香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。

Functional analysis of gpd-Le and ras-Le promoters from Lentinus edodes

The expression vectors pLg-gfp and pLr-gfp containing gpd-Le and ras-Le promoter fragments respectively from Lentinus edodes were constructed for detecting its functional activity by the fusion of promoter fragments to the reporter gfp gene.Co-transformation of plasmid pLe-gfp and pLr-gfp with plasmid pCc1001 respectively,which harbored the complementary gene trpl were conducted by the PEG-mediated protoplast transformation of the oidia of LT2,a tryptophan auxotrophic strain of Coprinus cinereus,The putative trp+ transformants were obtained from the selective regeneration medium and confirmed by PCR detection,Southern blotting and green fluorescence analysis. The results indicated that gfp gene expression in Coprinus cinereus. Strong green fluoescence acitivity was observed in the mycilia of pLg-gfp transformants of Coprinus cinereus LT2 under the fluorescent electronic microscope.However,the ras-Le promoter could not drive the gfp gene expression in Coprinus cinereus. No green fluorescence acitivity was observed in the mycelia of pLr-gfp transformants of Coprinus cinereus LT2.