真菌免疫调节蛋白基因LZ-8过表达载体的构建及转化灵芝原生质体的研究

摘要

Ganoderma lingzhi is a famous medicinal fungus in China.However,as one of the basidiomycetes with high nutritional and biological value,G.lingzhi still lacks a perfect method for transgenesis and a safe transgenic system.In this study,an overexpression system for regulating immune proteins was established,and a fungus-specific double-stranded T-DNA expression vector pSB130NG-LZ8 was constructed using fungus-specific promoter GPD,terminator NOS and target gene LZ-8.Protoplasts were isolated from G.lingzhi with lywallzymes,and protoplast activity of G.lingzhi was determined by FDA staining.Survival rate of protoplasts was about 80％.Protoplasts of G.lingzhi were successfully transformed by PEG-mediated transformation,and the transformed protoplasts grew on the plate with hygromycin resistance.Transformation rate was about 3-4/g pSB130NG-LZ8 + 107 protoplasts.Transformants were confirmed to be overexpressed in G.lingzhi by PCR and fluorescent quantitative PCR.%灵芝Ganoderma lingzhi是我国著名的药用真菌.但是,作为一种营养和保健价值都非常高的大型担子菌,灵芝还缺乏完善的转基因方法和安全转基因体系.本研究建立了免疫调节蛋白基因的过表达系统,并利用真菌特异性启动子GPD、终止子NOS和目的基因LZ-8构建真菌特异性双T-DNA表达载体pSB130NG-LZ8;利用溶壁酶提取灵芝原生质体,并用FDA染色法检测灵芝原生质体活性,原生质体成活率约为80％.通过PEG转化法对灵芝原生质体成功进行了转化,转化得到的原生质体在带有潮霉素抗性平板上长出,转化率为3-4μg pSB130NG-LZ8+107个原生质体.转化子通过PCR检测和荧光定量PCR检测,获得LZ-8在灵芝中的过表达.