grown as described previously [5].
A. convolutus was grown and maintained in Bold’s Basal
Medium enriched with 0.1% (w/v) of glucose. The culture
conditions were controlled at 25?C and 150 rpm agitation
in an incubator shaker under 12:12 h light–dark cycle.
Total RNA and mRNA isolation
Total RNA was extracted as described earlier [13]. Five
milliliter of RNA extraction buffer [100 mM Tris–HCl (pH
8.2), 1.4 M NaCl, 20 mM EDTA (pH 8.0), 2% (w/v)
CTAB] was prepared in the presence of 50 mM dithio-
threitol. The lyophilized cells were ground to fine powder
in liquid nitrogen and transferred to RNA extraction buffer
and chloroform/isoamylalcohol (24:1, v/v) in a ratio of 1:1.
The aqueous layer was collected and mixed with 0.3
volume of absolute ethanol. The upper aqueous phase was
transferred to a new tube and 0.25 volume of 12 M LiCl in
the presence of 1% (w/v) b-mercaptoethanol was added
prior to incubation at -80?C for 1 h. Upon centrifugation
and resuspension of nucleic acids in DEPC-treated water,
LiCl (12 M) was added to a final concentration of 2.4 M
before RNA precipitation at 4?C for 1 h. The RNA pellet
was then washed twice with 70% (w/v) cold ethanol and
dried before resuspension in 50 ll of DEPC-treated water.
The PolyATtract?mRNA Isolation Systems (Promega,
USA) was used for the purification of poly-A?mRNA
from total RNA. A cDNA library was constructed from
poly-A?mRNA by using a ZAP Express?cDNA Syn-
thesis Kit and ZAP Express?cDNA Gigapack?III Gold
Cloning Kit (Stratagene, USA) according to the manufac-
turer’s instructions.
Construction of cDNA library
The ZAP Express?cDNA synthesis kit (Stratagene, USA)
was used for cDNA library construction. First-strand
cDNA was synthesized from approximately 5 lg of mRNA
using a hybrid oligo(dT) linker-primer that contains an
XhoI restriction site. The mRNA was primed in the first-
strand synthesis with the linker-primer and was reverse
transcribed using AccuScript Reverse Transcriptase and
5-methyl dCTP. The second-strand cDNA was synthesized
with RNase H and DNA Polymerase I. The double-stran-
ded cDNA was blunt-ended using Pfu DNA Polymerase
and ligated to adapters with EcoRI recognition site by T4
DNA Ligase at 8?C overnight. After XhoI digestion, size
fractionation of cDNA was carried out by 1% agarose gel
electrophoresis. The cDNA fragments longer than 500 bp
were excised and purified using the QIAquick gel extrac-
tion kit (Qiagen, Germany). After purification, precipita-
tion and resuspension, the concentration of cDNA was
determined using ethidium bromide plate assay. After
ligation to the ZAP Express?vector, the recombinant DNA
was in vitro packaged using Gigapack?III Gold packaging
extract (Stratagene, USA) at 22?C for 2 h. Subsequently,
500 ll of SM buffer and 20 ll of chloroform were added to
the packaged product. The mixture was gently mixed,
briefly centrifuged and the supernatant containing the
phages stored at 4?C prior to titering. After the phage
titering, the primary cDNA library was amplified to obtain
a large quantity of high titer stock of the library.
Library screening and generation of expressed
sequence tags
For the primary screening of the library, an equivalent of
30,000 plaques forming units (pfu)/plate were screened
usingplaqueliftingmethod [14]. Afterovernight
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incubation at 37?C, nitrocellulose membranes (Hybond
N?, Amersham Bioscience) were placed onto the surface
of the NZY agar plates for 2 min, followed by step-by-step
submergence in denaturation solution (1.5 M NaCl, 0.5 M
NaOH) for 2 min, neutralization solution (1.5 M NaCl,
0.5 M Tris–HCl pH 8) for 5 min and finally rinsing solu-
tion (0.2 M Tris–HCl pH 7.5, 29 SSC) for 25 s. The
membranes were cross-linked by UV-crosslinker (Ultra-
Violet Products, UK) at a wavelength of 254 nm for 2 min
before hybridization at 65?C overnight with the cDNA
probe labeled with Biotin-14-dATP using a random primer
biotin labeling kit NEBlot?Phototape?Kit (New England
BioLabs, UK) following the manufacturer’s instructions.
