Tanguay Philippe, Bozza Siegfried, Breuil Colette
Department of Wood Science, University of British Columbia, Vancouver, BC, Canada.
Fungal Genet Biol. 2006 Dec;43(12):804-12. doi: 10.1016/j.fgb.2006.06.004. Epub 2006 Jul 21.
Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase (PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.
长喙壳菌属物种是一类在树木或木材中生长的具有经济重要性的腐生和致病真菌。像云杉长喙壳菌和絮状长喙壳菌这样的长喙壳菌会产生黑色素，这种色素会使木材和原木染色。我们将此类物种用作模式生物来表征真菌黑色素产生的分子机制。由于在长喙壳菌中同源重组很少见，因此鉴定该类群中的基因功能具有挑战性。我们通过评估RNA干扰（RNAi）作为基因替换的替代方法来解决这一问题。为此，我们构建了不同的反向重复转基因（IRT）构建体，以下调云杉长喙壳菌和絮状长喙壳菌中黑色素途径的聚酮合酶（PKS1）基因。用IRT-PKS进行转化降低了PKS1基因的mRNA水平，从而减少了转化体中的色素沉着。我们表明，PKS1 RNAi效率与从IRT构建体表达的dsRNA长度成正比。这些结果表明，RNAi是长喙壳菌基因功能分析的合适工具。
