In Silicon Cloning and Bioinformatics Analysis of the Raphanus Sativus WUS Gene

doi:10.4236/eng.2013.510B104

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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

Email: [email protected]

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

M. GUANG

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

M. GUANG

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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