The hybridized membrane was washed twice with 29 SSC,
0.1% (w/v) SDS for 15 min, and twice with 0.59 SSC,
0.1% (w/v) SDS for 30 min at 65?C. The membrane was
exposed to X-ray film (Kodak Medical X-ray Film, Kodak)
in an X-ray cassette before the film was developed for
signal visualization. The phage plaques were randomly
selected and excised in vivo according to the manufac-
turer’s instructions.
Plasmid DNA was prepared by using the alkaline
method [15], cDNA inserts were amplified by using T3 and
T7 universal primers, and DNA sequencing was carried out
using the sequencing service provided by First BASE
Laboratories Sdn. Bhd. (Selangor, Malaysia).
Sequence analysis
DNA sequences generated from each cDNA clone were
carefully edited to remove the vector sequence and the low
quality 30sequence. Generally, ESTs longer than 150 bps
and containing ambiguity of less than 4% were considered
usefulfordataanalysis[16].TheseESTsweretranslatedinto
six reading frames and searched against the non-redundant
(nr)peptidedatabaseatNCBI(http://www.ncbi.nlm.nih.gov
) using BLASTX Version 2.2.9 [17]. BLASTX results with
bit scores greater than 80 and E-values of less than 10-10
were generally regarded as significant match [18]. Multiple
sequence alignment between the amino acid sequences of
candidate clones and their homologues of the other species
were also analyzed by using CLUSTAL W [19].
Results
Construction of cDNA library
cDNA library of green microalga A. convolutus was con-
structed from approximately 5 lg of mRNA as starting
material. The result indicated that the size of the synthe-
sized double-stranded cDNA ranged from 300 bps to 4 kbs
(Supplementary Fig. 1a) while the concentration was about
50–100 ng/ll as determined by ethidium bromide plate
assay in which cDNA was spotted together with a serially
diluted DNA standard (Supplementary Fig. 1b). Approxi-
mately 150 ng of double-strand cDNA were ligated to the
ZAP Express vector. The titers of primary and amplified
library were 1.1 9 106pfu/ml (Fig. 1a) and 6.0 9 109pfu/ml,
respectively. The percentage of recombinants was deter-
mined to be 97% for primary library. PCR amplification
was carried out using T3 and T7 universal primers to
determine the presence and size of the cDNA insert. The
results showed that out of 415 randomly selected original
cDNA clones, 337 clones contained inserts ranging from
600 bps to 1.5 kbps (Fig. 1b). These cDNA clones were
selected for further analysis. Results of qualitative eval-
uations of the constructed cDNA library are summarized
in Supplementary Table 1.
Sequence analysis of 337 clones revealed the absence of
contaminating rRNA sequences in the constructed library.
The overall sequence success rate was approximately
81.2%. After eliminating clones with poor quality sequence
data (unreadable sequences, containing more than 4%
ambiguity, small inserts containing mainly poly-A, etc.),
retained clones were subjected to ESTs analysis.
Generation of expressed sequence tags and sequence
analysis
The primary cDNA library, instead of the amplified library,
was used for generation of ESTs to reduce the redundancy
of cDNA clones as only a small number of ESTs were
targeted through random selection. The result of EST
sequencing exercise on selected clones is summarized in
Table 1. After omitting clones with poor sequence results,
a total of 201 individual ESTs were analyzed and they
ranged from 390 to 1,038 nucleotides in length after
removal of vector sequences. The distribution of ESTs
from A. convolutus cDNA library based on BLASTX score
revealed that 72 (35.8%) of them were classified as strong,
the highest match with a BLASTX score greater than 200,
meanwhile 77 (38.3%) ESTs were nominal with BLASTX
score for the highest match between 80 and 200, and 52
(25.9%) ESTs were weak with BLASTX score for the
highest match less than 80 or no significant similarity to
sequences in the database (Fig. 2).
