Basic HTML Version
Genomics and Applied Biology
,
2012, Vol.3 No.3, 1
-
7
http://gab.sophiapublisher.com
1 Results and Analysis
1.1 The significant inhibition of “Hua-sha-ling WP1”
on the stamen development of
Brassica napus
L.
The morphology of the sterile flowers was almost the
same as that of the fertile flowers, including the size
of flowers, the color of petals and the development of
pistils, but the filaments were significantly shorter and
the anthers were empty compared with the fertile
flowers (Figure 1). The rates of sterile flowers were
more than 98%.
- 2 -
Figure 1 Effects of male sterilization of “Hua-Sha-Ling WP1”
Note: A, B: Untreated male-fertile flowers; C, D: Treated male-
sterile flowers
1.2 Separation and screening of the differentially
expressed fragments in the male sterile flower buds
The products of suppression subtractive hybridization
(SSH) were amplified by a second PCR reaction, and
connected to pMD19
-
T vector. After transforming, the
screened results indicated that 248 positive clones in
the forward subtraction and 359 positive clones in the
reverse subtraction were obtained. PCR detections
were conducted with a few randomly selected clones.
It was shown that the rate of monoclones was higher
than 95%. These fragments were clear, and all of them
were shorter than 1 000 bp (Figure 2). Two sets of
hybridization membrane were prepared for the forward
subtractive clones and the reverse subtractive clones.
Six positive clones, i.e., 1224, 1252, 1268, 1301, 1319,
1130, were screened out with the reverse northern dot
blotting method. These clones were all the expressed
fragments of the genes suppressed in the male sterilized
flower buds of R121. Figure 3 shows the fragment
of No.1319 screened with the reverse northern dot
blotting technology.
Figure 2 The PCR quality detection of the subtract libraries
Note: A: The forward subtract library; B: The reverse subtract library
Figure 3 Screening of the differentially expressed gene fragments
Note: A: The forward subtract library hybridized with R121A;
B: The reverse subtract library hybridized with R121B
1.3 Sequencing and homology analyses of the six
differentially expressed fragments
The 6 positive clones obtained in the section 1.2 were
sequenced and 4 effective cDNA fragments were obtained.
The homology and functional analyses were made
according to the NCBI database on internet (Table 1). The
fragment of No. 1319 was found to be 100% homologous
to the sequence of polygalacturonase gene
MF6
.
1.4 Quantitative expression analysis of
MF6
gene
With the results from the section 1.3, the quantitative
expression analysis of the
MF6
gene in the buds of
different fertility and lengths has been done in this
experiment. The reference gene
UBC21
showed
the most
stable expression (Table 2), and its amplification
specificity and efficiency also achieved the requirements
for reference genes by the method of 2
-ΔΔCt
(Figure 4).
The amplification specificity and amplification efficiency