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This report described an improved method for isolating
intact purified RNA from freezing organs of bread wheat plants. High-quality
RNA is important in studying gene expression. Common RNA extraction protocols
have produced poor yields because freezing leaves contain polysaccharides
and RNases. We used two methods for isolating RNA and comprised them.
CTAB (cetyltrimethylammonium bromide) method protocol is based on a guanidine
thiocyanate extraction combined with additional purification steps using
butanol and the ionic detergent CTAB. Using this protocol, RNA yields
ranged from 40-70 μg of total RNA 200 mg of fresh tissue. This method
can be adapted to large-scale isolations, allowing the recovery of larger
amounts of intact RNA (up to 250 μg g-1 of fresh tissue).
In this study, we exploit the useful described CODEHOP
primer design and RT-PCR strategy for targeted isolation of homologues
in large gene families. The method was tested with two different objectives.
The first was to apply CODEHOP strategy for design degenerate oligonucleotide
primers in a broad range of plant species. The second was to isolate an
orthologus of the transcription factor of dehydration-responsive element
binding protein (DREB) and to determine the complexity of gene family
in bread wheat. We used a new primer design strategy for PCR amplification
of unknown targets that are related to multiply-aligned protein sequences.
Each primer consists of a short 3` degenerate core region and a longer
5` consensus clamp region. Only 3-4 highly conserved amino acid residues
are necessary for design of the core, which is stabilized by the clamp
annealing to templates molecules. This provides the possibility of isolating
numerous additional DREB genes by Polymerase Chain Reaction (PCR) with
degenerate oligonucleotide primers. The relationship of the amplified
products to DREB genes was evaluated by several sequence and genetic criteria.
Present data show that expression of DREB and its homologues, is induced
by low temperature stress. Towards this step, it found that the expression
of DRE-regulated genes increased freezing tolerance in plants.
