Girke T, Schmidt H, Zähringer U, Reski R, Heinz E
Universität Hamburg, Institut für Allgemeine Botanik, Germany.
Plant J. 1998 Jul;15(1):39-48. doi: 10.1046/j.1365-313x.1998.00178.x.
The moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel delta 5- and delta 6-desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b5-related region and a C-terminal sequence with low similarity (27% identify) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of gamma-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a delta 6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC-MS. Only the transformed yeast cells were able to introduce a further double bond into the delta 6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.
小立碗藓含有高水平的花生四烯酸。要从亚油酸通过去饱和作用和延长作用合成花生四烯酸，需要新型的Δ5-和Δ6-去饱和酶。为了分离其中一种，采用了基于PCR的克隆方法，结果分离出了一个编码假定新去饱和酶的全长cDNA。推导的氨基酸序列有三个结构域：一个约100个氨基酸的N端片段，与数据库中的任何序列都没有相似性，接着是一个细胞色素b5相关区域和一个与酰基脂质去饱和酶低相似性（27%一致性）的C端序列。为了阐明该蛋白质的功能，我们通过用相应的线性基因组序列转化小立碗藓来破坏其基因，该线性基因组序列中插入了一个正向选择标记。对五个转化株系的分子分析表明，选择盒已插入到所有五个株系的相应基因组位点。基因破坏导致敲除植株的脂肪酸模式发生显著改变。所有敲除株系中亚油酸的大幅增加以及γ-亚麻酸和花生四烯酸的随之消失表明，新的cDNA编码一种Δ6-去饱和酶。这通过在酵母中表达该cDNA并通过GC-MS分析所得脂肪酸得到了证实。只有转化的酵母细胞能够在不饱和脂肪酸的Δ6位引入另一个双键。据我们所知，这是关于在多细胞植物中成功进行基因破坏导致特定生化表型的首次报道。