Among the ESTs with known putative function, 43
(21.4%) ESTs were found to be related to gene expression,
29 (14.4%) ESTs to photosynthesis, 22 (10.9%) ESTs to
metabolism, 11 (5.5%) ESTs to miscellaneous, and 4
(2.0%) ESTs to stress response. 40 (43.3%) ESTs were
‘‘unknown protein’’ with no significant matches (score B80
bits or E-value C10-10) and the other 47 ESTs have sig-
nificant matches (score C80 bits or E-value B10-10) with
hypothetical proteins, putative proteins and unknown
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proteins. Besides, 5 (2.5%) ESTs that have no significant
similarity to sequences in the public databases were cate-
gorized as ‘‘unclassified’’ (Fig. 3). The relative abundance
of ESTs with known or putative functions by BLASTX
analysis was shown in Supplementary Table 2. From the
analysis carried out, 90 of 201 ESTs were found to have
more than three copies and the rest ESTs have one to two
copies in green microalga A. convolutus cDNA library. The
most abundant ESTs were photosynthesis-related genes,
followed by ribosomal proteins, metabolism-related genes,
gene expression, miscellaneous and stress response-related
genes in descending order (Supplementary Table 2).
Due to their relatively high abundance, cDNA clones
categorized as photosynthetic can be strong candidates to
be selected for further in-depth studies on the isolation,
identification and characterization of strong native pro-
moters in the future. Within this category, three abundant
ESTs have significant sequence similarity to genes coding
for ribulose-1,5-bisphosphate carboxylase/oxygenase small
subunit, light-harvesting chlorophyll-a/b binding protein,
and oxygen-evolving enhancer protein which are respec-
tively homologous to genes involved in catalysis of the
primary step in carbon fixation of photosynthesis system
[20, 21], major components of light-harvesting antennae of
photosystem [22, 23], and photosynthetic oxygen evolution
[24, 25] (Supplementary Table 2).
Discussion
Composition of the cDNA library
Over the past years, cDNA library construction and anal-
ysis is considered to be an indispensable tool for functional
genomic analysis as it provides much more detailed
information on the genomic mechanisms underlying
diverse processes of the organism [26]. Due to the limited
volume and insufficient stability of the primary cDNA
library, it is necessary to amplify the primary library for
bulking and safe-keeping. A well constructed cDNA
library should contain at least 1.7 9 105independent
Fig. 1 Amplification of cDNA
inserts from randomly chosen
phage plaques. a Phage plaques
of primary library on the plate.
b Amplified inserts of cDNA
clones from the constructed
cDNA library were separated on
1% (w/v) agarose gel. Lane M,
2-log DNA ladder; lanes 1 to 8,
insert cDNA clones in phage
plaques randomly selected from
Plate a
Table 1 Summary of EST sequencing
Total of sequenced clones337
Successful ESTs sequencing reads 201
EST length ranged 390–1,038 bps
ESTs with significant matches156
ESTs with known or putative functions 109
Fig. 2 Distribution of ESTs from A. convolutus cDNA library
according to BLASTX score
Fig. 3 The classification of ESTs from A. convolutus cDNA library
based on their putative functions
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clones theoretically so that a clone derived from a low
abundance mRNA could be screened out with 99% prob-
ability from the library; furthermore, a desirable titer of
amplified cDNA library should be no less than 1 9 109
pfu/ml [15]. In this paper, the properties of the primary and
amplified cDNA library constructed from A. convolutus
were 1.1 9 106pfu/ml and 6.0 9 109pfu/ml, respectively;
which met all requirements of a successful cDNA library
construction. Using PCR amplification to analyse for the
presence of inserts and size determination, a large number
of cDNA clones contained inserts of longer than 600 bps
that will make them excellent candidates for subsequent
studies on gene cloning and expression analysis.
Earlier studies on large scale ESTs generation suggested
that a useful library should comprise at least 50% new
genes, a broad variety of transcripts and no more than 20%
uninformative sequences [27, 28]. The cDNA library
constructed from green microalga Ankistrodesmus convo-
lutus in this study meets these criteria.
Generation and analysis of ESTs
At the initial stage, the collection of ESTs were built by
single-pass sequencing of random cDNAs to discover new
genes at only fraction of the cost of genomic sequencing
while facilitating the identification of coding regions in
genomic sequences [29]. Generation of ESTs is an excel-
lent and unique approach in molecular studies as it allows
both expression and measurements, and the discovery of
new genes to be conducted at the same time. Consequently,
analysis of the expression of a large number of genes
combined with the knowledge of their functions can
facilitate the understanding and allows scientists to take a
glimpse of the overall picture of biological processes in
green microalgae cells.
