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Molecular Pathogens (online), 2011, Vol.2
ISSN 1925-1998 http://mp.sophiapublisher.com
33
Research Report Open Access
Cloning and Prokaryotic Expression of
Apple Stem Pitting Virus
CP Gene in Ya
Pear in
E. coli
Na Liu
,
Jianxin Niu
Department of Horticulture, Agricultural College of Shihezi University, Shihezi, 832003, P.R. China
Corresponding author email:
njx105@163.com;
Authors
Molecular Pathogens, 2011, Vol.2, No.5 doi: 10.5376/mp.2011.02.0005
Both authors contributed equally
Received: 25, Oct., 2011
Accepted: 30, Nov., 2011
Published: 13, Dec., 2011
This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Liu et al., 2011, Cloning and Prokaryotic Expression of
Apple Stem Pitting Virus
CP Gene in Ya Pear in
E. coli
, Molecular Plant Breeding, 9(6): 722-727 (doi:
10.3969/mpb.009.000722)
Abstract
In order to clarify evolutionary relationship and genetic diversity of
apple stem pitting virus
(ASPV), and preparation the
corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya (Y) pear which infected by ASPV
and used as template for cDNA synthesis. The coat protein (CP) gene of ASPV was cloned and sequenced. The CP gene of Ya pear
was cloned into expression vector PET-28a, and transformed into
E
.
coli
BL21 (DE3), and SDS-PAGE electrophoresis analysis. The
results showed that the complete CP gene consisted of 1 194 nucleotides and encodes a polypeptide of 397 amino acid (aa).
Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity.
Phylogenetic tree showed that all isolates of the coat protein (CP) genes of ASPV isolates at AA sequence revealed three groups. All
ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups
Ⅱ
(except NC_003462) and the Ya pear
were clustered into group
Ⅲ
. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the
inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data
for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.
Keywords
Apple stem pitting virus
(ASPV); Coat protein; Prokaryotic expression
Background
Apple stem pitting virus
(ASPV) is the type species of
the genus
Foveavirus
, the family
Flexiviridae
(Martelli and Jelkmann, 1998). ASPV has a worldwide
distribution and often remains symptomless. It has been
demonstrated that pear corky pit, pear stem pitting, pear
vein yellow, pear yellow etc. were caused by ASPV
(Jelkmann et al., 1992; Jelkmann, 1994; Deng and
Wang, 2002). ASPV was found worldwide and
frequently occurs in combination with other latent virus
such as
Apple chlorotic leafspot virus
(ACLSV) and
Apple stem grooving virus
(ASGV) (Lenoe et al., 1998).
This kind of co-infection is the main cause of a
decrease in yield quality and quantity, and even leads to
tremendous economic losses.
The genome of ASPV is a single-stranded positive-se-
nse RNA with molecular weight of 3.6×10
6
Da and
encompasses 44~48 kD coat protein (CP). Most
recently, there were only four complete genomic
sequences of ASPV isolates had been deposited in
GenBank, the accession numbers were NC_003462
(Jelkmann, 1994), AB045371 (Yoshikawa et al.,
2001), EU095327 and FR694186, respectively. Many
ASPV CP gene sequences from different regions have
been reported and the analyses have been
demonstrated that the sequences of CP gene were
highly variable amongst isolates (Schwarz and
Jelkmann, 1998; Nechinov et al., 1997; Li et al.,
2010a). ASPV content was low in plant tissues, and
difficulty to separation and purification. Jelkmann
(1994) prokaryotic expressed the ASPV CP gene and
preparation of ASPV antiserum, but not used for field
detection. Recently, there was not prepared high
efficiency antiserum for ASPV. In this study, the
complete CP gene of ASPV Ya isolates provided