A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens

The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gen e function. This technique has so far been difficult to apply to plants bec ause the frequency of gene disruption following transformation with constru cts containing DNA homologous to genomic sequences is low (0.01 to 0.1%). I t has recently been shown that high rates of gene disruption (up to 90%) ca n be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of ge ne disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isol ated segments of 13 other closely related members of the Cab gene family. I n the 199-bp stretch sequenced, the 13 new members of the Cab family show a n average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 30 4 silent substitutions and 16 substitutions that lead to a change in the am ino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab ge nes) into a vector containing a selectable hygromycin resistance marker, an d used this construct to transform P. patens. In three of nine stable trans formants tested, the construct had inserted in, and disrupted, the ZLAB1 ge ne. There was no discernible phenotype associated with the disruption. We h ave therefore shown that gene disruption is reproducible in P. patens and t hat the requirement for sequence homology appears to be stringent, therefor e allowing the role of individual members of a gene family to be analysed i n land plants for the first time.