Transcription of a maize cDNA in Lotus corniculatus is regulated by T-DNA methylation and transgene copy number

Lotus corniculatus plants transformed with a maize cDNA (G1L) encoding a su lphur-rich gamma-zein were obtained by using two fusion genes: one with the CaMV 35S promoter, the other with the ribulose bisphosphate carboxylase sm all subunit (rbcS) promoter. The highest expression of G1L mRNA was found i n plants transformed with G1L under the rbcS promoter. The steady state lev el of G1L mRNA in the leaves was generally directly correlated with the G1L copy number. However, due to a transcriptional block, no G1L mRNA was dete cted in some of the 35S-G1L multicopy transformants. Analyses with methylat ion-sensitive restriction enzymes revealed that the T-DNA of the silenced 3 5S-G1L transformants was methylated. T-DNA copy number, G1L silencing activ ity, and the state of methylation were strictly correlated in primary trans formants. A cross between two 35S-G1L transformed plants, one (S) with the T-DNA methylated and the other (NS) without, showed that: (1) the methylate d state of T-DNA passed through meiosis; and (2) when T-DNA copies from the two parents were combined in the progeny, the unmethylated T-DNA copies of parent NS became methylated at different levels and G1L mRNA production wa s dependent on the degree of methylation.