Basic HTML Version
Rice Genomics and Genetics 2012, Vol.3, No.8, 50
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54
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54
laboratory in November 2009. F
2
population was used
half-grain method. In other words, they were crosscut,
and divided into roughly two parts of the same length.
One half-grain with embryo was planted in MS
medium. (specific methods: Firstly, Shanghai Jiafeng
MS powder medium with 41.5 g/L ratio was dissolved
in water, added 0.5‰ bactericide of Yi Peilong, after
121
℃
high temperature sterilization 20 min, and then
put into ZP17-440 jar to reserve. Secondly, half-grain
was disinfected, 75% alcohol for 20 minutes, 0.1‰
mercuric chloride for 15 minutes, after distilled water
washing three times. Finally, put it into the jar with
MS medium. Placed into incubator with 30
℃
for
continuous light culture). The other part was used for
total protein SDS-PAGE analysis. Normal rice seeds
were directly cultured with water in culture dish.
3.3 Extraction of total proteins in rice seeds and
phenotype analysis by SDS-PAGE
Half-grain of F
2
without embryo and normal rice
seeds were used for total protein extraction. Protein
extraction was performed as described by Jiang
Shaomei (2003) method, slightly changed. Half-grain
was grinded into fine powder, and placed into a
1.5 mL Eppendorf tube, to which was added 150 µL
(65
℃
) SDS-Urea extract solution (5 mol/L Urea, 4%
SDS, 5% β-mercaptoethanol, 20% glycerol, 100 mmol/L
Tris-HCl buffer, pH 6.8). The tube was vortexed and
incubated overnight at room temperature (25
℃
).
Whole grain protein extraction used the same methods
as half-grain, but double reagent was added. After
centrifugation at 10 000 r/min for 10 min, 10 µL of
supernatant was loaded onto an SDS-PAGE gel (15%
polyacrylamide gel for separation, 7.5% polyacrylamide
gel for stacking). Following electrophoresis, gels were
stained with CBB-R250.
3.4 DNA extraction, PCR amplification and electro
phoresis
Tender leaves from rice were taken about 6~8 cm long.
Then genomic DNA was extracted following the
method described by Dellaporta et al (1983). The two
InDel markers, which were designed, analysis were
performed by PCR. Each 25 μL PCR reaction mixture
contained TaKaRa 2×PremixLA
Taq
12.5 μL, the
forward and reverse primers 1.5 μL (4 pmol/μL),
respectively; DNA 3.0 μL (50~100 ng/μL) and ddH
2
O
6.5 μL. The amplification procedure is as follows: 32
cycles of 10 s denaturation at 98
℃
, 30 s annealing at
56
℃
and 270 s extension at 72
℃
, followed by a final
10 min extension at 72
℃
. Amplification products
were electrophoresed on 1% agarose gels and stained
with ethidium bromide (EB).
Acknowledgements
This project was jointly funded by the National Transgenic
Crops Program (2008ZX08001-006), the Research Funds for
Public Benefit in Ministry of Agriculture (200803056), the Key
Support Program of Jiangsu Science and Technology
(BE2008354), the Self-directed Innovation Fund of Agricultural
Science and Technology in Jiangsu Province (CX[09]634)
and the natural science foundation of Jiangsu Province
(BK2008347).
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