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 (fad3a and fad3c) 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 (Brassica napus). The canola mutant line DMS100 carries a G-to-A base substitution at the 5' splice site, located at +1 G, of fad3c intron 6 and contains reduced C18: 3 content in oil seeds. Reverse transcription (RT) PCR analysis was used to amplify part of the fad3c 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 fad3c 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 fad3c transcript of the mutant. The impaired splicing could result in early termination of translation and synthesis of a shorter fad3c 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 fad3c mutant has significantly lower C18: 3 content (<3%) than the wild type (similar to 7%).