In this study, approximately 54.2% of the ESTs gener-
ated were sequences with known or putative functions,
while the remainder was unknown proteins or sequences
with no similarities to the databases. These unknown and
unclassified ESTs can become candidates for discovering
new interesting genes through functional analysis currently
being initiated in our laboratory. The ESTs identified to be
involved in photosynthesis were selected and used for our
follow-up study on the isolation and characterization of
highly-expressive native promoters for the establishment of
alternative expression system using green microalga
A. convolutus.
The results presented here represent our initial success
on the molecular studies of green microalga A. convolutus,
which, in our opinion, has great potentials to become an
interesting and excellent candidate for many biotechno-
logical applications. Preliminary analysis of the con-
structed cDNA library indicated that it was suitable for
ESTs generation and discovery of new useful genes.
However, in order to obtain a greater diversity of gene
sequences, additional libraries should be constructed from
green microalga cells grown under different conditions
such as by subjecting the cells to biotic/abiotic stresses.
Studies on stress-responsive genes from green microalgae
should be carried out to understand how its cells adapt to
harsh growing conditions, for example, in contaminated
ponds. The collection of ESTs presented in this study
should prove exceedingly useful for identifying A. convo-
lutus homologues to important genes from other organisms.
Nonetheless, the EST data presented herein were very
limited and efforts is underway to sequence more inserts
from our cDNA library to increase the number of candidate
sequences for the identification of new, interesting genes
and proteins that can be used in promoter studies as well as
alternative expression system. The initial database of
A. convolutus sequences will undoubtedly provide a foun-
dation for future research.
In conclusion, this study presented the construction of
cDNA library, preliminary generation and analysis of ESTs
from green microalga A. convolutus which provide both an
mRNA expression profile and a rapid, low-cost and effi-
cient way to identify new genes with known functions. This
is an effective alternative strategy for functional genomics
of green microalgae, particularly when genomic informa-
tion is absent like A. convolutus. The generation of ESTs
from A. convolutus has proved to be an effective approach
to isolate and characterize new genes from this species.
These ESTs also represented a significant contribution
to the extensive database of sequences from the green
microalgae.
Acknowledgments
number 02-01-04-SF0041 from the Ministry of Science, Technology
and Innovation, Malaysia. The first author is indebted to the Rubber
Research Institute of Vietnam on the financial support for the post-
graduate scholarship.
This work was supported by the MOSTI grant
References
1. Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998)
Primary production of the biosphere: integrating terrestrial and
oceanic components. Science 28:237–240
2. Day JG (1999) Conservation strategies for algae. In: Benson EE
(ed) Plant Conservation Biotechnology. Taylor and Francis Ltd,
London, pp 111–124
3. Hallmann A (2007) Algal transgenics and biotechnology.
Transgenic Plant J 1:81–98
4. Chu WL, Phang SM, Goh SH, Blakebrough N (1992) Promising
microalgae for production of useful chemicals. In: Shaari K,
Kadir AA, Ali ARM (eds) Proceeding of the Conference on
Medicinal Products from Tropical Rain Forests. Forest Research
Institute of Malaysia, Kuala Lumpur, pp 338–345
Mol Biol Rep (2011) 38:177–182181
123
215

Page 6

