The cDNA library was constructed from mRNA isolated from 2-3 week old seedlings that were induced for symptoms of SDS (Sudden Death Syndrome) disease by the translocation of culture filtrate of Fusarium solani f. sp. glycines (Plant Cell Report 18:375-380). Cultivar PI 567.374 is resistant to the disease SDS. Plant tissue (expanded leaves, folded leaves, and new shoots) were collected at 1, 6, 24, and 48 hrs after inoculation and their mRNA pooled equally for cDNA construction. The library was prepared using the Stratagene pBluescript II SK (+) library construction kit. Complementary DNA was synthesized from mRNA using a primer consisting of a poly(dT) sequence with an Xho I restriction site. Eco RI adaptors were ligated to the blunt-ended cDNA fragments followed by Xho I digestion. The cDNA insert is protected from XhoI digestion via methylation during first strand cDNA synthesis. The cDNA fragments were directionally cloned into the Eco RI-Xho I restriction site of the pBluescript vector. The ligated cDNA fragments were transformed into E. coli ElectroMax DH10B host cells. Plants were inoculated by Shuxian Li (Glen Hartman lab, University of Illinois). Library was constructed by Steve Clough (Lila Vodkin lab, University of Illinois).
Transformation efficiency: 1011
Percent white colonies: 60%
Average insert size of white colonies: 1100 bp
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