作　　者： ; ; ; ;

机构地区： 华南农业大学生命科学学院

出　　处： 《菌物学报》 2011年第3期442-452,共11页

摘　　要： 尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase,UGPase)是多糖生物合成过程中重要的酶,水稻基因组中存在两个UGPase同源基因分别命名为OsUgp1和OsUgp2。构建了由构巢曲霉3-磷酸甘油醛脱氢酶基因启动子驱动OsUgp2表达的真菌过量表达载体,并通过农杆菌介导法将OsUgp2基因转入紫芝中,获得了潮霉素抗性的转化菌株。PCR和Southern杂交结果显示OsUgp2基因成功整合到受体紫芝基因组中。半定量RT-PCR检测结果显示外源基因OsUgp2在紫芝转化菌株中有表达,且在3个独立的转化菌株中的表达量各有不同;UGPase酶活性测定结果显示:转基因菌株中UGPase酶活性或比活力均较野生型对照菌株有一定的增加,且在3个独立转化菌株中的增加趋势与RT-PCR检测结果相一致。采用恒重法和苯酚-硫酸法分别测定了转化菌株和野生型对照菌株的生物量、胞内多糖和胞外多糖的含量,结果显示:转基因菌株的生物量、胞内多糖和胞外多糖含量明显增加。采用了SPSS13.0数据统计分析软件分析了UGPase酶活力与多糖含量之间的相关性,结果表明紫芝菌丝体的多糖总量、胞内多糖含量和胞外多糖含量与UGPase的酶活力都存在高度的相关性,其中胞内多糖含量与UGPase酶活力的相关系数最高达0.98,说明过量表达的OsUgp2可能主要参与胞内多糖的合成。 UDP-glucose pyrophosphorylase （UGPase） plays an important role in the process of polysaccharide synthesis.Two UGPase homologous genes existing in rice genome were designated as OsUgp1 and OsUgp2.In this study,an over-expression vector,in which OsUgp2 gene was driven by a promoter of glyceraldehyde-3-phosphate dehydrogenase from Aspergillus nidulans,was constructed and introduced into Ganoderma sinense via Agribacterium tumefaciens mediated method.A number of hygromycin resistant transformants was obtained.PCR and Southern blot analyses showed that the exogenous OsUgp2 was integrated into the genome of Ganoderma sinense.The result of semi-quantitative RT-PCR indicated that OsUgp2 expressed and the expression level was different among the three independent transformants.The determined UGPase activity of transformants was found increased and its extent was correlated well with the mRNA level.The constant weight and phenol-sulfuric acid assays were utilized to determine the mycelial biomass and polysaccharide content respectively.The results showed that all of biomass,intracellular and extracellular polysaccharides were evidently increased.The positive correlation between UGPase activity and polysaccharide content was revealed by analysis using SPSS13.0 software.The correlation coefficient between UGPase activity and intracellular polysaccharide was the highest at 0.980.It is suggested that the overexpressed OsUgp2 is probably more concerned with intracellular polysaccharide synthesis.

关 键 词： 尿苷二磷酸葡萄糖焦磷酸化酶 过量表达 灵芝 多糖

领　　域： [农业科学]