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 sulphur-rich Þ-zein, were obtained by using two fusion genes: one with the CaMV 35S promoter, the other with the ribulose bisphosphate carboxylase small subunit (rbcS) promoter. The highest expression of G1L mRNA was found in plants transformed with G1L under the rbcS promoter. The steady-state level of G1L mRNA in the leaves was generally directly correlated with G1L copy number. However, due to a transcriptional block, no G1L mRNA was detected in some of the 35S-G1L multi-copy transformants. Analyses with methylation-sensitive restriction enzymes revealed that the T-DNA of the silenced 35S-G1L transformants was methylated. T-DNA copy number, G1L silencing activity and the state of methylation were strictly correlated in primary transformants. A cross between two 35S-G1L transformed plants, one (S) with the T-DNA methylated and the other (NS) without, showed that: 1) the methylated state of T-DNA passed unaltered through meiosis; 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 was dependent on the degree of methylation.