Basic HTML Version
Genomics and Applied Biology
,
2012, Vol.3 No.3, 1
-
7
http://gab.sophiapublisher.com
- 5 -
3.3 Sampling
Young flower buds were collected with RNA enzyme
free tweezers from the initial flowering stage plants.
The sample buds were collected from randomly
selected male fertile plants and male sterilized plants
and kept in liquid nitrogen in freezing tubes. The
sample buds were quickly taken to the laboratory and
separated into different size groups on ice in an
aseptic cabinet in order to meet the requirements of
SSH for peer-to-peer system rationality, and to avoid
artificial un-uniform abundance and false positive
results. The male sterile buds were first divided into
five size levels by according to the length:
①
0.00~
2.00 mm,
②
2.00~2.50 mm,
③
2.50~3.00 mm,
④
3.00~
3.50 mm and
⑤
3.50~4.00 mm. The fertile buds were
also divided into five levels:
⑥
0.00~2.00 mm,
⑦
2.00~
2.50 mm,
⑧
2.50~3.00 mm,
⑨
3.00~3.50 mm,
⑩
3.50~
4.00 mm. In order to detect the difference in gene
expression in a shorter time interval in the quantification
analysis of gene expression, the buds of both male sterile
plants (A) and male fertile plants (B) were divided into
seven size levels, namely, A1 (0.00~1.00 mm), A2 (1.00~
1.50 mm), A3 (1.50~2.00 mm), A4 (2.00~2.50 mm),
A5 (2.50~3.00 mm), A6 (3.00~3.50 mm), A7 (3.50~
4.00 mm); and B1 (0.00~1.00 mm), B2 (1.00~ 1.50 mm),
B3 (1.50~2.00 mm), B4 (2.00~2.50 mm), B5 (2.50~
3.00 mm), B6 (3.00~3.50 mm), B7 (3.50~4.00 mm). The
divided bud samples were put in freezing tubes, quickly
frozen in liquid nitrogen and stored in a
-
70
℃
fridge.
3.4 Extraction and detection of the total RNA
The bud samples were extracted using the Trizol Reagent
(Cat. No. 15596026) according to the product instructions.
The quality of the total RNA was detected by agarose
gel electrophoresis, and the purity and the concentration of
RNA were examined with a nucleic acid and protein
detecting instrument. When the 28S and 18S bands
were clearly seen and the ratio of OD
260
/OD
280
was
around 2.0, further experiments were continued.
3.5 Suppression subtractive hybridization
Total RNA of the five levels (
①
~
⑤
) of buds from the
male sterile plants was mixed into sample 1 (R121
male sterile buds), and the total RNA of the five levels
(
⑥
~
⑩
) of buds from the male fertile plants was
mixed into sample 2 (R121 male fertile). A forward
substractive hybridization was conducted using the
sample 1 as tester and the sample 2 as driver, and a
reverse substractive hybridization was conducted using
the sample 2 as tester and the sample 1 as driver,
following the product instructions of Subtraction Kit
PCR-SelectTM (Cat. No. 637401).
3.6 Construction of subtracted cDNA library and
PCR detection
After a second time PCR amplification the final products
of the forward and the reverse subtractive hybridization
were purified using the PCR Cleaning Kit (Cat. No.
AP-PCR). Cloning and transformation were made
following the instruction of pMD19
-
T vector (Cat. No.
D102A) of the Takara Company. A forward subtractive
cDNA library and a reverse subtractive cDNA library
were constructed, and the products were kept in tubes
at
-
70
℃
after loading 200 μL glycerol in the tubes.
Thirty tubes of bacteria suspension were randomly
taken from the forward subtractive library and the
reverse subtractive library. PCR amplification was
performed using 1 μL of the bacterium template DNA,
together with the nested primer l and the nested primer
2R (provided in the reagent box) as primers, in a 20
μL reaction system. The reaction conditions were the
following: predenaturing at 94
℃
for 20 s; followed by
27 cycles of denaturing at 94
℃
for 30 s, annealing at
68
℃
for 30 s, and extension at 72
℃
for 1 min; and a
final extension at 72
℃
for 10 min; finally stored at
12
℃
forever.
The rates of monoclones were calculated and the
fragment sizes of the clones were identified using 1%
agarose gel electrophoresis.
3.7 Screening of the positive clones and sequencing
The sample 1 and sample 2 RNA’s obtained in the
section 1.2.4 were reversely transcribed into the first
strand of cDNA, according to the specification of the
reverse transcriptase enzyme, M-MLV (Cat. No. D2639,
Takara Co.). The products were labeled R121A and
R121B, respectively, with digoxigenin according to
the Roche DIG High Prime DNA Labeling and
Detection Starter Kit I (Cat. No. 11745832910). Two
same sets of hybridization membrane were prepared
for the subtractive cDNA libraries. One set was used
to hybridize with the probe R121A, and another was
used to the probe R121B after they were baked and
pre-hybridized. The two sets of membrane were
hybridized over night at 42
℃
, and were washed and
immunologically colorized for comparison. The positive
clones on the membrane of the forward subtractive