Basic HTML Version
Molecular Plant Breeding 2012, Vol.3, No.3, 26
-
36
http://mpb.sophiapublisher.com
27
useful and efficient tools in function genomics and
comparative genomic studies. The worldwide grape
EST projects were initiated several years ago and
made a significant progress so far. In 2001, there were
less than 400
V. vinifera
ESTs in GenBank (da Silva et
al., 2005; Moser et al., 2005), with large margins of
increase from then on, this number has reached 352 730
ESTs according to the Release 7.0 (April 17, 2010;
http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?
gudb=grape) of the TIGR Grape Gene Index, which
consists of 34 154 putative different transcripts and 31
813 singleton ESTs. Ablett et al (2000) fulfilled the
V.
vinifera
sequences analysis of 2 479 ESTs and 2 438
ESTs from Chardonnay berry tissue and leaf respectively,
revealing that 2 330 distinct sequences were matched
with the non-redundant protein database. While Terrie
et al (2001) analyzed the
V. vinifera
L. berries (cv.
Shiraz) at various development stages, and 275
ESTs sequenced from their 3' ends were generated.
Pacey-Miller et al (2003) constructed a grape bud
cDNA library with 4 270 ESTs sequenced, indicating
that gene expression in the buds is high even in the
dormancy process. More recently, Moser et al (2005)
carried out the sequence project with 8 147 ESTs
generated from six different grape organs and 405
SSRs were identified. The available resource of EST
sequences and annotations were organized in the
IMAP-database. Peng et al (2007) constructed 11
cDNA libraries usd variety of tissues from two
grapevine (
V. vinifera
L.) cultivars, Cabernet Sauvignon
(wine grape) and Muscat Hamburg (table grape).
77 583 high quality ESTs were generated and analyzed,
with 2 725 novel grape unigenes discovered, which
may play the crucial roles in grape berry development
and regulation of berry composition important for
wine and table grape quality.
As mentioned above, in contrast with
V. vinifera
L.,
the molecular biological research of
V. amurensis
is
limited. The disease-resistance genes and the related
genes of anthocyanin biosynthesis pathway have been
cloned, such as
F3
'
H
, 3GT, and so on (Liu et al., 2009),
but the largest-scale EST sequencing project has a
great potential for further research aiming to discover
new genes and regulation mechanism of key enzymes.
As the raw material for wine making, selecting the
right ripening stage of the
V. amurensis
berry is
critical for its use in the wine-industry. Therefore, we
should focus on the berry development, so as to
elucidate the occurrence and process of the changes,
as well as the factors influencing it. In this research
we constructed the cDNA library of
V. amurensis
pericarps at veraison, illuminate the gene functions
through analyzing the gene expression and finding out
novel genes after veraison.
1 Results and Analysis
1.1 RNA extraction
The modified CTAB protocol was efficient for high-
quality RNA extraction from
V. amurensis
pericarp at
veraison stage. The concentration of total RNA was
1.256 μg/μL, with the A
260
:A
280
and A
260
:A
230
ratios
being 1.90 and 2.10 respectively. RNA examined
by electrophoresis on 1.1% agarose gel showed
the intact 28S and 18S rRNA bands with little
smearing, indicating that little or no RNA degradation
occurred during extraction (Figure 1). These parameters
demonstrated a high quality of the total RNA that
satisfied the needs of the cDNA library construction.
Figure 1 Agarose gel electrophoresis of total RNA
1.2 cDNA library quality evaluation
With the inverse transcription, the first-strand cDNA,
which was used as component in the double-strand
cDNA synthesis by LD-PCR was synthesized. Examined
by electrophoresis on 1.1% agarose gel (Figure 2), the
ds-cDNA size distribution appeared to have the range
of 200~2 000 bp, with moderately strong smear of
cDNA ranging from 500~1 000 bp, indicating that
some genes are highly expressed in this period.
After the drip procedure by the CHROMA SPIN
-
400
Column, the 7~10 fractions containing cDNA with
lengther no longer than 500 bp was collected (Figure 3).
To obtain a library of the desired complexity, three
separate ligations were performed for an optimal ratio
of cDNA to vector. The results showed that the ligation