Engineering, 2013, 5, 509-512
http://dx.doi.org/10.4236/eng.2013.510B104 Published Online October 2013 (http://www.scirp.org/journal/eng)
In Silicon Cloning and Bioinformatics Analysis of the
Raphanus Sativus WUS Gene
Ma Guang
Department of Life Sciences, Hengshui College, Hengshui, China
Received 2013
ABSTRACT
The complete coding sequence of Raphanus sativus WUS gene was obtained by using Arabidopsis thaliana gene se-
quence (NM_127349) as a probe in silico cloning. Then the hydrophilicity, secondary structure and advanced structure
of WUS protein in Raphanus sativus are analyzed by using bioinformatics methods. The results show that the cDNA
was 1298 bp, with no intron, contains an open reading frame of 936bp, encoding 312aa protein. The protein coded by
Raphanus sativus gene showed 74% similarity to Arabidopsis thaliana.
Keywords: Raphanus sativus; WUS; Bioninformatics; Stem Cell
1. Introduction
Recent years, many genes regulating the plant stem cell
development have been found in different tissues. WUS
(WUSCHEL) gene is one of those, which encodes a
transcription factor that specifies the adjacent cells to be
stem cell [1]. A negative feedback loop that couples the
antagonistic functions of the WUSCHEL and CLAVA-
TA loci controls stem cell fate in the shoot apical meris-
tem. In the embryonic meristem, the expression of CLV3
depends on W US only. Duri ng p os t -embryo development,
both WUS and STM are needed for CLV3 expression
and the triggering of organogenesis [2]. In floral meris-
tem, the expression of AG is activated by coexistence of
WUS and LFY, AG acts as a negative regulator of WUS
expression to down regulate the WUS level. The signal
system established by WUS is also involved in ovule
development [3]. The somatic embryogenesis can be
promoted efficiently by WUS, especially in the presence
of auxin [4]. The results of previous works indicated that
cell competence to WUS activity is related to microen-
vironment and the combination of WUS signal with dif-
ferent environmental factors could activate different down-
stream genes [5].
Because the WUS gene is important in plant stem cell,
it becomes necessary to clone the gene sequence and
elucidate the gene structure. To date, the WUS gene in
Arabidopsis thaliana was reported [6], but the cloning of
Raphanus sativu WUS gene has not been reported. In
this study, we cloned Raphanus sativu WUS based on in
the silico cloning strategy. The sequence of Raphanus
sativu WUS was analyzed by bioinformatics methods,
including open reading frame (ORF) analysis, BLAST,
protein structure, physical and chemical properties pre-
diction.
2. Materials and Methods
2.1. Bioinformatics Tools
http://www.ncbi.nlm.nih.g ov
http://mobyle.pasteur.f r/cgi-bin/portal.py?form=cap3
http://www.ncbi.nlm.nih.gov/gorf/gorf.html
http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://frodo.wi.mit.edu/p rimer3
http://www.expasy.org/tools/protparam.html
http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/
NPSA/npsa_sopma.html
http://www.expasy.org/cgi-bin/protscale.pl
http://swissmodel.expasy.org
2.2. BLAST Searching of Raphanus sa tivus EST
and Genomic Da t abases
The cDNA sequence of Arabidopsis thaliana protein
WUSCHEL (WUS) mRNA, complete cds (GenBank,
NM_127349) was used as a probe to search the Rapha-
nus sativus expressed sequence tag (EST) database
(http://www.ncbi.nlm.nih.gov/dbEST) for a homologous
clone, using the BLAST program. The EST sequence of
score ≥100 and length ≥100 bps selected from the blast
result, were generated contigs (http://pbil.univ-lyon1.fr/
cap3.php). The longer contig was used as second probe.
The above step was not repeated until the newly gener-
ated probe cannot be elongated. This approach led to a

