蓝色和黄色荧光蛋白在灵芝中的表达及黄色荧光蛋白在诱导型启动子Pcbh2活性分析中的应用

Expression of the blue and yellow fluorescent proteins in Ganoderma lingzhi and application of the yellow fluorescent protein in activity analysis of inducible promoter Pcbh2

本研究中通过密码子优化和内含子添加分别构建了蓝色荧光蛋白基因(bfp)表达载体PJW-EXP-intron-opbfp和黄色荧光蛋白基因(yfp)表达载体PJW-EXP-intron-opyfp,使用灵芝Ganodermalingzhi原生质体进行转化,筛选得到了表达BFP和YFP的工程菌株。工程菌株的菌丝在荧光显微镜下可分别检测到蓝色和黄色荧光信号,而野生型菌株(WT)的菌丝检测不到这2种荧光,结果显示我们在灵芝中实现了蓝色和黄色荧光蛋白的表达。基于本研究建立的荧光蛋白表达方法,我们评估了纤维二糖水解酶Ⅱ基因的启动子(Pcbh2)在微晶纤维素诱导下的活性。将黄色荧光蛋白基因插入到Pcbh2启动子的下游构建yfp-Pcbh2表达载体,经过转化获得了YFP-Pcbh2灵芝工程菌株。在微晶纤维素的诱导下,检测到YFP-Pcbh2菌丝体可发出黄色荧光信号,而不加微晶纤维素诱导时则没有检测到荧光,结果表明Pcbh2启动子在微晶纤维素诱导下具有转录活性。

The blue fluorescent protein expression plasmid PJW-EXP-intron-opbfp and yellow fluorescent protein expression plasmid PJW-EXP-intron-opyfp were constructed and transformed into protoplasts of Ganoderma lingzhi.Transformants were selected by PCR and subsequently microscopically screened for fluorescent signals.The pJW-EXP-intron-opbfp and pJW-EXPintron-opyfp transformants respectively exhibited blue and yellow fluorescence in the mycelia.However,no fluorescence was detected in wild-type(WT)strains.These results showed that blue and yellow fluorescent proteins were successfully expressed in G.lingzhi.Moreover,the activity of the cellulaseⅡgene promoter(Pcbh2)was evaluated in G.lingzhi using yfp as a reporter gene.The yfp-Pcbh2 transformants were obtained by genetic transformation of the plasmid yfp-Pcbh2 into protoplasts of G.lignzhi.Under induction of inducer avicel,the mycelia of the yfp-Pcbh2 transformants emitted yellow fluorescence signals,while no fluorescence was detected in the mycelia without the inducer.These results showed that Pcbh2 was successfully activated by avicel in G.lingzhi.