【摘要】 利用低能氮离子注入介导外源DNA转化酵母菌,获得了遗传稳定的产醌类物质的重组酵母菌株N6076。利用抑制差减杂交(SSH)技术从重组菌N6076的DNA中分离获得了14个差异表达基因片断,其中由于碱基突变造成其蛋白质氨基酸序列变化的差异表达基因有4个。这些差异表达基因的功能分别涉及遗传信息的转录和翻译。应用2-D荧光差异双向电泳-质谱(2-D DIGE-MS)技术,在重组菌N6076中共检测到56个蛋白质表达量变化的差异点(p<0.05),其中26个蛋白质表达量上调,编号为273和1 294的蛋白质可信度指数分别为81.37%和12.86%。本研究为离子束重组酵母菌生物合成醌类物质的代谢通路研究提供了一定的帮助。更多还原

【Abstract】 The original yeast strain Saccharomyces cerevisiae KH08 was implanted by low-energy nitrogen ion and the recombined mutant strain N6076 was obtained.The gas chromatography-mass spectrography(GC-MS) were used to detect the newly generated quinone compound produced by strain N6076 and to evaluate the change of metabolic pathways.In order to explore the change in metabolic pathways of strain N6076,a suppression subtractive hybridization(SSH) technology and two-dimensional fluorescence difference gel electrophoresis(2-D DIGE) with mass spectrometry(MS) were used to discuss the gene expression and proteomic change of the strain N6076.Through sequencing analysis,a total of 14 gene fragment sequences with high expression levels were obtained from the strain N6076,among these,4 genes with base mutation resulted in the change of protein sequences.56 proteins differentially expressed(p<0.05) were found in the strain N6076 when cultured in liquid medium for 96 h compared with the original strain.Of these,26 protein spots were upregulated.In total,56 protein spots were identified by MALDI-TOF and tandem(TOF-TOF) mass spectrometry.By bioinformatics analysis,the protein score confidence interval of protein profiling in the down-regulated protein spots 273 and 1 294 were 81.371% and 12.864%,respectively.This was probably due to the irradiation by nitrogen ions contributing to the mutation of these two proteins.The results provided some help for studying on metabolic pathway of quinones biosynthesis in ion-beam-recombinant yeast.更多还原