Kim Dae-Hyuk, Ko Yo-Han, Chun Jeesun
Department of Molecular Biology, Institute for Molecular Biology and Genetics, Jeonbuk National University, 567 Baekje-daero, Jeonju, Chonbuk 54896, Korea.
J Fungi (Basel). 2021 Apr 25;7(5):332. doi: 10.3390/jof7050332.
Functional analysis of a GSP1/Ran ortholog, , from was conducted. Genotype analysis revealed that the putative -null mutant was a heterokaryotic transformant harboring two different types of nuclei, one with the wild-type allele and the other with the -null mutant allele. The mycelial growth and colony morphology of the heterokaryotic transformant was normal. Microscopic analysis of the resulting conidia (aseptate and monokaryotic asexual spores) demonstrated that although normal germinating spores were observed from conidia harboring a nucleus with the wild-type allele, a number of residual conidia that did not germinate existed. Complementation analysis using protoplasts from the heterokaryon with the wild-type allele confirmed that the gene is essential to . Complementation analysis using the various chimera constructs allowed us to perform a functional analysis of essential amino acids of the . Among the four suggested essential amino acids, Lys-97 for ubiquitination was determined to not be an essential residue. Moreover, the -null mutant allele was successfully complemented with mouse Ran gene, which suggested that the biological function of Ran gene is evolutionary conserved and that our heterokaryon rescue can be applied for the functional analysis of heterologous genes.
对来自[具体来源]的GSP1/Ran直系同源基因进行了功能分析。基因型分析表明，推定的[基因名称]缺失突变体是一种异核转化体，含有两种不同类型的细胞核，一种带有野生型[基因名称]等位基因，另一种带有[基因名称]缺失突变等位基因。该异核转化体的菌丝生长和菌落形态正常。对产生的分生孢子（无隔单核无性孢子）进行显微镜分析表明，虽然从含有野生型[基因名称]等位基因细胞核的分生孢子中观察到正常萌发的孢子，但存在许多未萌发的残留分生孢子。使用来自带有野生型[基因名称]等位基因的异核体的原生质体进行互补分析证实，[基因名称]基因对[相关过程或功能]至关重要。使用各种[基因名称]嵌合构建体进行互补分析使我们能够对[基因名称]的必需氨基酸进行功能分析。在四个推测的必需氨基酸中，用于泛素化的赖氨酸97被确定不是必需残基。此外，[基因名称]缺失突变等位基因成功地被小鼠Ran基因互补，这表明Ran基因的生物学功能在进化上是保守的，并且我们的异核体拯救可用于异源基因的功能分析。
