Biology Reference
In-Depth Information
RNA In Situ Hybridization
Floral tissue for in situ hybridization was fixed, sectioned and hybridized to digox-
ygenin-labeled probes of OpdCYC1C, OpdCYC1D, OpdCYC2A and OpdCY-
C2B with reference to described methods [52]. Four gene-specific fragments of
OpdCYC1C, OpdCYC1D, OpdCYC2A and OpdCYC2B in the coding region
were amplified, respectively, and then were purified and cloned into pGEM®-T
Easy vectors. Digoxygenin-labeled probes of OpdCYC1C, OpdCYC1D, Opd-
CYC2A and OpdCYC2B were prepared from linearized templates amplified us-
ing primer Yt7 and Ysp6 from pGEM®-T plasmids [53].
Gene-Specific Semiquantitative RT-PCR
Flowers of different stages were collected as follows: Flower buds of middle-stage
(less than 1 cm long) and flowers of late-stage (3-4 cm long) were collected sepa-
rately. Sepals were removed from the outer whorl. The petals with corresponding
corolla-tube were dissected into dorsal (including the attached dorsal staminode),
lateral, and ventral regions. Lateral stamens and ventral staminodes were dissected
from the corolla-tube and collected each for RT-PCR. All materials were frozen in
liquid nitrogen immediately after collection for ribonucleic acid (RNA) isolation.
The extraction of total RNAs, purification of poly (A) mRNAs, and synthesis
of the first-strand cDNAs were performed according to the methods described
above. The template quantity was regulated to be uniform using the ACTIN gene
[54]. PCR was performed by using gene-specific primers of OpdCYC1C, Op-
dCYC1D, OpdCYC2A, OpdCYC2B, OpdcyclinD3a and OpdcyclinD3b. To
make sure that each pair of primers was suitable, we first used them to amplify
genomic DNA of O. dinghushanensis. The PCR products were then cloned. At
least 20 clones of each PCR product were sequenced, and all the primers used
could amplify the specific copies of OpdCYC and OpdcyclinD3 genes. The fol-
lowing thermocycling conditions were employed: initial denaturation at 96°C
for 3 min, 30 cycles of 96°C for 30 s, 55-60°C (depending on the Tm value of
primer pairs) for 30s, and 72°C for 1 min, and a final extension at 72°C for 10
min. The amplified products were separated on a 1.5% agarose gel, and the den-
sity of ethidium bromide-stained bands was determined using a Bioimaging Sys-
tem (Gene Tools Program, Syngene, UK). We repeated the RT-PCR experiments
five times independently with a new RNA extraction each time. In addition, all
RT-PCR products were cloned into pGEM-T Easy-vector, and at least 20 clones
from each product were sequenced to test the gene specificity of RT-PCR. The
OpdCYC/ACTIN and Opdcyclin/ACTIN ratios represented the relative level of
OpdCYC and OpdcyclinD3 mRNA expression. Data are presented as the mean
± SD of independent RT-PCR experiments, and one-way analysis of variance was
