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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 (UDP-glucose pyrophosphorylase, UGPase) is an important enzyme in polysaccharide biosynthesis. There are two UGPase homologous genes in rice genome named OsUgp1 and OsUgp2, respectively. An overexpression vector for OsUgp2 driven by the promoter of Aspergillus nidulans 3-glyceraldehyde dehydrogenase gene promoter was constructed and OsUgp2 gene was transferred into G. purpurea by Agrobacterium tumefaciens, and hygromycin resistance was obtained Strain. Results of PCR and Southern hybridization showed that the OsUgp2 gene was successfully integrated into the receptor genome of R. solanacearum. The results of semi-quantitative RT-PCR showed that the expression of exogenous gene OsUgp2 was expressed in the transformants of Ganoderma lucidum, and the expression levels in three independent transformed strains were different. The results of UGPase activity assay showed that the activity of UGPase or Specific activity than the wild-type control strains have increased to some extent, and in three independent transformed strains increased trend consistent with the RT-PCR test results. The biomass, the contents of intracellular polysaccharides and extracellular polysaccharides of the transformed and wild-type control strains were measured by constant weight method and phenol-sulfuric acid method. The results showed that the biomass, the contents of intracellular polysaccharides and extracellular polysaccharides obviously increase. SPSS 13.0 data statistical analysis software was used to analyze the correlation between UGPase activity and polysaccharide content. The results showed that total polysaccharide content, intracellular polysaccharide content, extracellular polysaccharide content and enzyme activity of UGPase The correlation coefficient between intracellular polysaccharides and UGPase activity was up to 0.98, indicating that overexpression of OsUgp2 may be mainly involved in the synthesis of intracellular polysaccharides.