Cloning the Coprinus bilanatus TRP2 gene and its use as a selectable marker in transformation

Abstract

A Coprinus bilanatus tryptophan biosynthesis gene ( TRP2 ) has been cloned and used to develop homologous transformation for the species. Transformation of another Coprinus species, C. cinereus , was used to screen a C. bilanatus cosmid genomic library for the TRP2 gene and identify a cosmid clone LX14B5. Some transformants were identified in which the cosmid vector had not integrated with the TRP2 gene; this type of transformation even could account for an earlier failure to clone a resistance gene. The C. cinereus trp-2 gene was used to probe and identify putative functional fragments in Southern blots of LX14B5. A TRP2 subclone, pCBT2-S5, which contained a 6·2 kb Sac I fragment, was used to develop homologous transformation of C. bilanatus , and up to 1355 transformants μg −1 of DNA were recovered. A 3·5 kb Xba I fragment or a 2·5 kb Cla I fragment were also shown to transform for TRP2 . Rates of transformation achieved with the smaller fragments were lower than those for the larger Sac I fragment, suggesting that regulatory sequences may have been lost during subcloning. Transformants of both C. bilanatus and C. cinereus were integrative and stable through mitosis. Meiotic transmission of the heterologous TRP2 gene was demonstrated in progeny of C. cinereus .