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银耳(Tremella fuciformis)属于高等担子菌,其担孢子芽殖产生酵母状分生孢子称为银耳芽孢.银耳芽孢单核,能像酵母那样快速生长且容易培养,具备优良外源基因表达宿主的特点.本研究以gpd-Gl启动子分别与绿色荧光蛋白基因gfp和潮霉素抗性基因hph连接构建表达载体pGlg-gfp和pGlq-hph;设置潮霉素浓度梯度在3种培养基中对银耳芽孢的敏感性进行测定,结果表明银耳芽孢在不同的培养基上对潮霉素的敏感性不同,在MA培养基上其最低敏感浓度为5μg/mL;采用电击法把pGlq-hph质粒转化进银耳芽孢完整细胞,假定转化子经MA筛选培养基筛选,结果表明银耳芽孢完整细胞电击转化的最佳参数为:STM电击缓冲液、银耳芽孢浓度1.0×108个/mL,电击体积200μL,表达质粒6μg,电击电压2.0kV/cm,电击后采用MB液体培养基静置预培养48h,转化率达277个/μgDNA.采用最佳电击参数把质粒pGlg-gfp和pGlg-hph按1︰1共转化银耳芽孢,转化子经过筛选培养基筛选、PCR鉴定及Southern杂交验证,结果表明受鉴定的8个转化子中有3个整合了gfp基因,其共转化率为37.5%.这3个gfp基因转化子的芽孢在激光荧光显微镜下可观察到发出强烈的荧光,表明了外源gfp基因能够在银耳芽孢中获得高效率的表达.
Tremella fuciformis belongs to the higher basidiomycetes, and its basidiospore produces yeast-like conidia, which is called as Tremella fuciformis. The single nucleus of the white fungus, like yeast, grows rapidly and is easy to grow with the characteristics of excellent exogenous gene expression host In this study, the expression vectors pGlg-gfp and pGlq-hph were constructed by gpd-Gl promoter linked to gfp and hygromycin resistance gene hph, respectively. The concentration of hygromycin The results showed that the sensitivity of the fungus to different concentrations of hygromycin on hygromycin was different. The minimum sensitivity was 5μg / mL on MA medium. The plasmid pGlq-hph was electroporated into The results showed that the optimal parameters of electroporation of whole cells of Tremella mesenterha were STM shock buffer, the concentration of Tremella spores was 1.0 × 108 / mL, the volume of electric shock was 200μL, the expression plasmid 6μg, the electric shock voltage 2.0kV / cm, the MB liquid medium after shock shock pre-cultured 48h, conversion rate of 277 / μg DNA using the best electric shock parameters pGlg-gfp and pGlg-hph plasmid by a total of 1: 1 The results showed that three of the eight transformants were identified as gfp, with a total conversion rate of 37.5%. The three gfp genes The transformant spores under the laser fluorescence microscope can be observed to emit a strong fluorescence, indicating that the exogenous gfp gene can be highly efficient expression in Tremella fuciformis.