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White lupin seeds (Lupinus albus v Lupro 2085) were scarified, germinated in a coarse sand and turface mixture and grown under greenhouse conditions (temperature, light cycle). Roots of white lupin seedlings were harvested at 5, 10, 15 and 20 days post-emergence, immediately frozen in liquid nitrogen and stored at -80oC prior to analysis. Total RNA was extracted from the root tissue using TRI reagent (Molecular Research Center, Cincinnati, OH). Equal amounts of RNA sample from each time point were pooled and the combined total RNA was used for construction of a cDNA library. Roots for protein analysis were grown under the same greenhouse conditions, except that they were harvested at 17 days post emergence. A developing white lupin root cDNA library was prepared from total RNA using the Creator Smart cDNA library construction kit (BD Bioscience, Palo Alto, CA) following the manufacturer'¯s instructions, and was transformed into E. coli. Eight thousand bacterial clones were randomly picked and used for inoculation of liquid culture. Plasmid DNA was purified from the overnight liquid culture and sequenced with the M13 forward primer.
Nucleotide
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