作　　者： ; ;

机构地区： 中国农业大学生物学院农业生物技术国家重点实验室

出　　处： 《微生物学报》 2010年第5期593-600,共8页

摘　　要： 【目的】通过构建26S蛋白酶体的3个亚基RPN4、RPN7、RPN10的缺失突变菌株,研究这些缺失突变体的表型,进而探究这3个亚基在蛋白酶体中的作用。【方法】采用同源重组基因敲除技术、电转化、粗糙脉胞菌杂交、子囊孢子萌发及PCR鉴定等方法分别获得3个调节亚基的基因缺失突变体。利用racetube和平板生长法进行突变体表型检测。【结果】得到rpn4和rpn10的缺失突变纯合体及rpn7缺失突变异核体菌株。【结论】与野生型相比,rpn7KO(ku70RIP背景)突变体的菌丝生长及产生分生孢子的能力显著减弱;rpn4KO突变体在生长初期的菌丝生长缓慢,而后期的产孢能力与野生型无显著差异;rpn10KO突变菌株的表型介于上述两种突变体的表型之间。这些结果表明26S蛋白酶体的这3个亚基对脉胞菌的生长和发育至关重要。 [Objective]In this study,we generated strains with knockout rpn4,rpn7 and rpn10 genes respectively in Neurospora crassa,and studied their phenotypes to uncover their functions in the 26 S proteasome pathway.[Methods] We used gene-replacement strategy to make knockout mutants.After electrotransformation,the resulted transformants were examined by PCR.To get homokaryotic mutants,the heterokaryotic strains were crossed with the wild-type strain.The ascospores were germinated after heat shock,and the resulting strains were examined by PCR to confirm the homokaryotic strains.[Results]Homokaryotic rpn4 and rpn10 knockout strains,and heterokaryotic rpn7^KO strains were obtained.[Conclusion]Compared to the wild-type strain,rpn7^KO strains exhibited severe defects in hyphal growth and conidial formation;rpn4^KOstrain had defect in hyphal growth too,but displayed normal conidiation;the growth and developmental phenotypes of rpn10^KOstrain were slightly affected.Taken together,these data demonstrated that these three genes were important for the growth and developmental phenotypes of Neurospora crassa.

关 键 词： 粗糙脉孢菌 泛素 蛋白酶体途径 蛋白酶体 调节亚基

领　　域： [生物学]