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510
sequence as a putative Raphanus sativu WUS cDNA.
2.3. Bioinformatics Analysis of the Raphanus
sativus WUS Gene
The putative Raphanus sativu WU S cDNA was analyzed
by on-line bioinformatics softwares. The open reading
frame (ORF), protein structure, physical and chemical
properties prediction Raphanus sativu WUS was studi ed.
3. Results
3.1. Identification of EST Sequence Containing
Part of Putative Raphanus sativus WUS
With the cds of Arabidop sis thaliana protein WUSCHEL
(WUS) mRNA (GenBank, NM_127349) as probe, 16
EST sequences (score ≥100 and length ≥100) were
found by Blastn searching the EST database, in NCBI
(Figure 1).
The sequences were selected saved in a file with FA-
STA format. The file was submitted to on -line CA P soft -
ware. Three contigs were obtained by assembling EST
sequences. The longest contig of 1298 bp was selected as
putative Raphanus sativu WUSCHEL (WUS) cDNA
(Figure 2). Searching the cDNA sequence for potential
coding regions by ORF finder (NCBI), an entire open
reading frame (ORF) of 312 amino acids was detected
with a potential start codon at the 132rd site and a stop
codon at the 1069th site (Figure 2)
3.2. Gene Comparison between Arabidopsis
Thaliana and Raphanus sativu WUS cDNA
by ClustalW2
The alignment of Arabidopsis thaliana and Raphanus
sativu WUS amino acid sequences, constructed using
ClustalW program, sugges ted that Raphanus sativu WUS
amino acid sequences were very similar to Arabidopsis
thaliana amino acid sequences. There were same 74.0%
amino acid in Arabidopsis thaliana and Raphanus sativu
WUS (Figure 3).
3.3. Secondary Structure of Raphanus sativu
WUS
The secondary structure of Raphanus sativu WUS was
Figure 1. BLAST result of wus cDNA in the Raphanus sati-
vu EST databank.
Figure 2. Nucleotide sequence and deduced amino acid se-
quence of WUS of Raphanus sativu. The asterisk indicates
the stop codon.
predicted utilizing online service (http://npsa-pbil.ibcp.fr/
cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html).
The results showed that Raphanus sativu WUS of 19.87%
alpha helix, 10.58% extended strand, 66.03% random
coil and 3.53% beta turn.
3.4. Hydrophilicity Prediction of Raphanus
Sativu WUS
Hydrophilicity of Raphanus sativu WUS was predicted
utilizing Program of ProtScale (Kyte and Doolittle). The
results showed that most sites of Raphanus sativu WUS
are in the hydrophilic region (Score: 0 ~ −0.35) (Figure
4). It was concluded that the Raphanus sativu WUS is a
hydrophilic protein.
3.5. Advanced Structure of Raphanus sativu
WUS
The structure prediction from primary to advanced

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511
Figure 3. Homology of Arabidopsis thaliana and Raphanus sativu WUS.
Figure 4. Hydrophilicity profile of Raphanus sativu WUS.
structure is an important task in field of the protein re-
search. The three-dimensional structure model of Ra-
phanus sativu WUS was predicted by the Swiss-Model
server, by homology modeling, based on the available
structures. The result showed that there were 3 α-helices
some irregular coiled peptides Raphanus sativu WUS

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512
protein (Figure 5).
4. Disscusion
In silico cloning is a method developed in recent years
for functional gene identification by using genome and
EST database. Compared to the traditional methods, such
as molecular hybridization, sceening of genomic or
cDNA library, it is advanced for low cost, high effici e ncy,
easy operation, etc [7]. With more and more EST and
genome sequencing data were r eported, it would beco me
possible and feasible to isolate and identify the functional
genes by in silico cloning. Many successful examples
strongly support the fact that in silico cloning is abso-
lutely a feasible tool for gene cloning and presents some
advantages, compared to the traditional methods [8]. In
this study, the full cDNA of Raphanus sativu WUS was
obtained primarily by searching and splicing the EST
sequences. The structure and function were analyzed and
predicted using bioinformatics methods successfully. The
results revealed that it is a convenient technique for
cloning novel gene by searching EST database with ho-
mologous gene of model living things. To our knowledge,
it was the first report about cloning of Raphanus sativu
WUS cDNA with in silico cloning. This research
Figure 5. The predicted three-dimensional structure of Ra-
phanus sati vu WUS.
achievement will provide theory and reference for plant
stem research in Raphanus sativu.
5. Acknowledgements
The research is supported by fund of Hebei province
colleges and universities (No. Z2010159) and fund of
Hengshui city science and technology research (No.
09055Z).
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