粗糙脉孢菌26 S蛋白酶体三个亚基缺失菌株的构建及表型分析

Construction of deletion mutants for three proteasome protein coding genes and characterization of their phenotypes in Neurospora crassa

摘要:

摘要：【目的】通过构建26 S蛋白酶体的三个亚基RPN4、RPN7、RPN10的缺失突变菌株，研究这些缺失突变体的表型，进而探究这三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三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26 S蛋白酶体的这三个亚基对脉胞菌的生长和发育至关重要。

Abstract:

Abstract: [Objective] In this study, we generated knockout strains of rpn4, rpn7, and rpn10 genes respectively in Neurospora crassa, and studied their phenotypes to uncover their functions in the 26 S protesome pathway. [Methods]We used gene-replacement strategy to make knockout strains. 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 rpn7KO strains were obtained. [Conclusion] Compared to the wild-type strain, rpn7KO strains exhibited severe defects in hyphal growth and conidial formation; rpn4KO strain had defect in hyphal growth too, but displayed normal conidiation; the growth and developmental phenotypes of rpn10KO strain were slightly affected. Taken together, these data demonstrated that these three genes were important for the growth and developmental phenotypes of Neurospora crassa.