Intron splicing in 5′ untranslated region of the rolA transcript in transgenic apple

The rolA gene encoded on the Ri plasmid of Agrobacterium rhizogenes causes developmental alterations, including dwarfing characteristics in the transgenic plants. In an attempt to introduce dwarfing characteristics into apple rootstocks for breeding purposes, the rolA gene was incorporated into the apple rootstock M26 and obtained four transgenic clones. All the clones exhibited reduced growth compared to untransformed control plants but different degree of dwarfing and wrinkled leaves. In the present study, expression of the rolA gene was further investigated by analysing the structure of the rolA transcript and the levels of the rolA mRNAs from these clones. The nucleotide (nt) sequence of the rolA transcript showed two forms of the transcript: one, the unspliced form, was co-linear with the rolA sequence in the genomic DNA; the other was spliced mRNA in which an 85-base pair (bp) intron sequence in the 5′ untranslated region (5′UTR) was spliced out. The position of splicing is different from that in Arabidopsis thaliana but similar to the splicing site found in tobacco. The transcription start region of the rolA gene in apple was 206 bp upstream of that in Arabidopsis and 277 bp upstream to Nicotiana tabacum transcription start. A hairpin-like secondary structure and an upstream open reading frame (uORF) were revealed in the rolA 5′UTR. The levels of the rolA mRNA in the apple transgenic clones were analysed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed slight variation in the shoot tissues of the transgenic clones.