Biology Reference
In-Depth Information
(Airam, Kerava, Finland) and natural sunlight. After two to three leaves had de-
veloped per plant, shoot apex samples (tip of the shoot containing the meristem
as well as two to three leaf initials) were collected under a stereomicroscope at
ten different time points with three days intervals. Samples from each time point
were pooled and used for the construction of cDNA libraries and real-time RT-
PCR. WT samples contained shoot apices of the main crown, collected from 50
plants per time point. Also in EB genotype, shoot apices of the main crown were
collected until the sepal initials became visible in the meristems. After this time
point, the apices from one to three side shoots per plant were collected, altogether
from 40 plants per sampling. In addition, leaf samples were collected from the
same plants at four leaf stage for real-time RT-PCR analysis. Moreover, separate
shoot apex samples were collected from WT and EB genotypes at one, two, three
and four leaf stages. Control plants were grown in LD and their flowering time
was determined by counting the number of leaves in the main crown before the
terminal inflorescence. All samples were collected in July - August 2006 - 2008.
Preparation and Sequencing of Subtracted cDNA Libraries
Total RNA from pooled shoot apex samples was extracted with a pine tree meth-
od for RNA isolation [74]. The cDNA was synthesized with BD SMART cDNA
Synthesis kit (Clontech, Palo Alto, US), amplified with PCR as instructed for
subtraction, purified with Chroma Spin-1000 DEPC-H2O Columns (Clontech),
extracted with chloroform:isoamylalcohol (24:1) using Phase Loch Gel Heavy 1.5
ml tubes (Eppendorf, Hamburg, Germany), digested with RsaI (Boehringer Man-
nheim, Mannheim, Germany), and purified with High Pure PCR Product Puri-
fication kit (Roche Diagnostics, Indianapolis, US). The cDNAs were subtracted
using BD PCR-Select cDNA Subtraction Kit (Clontech) in both forward and
reverse directions. The forward and reverse PCR mixtures were digested with RsaI
(Boehringer Mannheim) and purified with High Pure PCR Product Purification
Kit (Roche). After digestion, A-tailing was done as instructed in the technical
manual of pGEM-T and pGEM-T Easy Vector Systems and PCR mixtures were
ligated to pGEM-T Easy Vector (Promega, Wisconsis, US), and electroporated
to TOP10 cells. The libraries were sequenced at the Institute of Biotechnology,
University of Helsinki, as described earlier [75].
Bioinformatics Analysis
Raw EST sequences were quality checked before annotation. Base calling, end
clipping and vector removal were performed by CodonCodeAligner-software
(CodonCode Corporation, US). After this the ESTs were manually checked and
