Nakayashiki Hitoshi, Hanada Shugo, Nguyen Bao Quoc, Kadotani Naoki, Tosa Yukio, Mayama Shigeyuki
Laboratory of Plant Pathology, Kobe University, 1-1 Rokkodaicho, Nada, 657-8501 Kobe, Japan.
Fungal Genet Biol. 2005 Apr;42(4):275-83. doi: 10.1016/j.fgb.2005.01.002.
We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.
我们已经为子囊菌开发了一种类似pHANNIBAL的沉默载体pSilent-1，它携带潮霉素抗性盒和用于发夹RNA表达的转录单元，其间隔区来自稻瘟病菌Magnaporthe oryzae的角质酶基因内含子。在稻瘟病菌中，带有角质酶内含子间隔区（147 bp）的沉默载体在沉默eGFP基因方面比带有GUS基因片段间隔区或几丁质结合蛋白基因较长内含子（850 bp）的沉默载体效率更高。将pSilent-1应用于稻瘟病菌的两个内源基因MPG1和聚酮合酶样基因，在70-90%的转化子中导致了不同程度的基因沉默。基于pSilent-1的载体在黄瓜炭疽病菌中也诱导了RNA沉默，但其效率略低于在稻瘟病菌中的效率，这表明该沉默载体应为子囊菌提供一种有用的反向遗传工具。
