Page 10 - Molecular Plant Breeding

Deng et al., 2011, Ectopic expression of an AGAMOUS homolog NTAG1 from
Chinese narcissus accelerated earlier flowering and senescence in Arabidopsis, Molecular Plant Breeding Vol.2 No.3 (doi: 10.5376/mpb.2011.02.0003)
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3 Materials and methods
3.1 Plant materials and growth conditions
Plants of narcissus Yulinglong and Jinzhanyutai used
for this study were grown in the field in Chongming,
Shanghai. Arabidopsis thaliana of ecotype
Columbia-0 (Col) and Lersberg (Ler) plants were
grown in the green house under constant illumination
(-80 μ mol·m–2·s–1) at 22±2ºC. For transgenic plants to
be screened, seeds were surface sterilized, stratified at
4ºC for 3 d, plated on 1/2 Murashige and Skoog
(Murashige and Skoog, 1962) medium (MS)
containing 50 μg.mL-1 kanamycin, and grown under
continuous fluorescent light at 22ºC.
3.2 Scanning electron microscopy (SEM)
Plants were fixed in FAA (70% ethanol 89%,
formaldehyde 5%, acetic acid 6%) at 4ºC overnight,
dehydrated in an ethanol series, critical-point dried in
liquid CO2, sputter-coated with gold palladium,
analyzed and photographed with a Philips XL 30 FEG
SEM.
3.3 Determination of malonaldehyde (MDA) levels
MDA determination was followed the method
described by Zhang (Zhang et al., 2009). Fresh leaves
of 20-d-old plants (0.2 g) were homogenized in 6 mL
10% (w/v) trichloroacetic acid. The homogenates
were centrifuged at 5000×g for 10 min. A reaction
mixture of the supernatant (2 mL) and 2 mL
thiobarbituric acid (0.6%) was incubated in a boiling
water bath for 15 min, then cooled immediately before
centrifugation. Absorbance of the supernatants was
determined at 450, 532 and 600 nm, respectively.
Calculation of MDA was based on the following
formula: C (μ mol/L) = 6.45(A532- A600)-0.56 A450.
3.4 Total RNA and genomic DNA extraction
Total RNA was isolated using Trizol reagent (Tiangen,
Shanghai) plus 5 mol/L NaCl to reduce polysaccharide
level in the RNA. For Arabidopsis RNA isolation,
total RNA was extracted from the above ground
tissues. And RNA was pretreated with RNase-free
DnaseI (Takara, Japan) to remove contaminating
genomic DNA. Arabidopsis and narcissus genomic
DNA was isolated by using the lysis buffer (0.2 mol/L
Tris, 0.05 mol/L EDTA, 7 mol/L Urea, 2% Sarkosyl,
pH 8.0), employing phenol/chloroform followed by
precipitation of DNA with iso-propyl alcohol.
3.5 Isolation and sequence analysis of NTAG1
To obtain the main fragment of the C-lineage genes
from both narcissus varieties, primers AGAMOUS F
(5'-ctc gag ATG GGG AGG GGT AAG ATA GAG
ATC AA-3') and AGAMOUS R (5'-ctc gag TCA
TCC CAG TTG AAG GGT AGT- 3') were designed
according to the NAG gene reported by Wang (Wang
et al., 2006). Both the specific 5' and 3' primers for
NTAG1 contained the generated KpnI recognition site
(5'-ctc gag-3', a lowercase letter) to facilitate the
cloning of this gene. cDNA was synthesized from 500
μg total RNA using a cDNA synthesis kit (TOYOBO,
Japan). PCR reactions were performed using the pfu
PCR kit (Takara, Japan) and the amplified fragments
were purified with the DNA Gel Extraction kit
(Tiangen, Shanghai), cloned into the pMD18-T vector
(Takara, Japan), and verified by sequencing.
3.6 Plant transformation and transgenic plants
analysis
A KpnI fragment containing the full-length cDNA for
NTAG1 gene was introduced into the binary T-DNA
vector pMon530 (Monsanto, USA), driven by the 35S
promoter. The sense orientation construct for this gene
was determined using digestion and sequencing. The
construct was transferred into Agrobacterium
tumefaciens GV3101 (Koncz and Schell, 1986), and
then introduced into Arabidopsis plants using the
Floral dip method (Clough and Bent, 1998).
Transformants that survived in the medium containing
kanamycin (50 μg·mL-1) were further verified by PCR
and reverse transcriptase-PCR analyses.
The phenotypic effects of NTAG1 in transgenic plants
were analyzed in the T1 generation and in the
homozygous kanamycin-resistant T3 generation plants
through photographing with a digital camera or SEM.
To compare the phenotypes of the transgenic and wild
type plants, at least 20 transgenic plants from 5
independent lines respectivelly and 20 wild-type
plants were grown under the same conditions. Plant
height, silique length, branch number, leaf number etc.
were recorded.