Rice Genetics IV - IRRI books - International Rice Research Institute

Transgene integration,organization, and expressionin cerealsP. Christou, R.M. Twyman, Xiangdong Fu, E. Wegel, A. Kohli, and E. StogerAs in other direct DNA transfer systems, transgenes delivered by particlebombardment integrate into the cereal genome by illegitimate recombinationassisted by short regions of homology. Exogenous plasmid DNA tends toconcatemerize prior to integration, resulting in tandemly arranged contiguouscopies. However, recent research has shown that such concatemers maypositively influence the integration of further transgenes nearby, perhaps byrecruiting DNA repair complexes to the site of the original break. Transgenicloci therefore tend to comprise clusters of contiguous copies interspersedwith short regions of genomic DNA. The analysis of transgenic wheat chromosomesby fluorescence in situ hybridization (FISH) indicates a further level oforganization, where several transgene clusters integrate in the same regionof the chromosome arm, but individual clusters produce separable FISH signalsat metaphase, suggesting they are interspersed by large segments ofgenomic DNA. A segregating transgene locus therefore has a hierarchicalstructure, showing up to three levels of organization.The analysis of transgene integration shows that recombination plays astrong role in determining integrated transgene structure. Particularly, certainsites in the transformation plasmid can provide hotspots for recombination,leading to particular types of transgene rearrangement. Since the failureof transgene expression often reflects such rearrangements, the modificationof transformation vectors to eliminate troublesome sequences shouldimprove the efficiency of transformation. The “clean DNA” system takes thisstrategy to its logical extreme by removing all unnecessary backbone elementsand using just minimal linear cassettes (promoter, coding region, terminator)for transformation. Such experiments consistently generate transgenicplants with simple integration patterns, lower transgene copy numbers, fewerrearrangements, and stable transgene expression. The clean DNA systemalso allows direct transformation with multiple genes, but the integrationpatterns remain simple and silencing occurs very rarely.Silencing often correlates with DNA methylation, and it is of particularinterest to identify sequences in plant transgenes that induce methylationand result in unstable expression. There is evidence that methylation can beinduced by interactions between homologous transgene copies (or betweena transgene and homologous endogene) or may reflect genomic position ef-Transgene integration, organization, and expression . . . 449