5. Chu WL, Phang SM, Goh SH (1995) Influence of carbon source
on growth, biochemical composition and pigmentation of
Ankistrodesmus convolutus. J Appl Phycol 7:59–64
6. Ying SY (2004) Complementary DNA libraries: an overview.
Mol Biotechnol 27:245–252
7. Rudd S (2003) Expressed sequence tags: alternative or comple-
ment to whole genome sequence. Trends Plant Sci 8:321–329
8. Adams MD, Kelley JM, Gocayne JD et al (1991) Complementary
DNA sequencing: expressed sequence tags and human genome
project. Science 252:1651–1656
9. Zhuang J, Xiong AS, Peng RH, Gao F, Zhu B, Zhang J, Fu XY,
Jin XF, Chen JM, Zhang Z, Qiao YS, Yao QH (2009) Analysis of
Brassica rapa ESTs: gene discovery and expression patterns of
AP2/ERF family genes. Mol Biol Rep. doi: 10.1007/s11033-009-
9763-4
10. Shrager J, Hause C, Chang C et al (2003) Chlamydomonas
reinhardtii genome project. A guide to the generation and use of
the cDNA information. Plant Physiol 131:401–408
11. Liu Y, Qiao DR, Zheng HB, Dai XL, Bai LH, Zeng J, Cao Y
(2008) Cloning and sequence analysis of the gene encoding 19-
kD subunit of Complex I from Dunaliella salina. Mol Biol Rep
35:397–403
12. Henry IM, Wilkinson MD, Hernandez JM, Schwarz-Sommer Z,
Grotewold E, Mandoli DF (2004) Comparison of ESTs from
juvenile and adult phases of the giant unicellular green alga
Acetabularia acetabulum. BMC Plant Biol 4:3
13. Thanh T, Omar H, Abdullah MP, Chi VTQ, Noroozi M, Ky H,
Napis S (2009) Rapid and effective method of RNA isolation
from green microalga Ankistrodesmus convolutus. Mol Biotech-
nol 43:148–153
14. Benton WD, Davis RW (1977) Screening kgt recombinant clones
by hybridization to single plaques in situ. Science 196:180
15. Sambrook J, Russell DW (2001) Molecular Cloning: a Labora-
tory Manual, 3rd edn. Cold Spring Harbor Lab Press, New York
16. Franco GR, Adams MD, Soares MB, Simpson AJG, Venter JC,
Pena SDJ (1995) Identification of new Schistosoma mansoni
genes by the EST strategy using a directional cDNA library. Gene
152:141–147
17. Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller V,
Lipman D (1997) Gapped BLAST and PSI-BLAST: a new gen-
eration of protein database search programs. Nucleic Acids Res
25:3389–3402
18. Nguyen PD, Ho CL, Harikrishna JA, Wong MCVL, Rahim RA
(2006) Generation and analysis of expressed sequence tags from
the mangrove plant, Acanthus ebracteatus Vahl. Tree Genet
Genomes 2:196–201
19. Thompson JD, Higgin DG, Gibson TJ (1994) Clustal W
improving the sensitive of progressive multiple sequence align-
ment through sequence weighting, position specific gap penalties
and weigh matrix choice. Nucleic Acids Res 22:4673–4680
20. Manzara T, Gruissem W (1988) Organization and expression of
the genes encoding ribulose-1, 5-bisphosphate carboxylase in
higher plants. Photosynth Res 16:117–139
21. Dean C, Pichersky E, Dunsmuir P (1989) Structure, evolution,
and regulation of RbcS genes in higher plants. Annu Rev Plant
Physiol Plant Mol Biol 40:415–439
22. Teramoto H, Nakamori A, Minagawa J, Ono T (2002) Light-
intensity-dependent expression of Lhc gene family encoding
light-harvesting chlorophyll-a/b proteins of photosystem II in
Chlamydomonas reinhardtii. Plant Physiol 130:325–333
23. Chen C, Bai LH, Qiao DR, Xu H, Dong GL, Ruan K, Huang F,
Cao Y (2008) Cloning and expression study of a putative carotene
biosynthesis related (cbr) gene from the halotolerant green alga
Dunaliella salina. Mol Biol Rep 35:321–327
24. Mayfield SP, Rahire M, Frank G, Zuber H, Rochaix JD (1987)
Expression of the nuclear gene encoding oxygen-evolving
enhancer protein 2 is required for high levels of photosynthetic
oxygen evolution in Chlamydomonas reinhardtii. Proc Natl Acad
Sci USA 84:749–753
25. Sharma K, Mishra AK, Misra RS (2009) Identification and
characterization of differentially expressed genes in the resistance
reaction in taro infected with Phytophthora colocasiae. Mol Biol
Rep 36:1291–1297
26. Shao ZT, Cong X, Yuan JD et al. (2009) Construction and
characterization of a cDNA library from head kidney of Japanese
sea bass (Lateolabrax japonicus). Mol Biol Rep. doi:10.1007/
s11033-009-9536-0
27. Adams MD, Kerlavage AR, Fleischmann RD et al (1995) Initial
assessment of human gene diversity and expression patterns
based upon 83 million nucleotides of cDNA sequence. Nature
377:3–174
28. Franco GR, Rabelo EM, Azevedo VA et al (1997) Evaluation of
cDNA libraries from different developmental stages of Schisto-
soma mansoni for production of expressed sequence tags (ESTs).
DNA Res 4:231–240
29. Lievens S, Goormachtig S, Holsters M (2001) A critical evalu-
ation of differential display as a tool to identify genes involved in
legume nodulation: looking back and looking forward. Nucleic
Acids Res 29:3459–3468
182 Mol Biol Rep (2011) 38:177–182
123
216