Fig. 5

Mature trnK ^(UUU) product from three orchid species. MatK activity in orchids was determined by examining whether the trnK ^(UUU) intron was effectively removed from precursor transcript. Total RNA was isolated from three orchid species, Phaius tancarvilleae, Spiranthes cernua and S. vernalis and subsequently converted to cDNA by first strand synthesis using an oligo dT₍₂₀₎ primer. Mature trnK ^(UUU) product resulting from removal of intervening group IIA intron and ligation of surrounding trnK exons resulted in a RT-PCR product range from 50 to 61 bp depending on species based on binding position of primers NITtrnkRev and trnK 3’end primer (this study). Precursor RNA still containing intron would result in a band of ~2800 bp based on these same primers. (a) Annotation of primers to the trnK/ matK gene region. (b) RT-PCR products using primers specific to the trnK ^(UUU) exons resolved on 1.7 % agarose gels. Lanes: L1, 1 Kb ladder (New England Biolabs), L2, BenchTop PCR markers (Promega), 1–3 represent products from RT-PCR of three biological replicates for each orchid species examined, neg is the negative control. Sizes of relevant DNA standards based on expected or identified PCR products are shown. Arrows indicate mature trnK ^(UUU) product. Primer dimer due to secondary structure of primers used in PCR is evident as a faint disperse band below the 50 bp standard in negative controls