少孢节丛孢中CRISPR/Cas9基因编辑系统的建立及6-甲基水杨酸合酶新活性位点的发现

Establishment and application of the CRISPR/Cas9 system in Arthrobotrys oligospora to identify new active sites of 6-methylsalicylate synthase

以捕食线虫真菌少孢节丛孢Arthrobotrys oligospora YMF 1.03170为研究材料,通过优化sgRNA表达驱动体系tRNAGly,构建CRISPR/Cas9基因编辑系统,成功获得基因定点编辑菌株。将该CRISPR/Cas9系统与同源重组相结合,可精确地对两个目的氨基酸编码基因同时进行定点置换。结合代谢图谱及前体化合物饲喂实验,发现6-甲基水杨酸合酶编码蛋白新的活性位点Arg17、Arg18、His33和His34。本研究将CRISPR/Cas9基因编辑系统应用在少孢节丛孢中,并成功建立基因编辑精细体系,为快速构建少孢节丛孢的遗传转化体系和研究该菌的基因功能提供有效方法。

A CRISPR/Cas9 gene editing system was successfully constructed in nematode-trapping fungus Arthrobotrys oligospora YMF 1.03170 by optimizing the sgRNA expression driving system tRNAGly. The CRISPR/Cas9 system combined with homologous recombination led to the precise replacement of the two target amino acid coding genes at the same time. Combined with metabolic profile and feeding experiment of precursor compounds, new active sites Arg17, Arg18, His33 and His34 in 6-methylsalicylate synthase 283 were found. The fine gene editing system CRISPR/Cas9 established in Arthrobotrys oligospora provided an effective method for rapidly constructing the genetic transformation system of Arthrobotrys oligospora and studying the gene function of this fungus.