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

• Published in: Journal of plant physiology Vol. 165; no. 5; pp. 544 - 552
• Main Authors: Xue, Zhong-Tian, Holefors, Anna, Welander, Margareta
• Format: Journal Article
• Language: English
• Published: Jena Elsevier GmbH 01-01-2008; Elsevier
• Subjects: 5'-untranslated region; 5′ untranslated region (5′UTR); Agrobacterium rhizogenes; Agronomy. Soil science and plant productions; apples; Arabidopsis thaliana; Bacterial Proteins - genetics; Base Sequence; Biological and medical sciences; Dwarfing phenotype; Fundamental and applied biological sciences. Psychology; gene expression regulation; Genetic engineering applications; Genetics and breeding of economic plants; introns; Introns - genetics; Malus; Malus - genetics; Malus domestica; messenger RNA; Molecular Sequence Data; Nicotiana tabacum; nucleotide sequences; Plant breeding: fundamental aspects and methodology; Plants, Genetically Modified - genetics; Reverse Transcriptase Polymerase Chain Reaction; Rhizobium rhizogenes; RNA Splicing; RNA, Messenger - genetics; RNA, Messenger - metabolism; rolA transcript; Semi-quantitative RT-PCR; sequence analysis; Sequence Homology, Nucleic Acid; transcription start region; Transcription, Genetic; Transgenic apple; transgenic plants; Untranslated Regions - genetics; Semi-quantitative RT-PCR; DIG; GUS; nt; dNTP; bp; UTR; Dwarfing phenotype; RT-PCR; uORF; rolA transcript; Transgenic apple; 5′ untranslated region (5′UTR); 5' untranslated region (5'UTR); Splicing
• ISSN: 0176-1617; 1618-1328
• DOI: 10.1016/j.jplph.2006.11.010

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.