XVI International Botanical Congess

Glucose-6-phosphate dehydrogenase (G6PDH) was detected in total RNA of tissue cultured soybean cells using RT-PCR with primers modified from sequence homologies to potato and other published G6PDH sequences. The soybean PCR product was cloned and sequenced to verify it was specific for a G6PDH. Cloned cDNA's of G6PDH were captured from phagemids excised from a soybean lambda ZapII (Stratagene)cDNA library. Capture was effected by hybridization of ss phagemids with a biotin labeled G6PDH specific oligonucleotide based on the internal soybean PCR product sequence, and magnetic separation after interaction of the biotin-oligo-cDNA complex with streptavidin coated magnetic particles. An ExoIII deletion series was generated and deletions sequenced by PCR dideoxy sequencing with fluorescent labeled dNTP's and separation by capillary electrophoresis(PE/ABI).