Basic HTML Version
Molecular Pathogens (online), 2011, Vol.2
ISSN 1925-1998 http://mp.sophiapublisher.com
34
additional baseline data for further study of ASPV
differentiation and molecular variation, and prokaryot-
ic expression vector of Ya pear provided some insights
for preparation recombinant polyclonal antibody of
ASPV and further study of ASPV molecular biology.
1 Results and Analysis
1.1 Cloning and Sequencing of ASPV CP gene
Taken the phloem of pear (Y) which were infected
with ASPV as materials to extract total RNA, first
strand cDNA synthesis was obtained by reverse
transcription using specific primer, a 1 392 bp
fragment was amplified by PS/PA primers as shown in
Figure 1. The purified DNA fragments were ligated
into the PMD19-T vector and transform into
E
.
coli
DH5α. The positive clones were confirmed by PCR
and restriction enzyme digestion and designated
Y/PMD-T before sequencing (Figure 2).
Figure 1 Detection of ASPV CP gene by RT-PCR
Note: M: D514A 200 marker; 1: Y RT-PCR product; 2:
Negative control
Figure 2 Identification of recombined plasmid by PCR and
restriction enzyme
Note: M: D514A 200 marker; 1~3: Identification recombined
plasmid by PCR; 4~9: Identification recombined plasmid by
restriction enzyme
1.2 Analysis of ASPV isolates
The complete CP gene of Y isolate, which has been
deposited in the GenBank and allocated the accession
numbers of JF946774, was consisted of 1 194 bp and
potentially encoded a protein of and molecular mass
(Mr) of 42 kD, consisting of 397 amino acids. The
base compositions of CP gene were found to be
adenine 27%, cytosine 25%, guanine 24%, and uracil
24%, respectively.
To include a wider range of isolates and thus obtain
more representative results in the phylogenetic
analysis of ASPV, we further analyzed the complete
CP gene sequences of 28 ASPV isolates including the
determined in this research. Y isolate CP gene at aa
sequences showed approximately 70% identities with
the CPs of ASPV isolates (Table 1).
Phylogenetic analysis of the CP genes of 28 ASPV
isolates at aa sequence revealed three groups. All
ASPV isolates from apple were clustered to group I,
whereas those of pear were clustered to group
Ⅱ
which included nine isolates (except NC_003462,
apple) and Y isolate was clustered to group
Ⅲ
(Figure
3). Furthermore, group I and group II both can be
further divided into five sub-groups. However, the
formation of these sub-groups had not certain
regularity.
1.3 Cloning and c
onstruction of theASPV-CP-Y/PET
A predicted 1 194 bp product containing the entire CP
gene of ASPV was amplified using PCR from Y
isolate. The sequence result of the resulting clone
CP-Y/PMD was consistent with the sequence of
previously study. The constructed CP-Y/PMD-T was
digested with
Sal
I and
Nco
I, and the insert was ligated
into PET-28a vector precut with the same enzymes.
The resulting ASPV-CP-Y/PET-28a was verified by
PCR and restriction enzymes analysis (Figure 4).