Molecular and genetic analysis of transgenic rice plants expressing the maize ribosome-inactivating protein b-32 gene and the herbicide resistance bar gene

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zm-crip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R-0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R-1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene cosegregated with the phosphinothricin-resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R-1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5-1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R-2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize.