Page 10 - Molecular Plant Breeding

Huang et al., 2011, Integrating the hrap Gene from Sweet Pepper into Potato Enhances Resistance to Phytopthora infestans, Molecular Plant Breeding Vol.2
No.5 (doi: 10.5376/mpb.2011.02.0005)
35
subcultured and others were transplanted into the
nutrition pots. Except pre-culture and co-culture, all
the cultivation conditions were 12000 LX light
intensity, 12 h/12 h (light/dark) and temperature of
(22±1)℃ .
3.4 PCR detection
After four to five weeks of the regenerated transgenic
plants growing in a pot, the lamina of different strains
were collected for DNA extraction according to the
method reported by Fulton et al (1995), followed by
polymerase chain reaction (PCR) trace analysis. DNA
was used as the template, with the plasmid pBIHRAP
with the gene hrap as positive control (about 720 bp),
and the non-transformed plant as negative control. The
PCR system included 25 µL of reaction liquid
containing 10×Buffer 2.5 μL , dNTPs (10 mmol/L) 0.5
μL , Taq polymerase (2.5 U/ μL ) 0.5 μL, DNA
template 1~2 μL, primer I (10 μmol/L) 0.5 μL
(5’-GTTGGAGTTGGAGGACGAGG -3’), primer II
(10 μmol/L) 0.5 μL (5- CGCGGATCCATGAAAATG
AAGAACCTCTC-3’) , add ddH2O to 25 μL final
volume. PCR was conducted under the following
conditions: an initial step of 7 min at 94℃, followed
by 30 cycles of 50 s at 94℃ , 50 s at 53℃ and 60s at
72℃ , and a final extension step of 10 min at 72℃.
The products were stored at 4℃ .
3.5 Southern Blot analysis
The CTAB method was used to extract DNA (Doyle
and Doyle, 1987), followed by restriction digestion of
genomic DNA with BglII (Promega Corp., Madison,
WI, USA). After complete digestion, the sample was
subjected to 1% agarose gel electrophoresis and
transferred to a nylon membrane for Southern blot.
Hybridization (with color) and probe preparation were
performed following the instructions specified in the
PCR DIG Labeling Mix (Roche Corp., Basel,
Switzerland).
3.6 RT-PCR analysis
Extraction of total RNA: A leaf from each of 10
putatively-transformed potato plants from 10
independent transformation lines was ground into a
fine powder in liquid nitrogen. Total RNA was
isolated by using the TRIzol® reagent Kit (Tiangen
Biotech Co., Ltd, Beijing, China).
RNA retrovirus reaction system: Using RNA
retrovirus Kit (1st Takara Strand cDNA bio-synthesis
Kit) according to use instructions. Reaction conditions:
30 ℃ 10 min; 42 ℃ 60 min; 70 ℃ 15 min; 8 ℃
preservation.
cDNA template amplification hrap gene reaction
system (25 μL): Taq PCR MasterMix 12.5 μL,
forward and reverse primer each 1 μL, cDNA template
3 μL, ddH2O 7.5 μL. PCR was conducted under the
following conditions: an initial step of 3 min at 95 ℃,
followed by 34 cycles of 50 s at 94℃ , 50 s at 53℃
and 60 s at 72 ℃, and a final extension step of 10 min
at 72℃ . The product was preserved at 8°C.
PCR products were subjected to electrophoresis in a
1.2% agarose gel and photo-documented under UV
light. The Products were purified using the Tiangel
Mini/Midi Purification kit (Tiangen), cloned into
pMD18-T vector (Takara) and then transformed into
DH5α-competent cells (Invitrogen, Carlsbad, CA,
USA). Inserts from independent clones for each gene
were sequenced at Beijing Sunbiotech Co., Ltd
(Beijing, China). The sequence of hrap genes isolated
from transformation plants were compared with hrap
genes form GenBank.
3.7 Assay of disease resistance
3.7.1 Identification of the resistance of P. infestans
The purified test strains of P. infestans (ZY15, LSX18
and XH05-5-4) were cultured in rye-tomato juice
media. After 10~15 days, the culture dish was covered
with mycelia. A small volume of water was added into
the dish and the surface of the mycelia was lightly
scraped in order to make the sporangium fall into
water. The solution was then filtered applying
270-mesh filter, and the number of sporangium in the
filtrate was determined. The target concentration was
1000~2000 sporangium/mL of filtrate. The potato
test-tube plantlets were grown in nursery pots, with
1~4 plants per pot at room temperature. The plants
were inoculated when they had six to nine leaves. The
inoculum of the sporangium suspension was sprayed
on the potato plants, which were maintained in a
humid environment for 24 h and monitored for the
presence of disease.