摘要
以银耳芽孢为材料,由载体pEGFP、pCAMBIA1300经过HindIII和EcoRⅠ双酶切、连接、转化,重组质粒酶切验证,成功构建了由双孢蘑菇gpd启动子调控EGFP基因的银耳真核表达载体,命名pCB-BEGFP。采用电击转化法将携带EGFP基因的银耳表达载体pCB-BEGFP转化到银耳芽孢。提取4个转化子基因组DNA,用EGFP基因特异引物PCR扩增,电泳结果表明有与EGFP基因大小一致的特异带出现;荧光显微镜观察在再生培养基上的转化子可看到很强的绿光;其中一个转化子蛋白SDS-PAGE电泳,结果发现在大约27kD处有一明显的蛋白条带出现,与预期的蛋白分子量相近。以上结果证明EGFP基因转入银耳芽孢中,双孢蘑菇启动子可以调控外源基因的在银耳芽孢中正确表达。
Yeast-like conidia of Tremella fuciformis were used as the material. Plasmid pEGFP and pCAMBIA1300 were digested by Hind III and EcoRⅠ restriction enzymes and ligated to transform. We constructed Tremella fuciformis expressed vector pCB-BEGFP contained enhanced green fluorescent protein gene which was regulated by gpd promoter of Agaricus bisporus. Electroporation was performed to transfer plasmid DNA of pCB-BEGFP into yeast-like conidia from Tremella fuciformis. The chromosomal DNAs of the four transformants and the nontransgenic strain as the control were used as the templates for the PCR using the EGFP-specific primers. When the products were analyzed by the agarose gel electrophoresis, the expected 750bp amplified bands appeared. By fluorescence microscopy, distinct green fluorescence could be observed from the colonies of YLCs grown on RM agar plates and could not be observed from the colonies of YLCs which did not contain plasmid pCB-BEGFP. Expression of EGFP was detected by SDS-PAGE analysis of TCP of one transformants, as expected, a dark band of the protein was visualized at 27kD by Coomassie Brilliant Blue staining. These results indicated the EGFP gene had been integrated into the genome of transgenic Tremella fuciformis strains and was expressed successfully by regulation of gpd promoter of Agaricus bisporus.
出处
《中国农学通报》
CSCD
2008年第4期42-46,共5页
Chinese Agricultural Science Bulletin
基金
国家卫生部课题<银耳生物反应器生产人胰岛素研究>(WKJ2005-2-009)
(国家自然基金)银耳生物反应器基础研究(编号:30471210)
国家自然基金"银耳生物反应器基础研究"(30471210)
关键词
银耳
绿色荧光蛋白基因
表达
Tremella fuciformis, EGFP gene, expression
