Host for highly efficient gene targeting in filamentous fungi

为了研究基因的功能，最理想的方法是基因打靶。然而，在许多生物体中，基因打靶的频率非常低。为了提高频率，人们进行了一些试验，但还没有找到确凿的技术。双链DNA断裂的修复有两种不同的重组机制：同源重组和非同源末端连接。从我们对粗枝脉孢菌的研究中，我们推测如果非同源末端连接功能被阻断，同源重组频率会增加。我们克隆了脉孢菌的ku70和ku80同源基因，并将它们作为宿主进行了转化实验。在本研究中，观察到DNA的高度同源整合(Ninomiya，et.艾尔为了开发更方便的宿主，我们中断了涉及非同源末端连接的其他脉孢子菌基因；Ligase IV和XRCC4同源基因。即使导入的DNA的同源长度很短，这些突变体也表现出很高的靶向性。我们还测试了MRX缺陷菌株的靶向频率。MRX是Mre11-Rad50-Xrs2的复合体，在双链断裂修复中发挥作用。许多转化子来自同源整合，但转化频率较低，为了了解该系统在其他生物中是否适用，选择了米曲霉进行测试。在米曲霉中构建了LigD(人Lig4同源基因)突变体。在许多其他真菌中，世界各地的研究人员都进行了类似的实验，证实了利用ku突变体进行了高的靶向效率。然而，由于在转化、选择标记等方面存在意想不到的问题，我们在担子菌上的试验没有进入最后一步。我们将继续进行这些实验，以构建更便捷的靶向系统。

To investigate gene function, most desirable method is gene targeting. However, gene targeting frequency is extremely low in many organisms. To raise the frequency, some trials have been carried out, but conclusive technique was not found.Double strand DNA breaks are repaired by two different recombination mechanisms: homologous recombination and nonhomologous end joining. From our study in Neurospora crassa, we speculated that homologous recombination frequency is increased if function of nonhomologous end joining is blocked. We disrupted ku70 and ku80 homolog genes of Neurospora and used them as a host in transformation experiments. In this study, high homologous integration of DNA was observed (Ninomiya, et. al. 2004).To develop more convenient host, we disrupted other Neurospora genes involving in nonhomologous end joining; Ligase IV and XRCC4 homolog genes. These mutants showed high targeting frequency even if homologous length of introduced DNA is short. We also tested targeting frequency in MRX-defective strains. MRX is complex of Mre11-Rad50-Xrs2 and functions in double strand breaks repair. Many transformants were from homologous integration, though transformation frequency was low.To know whether this system works in other organisms, Aspergillus oryzae was selected for test. Mutant of LigD ( human Lig4 homolog) was constructed in A. oryzae. Targeting experiments using ligD mutant as a host indicated high homologous integration.In many other fungi, similar experiments have been coducted by researchers all over the world and they confirmed high targeting efficiency by using ku mutants. as we presented.However, our trials in basidiomycetes did not reached to final step since there were unexpected problems in transformation, selection marker, so on. We will continue these experiments for construction of more convenient targeting system.