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Host for highly efficient gene targeting in filamentous fungi

丝状真菌中高效基因靶向的宿主

基本信息

批准号：
18370001
负责人：
INOUE Hirokazu
金额：
$ 6.03万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370001/
关键词：
Nonhomologous end joining Gene targeting KU70 KU80 Ligase IV MRX Neurospora Aspergillus 多目同末端結合遺伝子ターゲッティング Liase lV Neurosora Aserillus 非相同組換え糸状菌

项目摘要

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.

研究基因功能最理想的方法是基因打靶。然而，基因打靶在许多生物体中的频率极低。为了提高双链DNA断裂的发生率，人们进行了一些试验，但还没有找到确切的技术。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。从我们在粗糙脉孢菌中的研究中，我们推测，如果非同源末端连接的功能被阻断，同源重组频率会增加。我们将脉孢菌的ku 70和ku 80同源基因破坏，并将它们用作转化实验的宿主。在该研究中，观察到DNA的高度同源整合（二宫，et.为了开发更方便的宿主，我们破坏了涉及非同源末端连接的其他脉孢菌基因;连接酶IV和XRCC 4同源基因。这些突变体显示出高的靶向频率，即使导入的DNA同源长度很短。我们还检测了MRX缺陷菌株的靶向频率。MRX是Mre 11-Rad 50-Xrs 2的复合物，在双链断裂修复中起作用。为了了解该系统在其他生物中是否有效，本研究选择了黑曲霉作为实验材料。在A.米。利用ligD突变体作为宿主进行的靶向实验表明其具有很高的同源整合率，在许多其他真菌中，国内外的研究人员也进行了类似的实验，证实了利用ku突变体的靶向效率高。然而，由于在转化、选择标记等方面存在意想不到的问题，我们在担子菌中的试验还没有进行到最后一步，我们将继续这些试验，以构建更方便的靶向系统。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

麹菌(A. oryzae)のligD遺伝子破壊による高頻度相同組換え宿主の造成

破坏米曲霉ligD基因构建高频同源重组宿主

DOI：
发表时间：
2006
期刊：
影响因子：
0
作者：
Kusaba M;Ito H;Morita R;Iida S;Sato Y;Fujimoto;M.;Kawasaki S;Tanaka R;Hirochika H;Nishimura M;Tanaka A;井上弘一;Tanaka,A;工藤洋平
通讯作者：
工藤洋平

麹菌のDNAligase (lig4)遺伝子破壊による高頻度組換え宿主の造成

通过破坏米曲霉中的 DNA 连接酶 (lig4) 基因创建高度重组宿主