G-to-A mutation at a 5′ splice site of fad3c caused impaired splicing in a low linolenic mutant of canola (Brassica napus L.)
Two genes (<i>fad3a and fad3c</i>) encode for endoplasmic delta-15 linoleate desaturase, which is responsible for the desaturation of linoleic acid (C18:2) into linolenic acid (C18:3) in canola (<i>Brassica napus</i>). The canola mutant line DMS100 carries a G-to-A base substitution at the 5′ splice site, located at +1 G, of <i>fad3c</i> intron 6 and contains reduced C18:3 content in oil seeds. Reverse transcription (RT) PCR analysis was used to amplify part of the <i>fad3c</i> transcript spanning intron 5, exon 6 and intron 6 using the primers specific to the exon 5 and 7 to investigate whether the mutation caused any abnormal splicing. The RT-PCR amplified a fragment of larger size in the <i>fad3c</i> mutant than in wild-type. Sequencing of the RT-PCR fragments revealed that the mutation activated an impaired splicing which retained the entire intron 6 in the mature <i>fad3c</i> transcript of the mutant. The impaired splicing could result in early termination of translation and synthesis of a shorter <i>fad3c</i> polypeptide because the intron 6 contained stop codons in all three possible reading frames. The incomplete translation could produce an inactive enzyme which blocked the desaturation of linoleic acid (C18:2) to linolenic acid (C18:3), resulting in the decrease of C18:3 accumulation in canola seeds. This is consistent with the observation that the <i>fad3c</i> mutant has significantly lower C18:3 content (<3%) than the wild type (~7%).
- Plant biotechnology
Plant biotechnology 24(4), 397-400, 2007-09-01
Japanese Society for Plant Cell and Molecular Biology