【摘要】 为建立适用于研究香菇(Lentinula edodes)分化中基因表达差异的银染差异显示方法,以香菇突变株fbd1及其出发菌株98411的菌丝体和子实体为材料,采用Trizol法提取总RNA,优化了反转录和PCR扩增中5项反应参数,再经6%变性聚丙烯酰胺凝胶电泳分离和银染显示差异条带。试验结果表明,适宜分析香菇mRNA差异的反转录体系中RNA投入量为0.5~1μg、dNTP浓度为20μmol/L,PCR扩增反应MgCl2、dNTP和Taq酶分别为2mmol/L、200μmol/L和1.0U,此条件下,目标条带清晰且重复性好。更多还原

【Abstract】 An effective and feasible silver staining-based display method for studying differentially expressed genes in Lentinula edodes is described.Five intrinsic factors(amount of template RNA,dNTP concentrations used in reverse transcription,and MgCl2,dNTP and Taq polymerase concentrations used in PCR amplification)affecting differential display were optimized using high-quality total RNA extracted from mycelia and fruiting bodies of L.edodes strain 98411 and its mutant strain fbd1.RNA extraction using the Trizol method,followed by separation of amplified cDNA fragments using 6%(w/v)denaturing polyacrylamide gel electrophoresis and visualization by silver staining,produced clear target bands and good reproducibility under the following optimal conditions:0.5~1 μg template RNA,20 μmol/L dNTP(reverse transcription),2 mmol/L MgCl2,200 μmol/L dNTP(PCR amplification)and 1 U Taq polymerase.更多还原