| 摘要: |
| 【目的】以增强型绿色荧光蛋白为报告基因，构建斜卧青霉转录调控蛋白Hac1激活形式基因（Hac1i）随机插入过表达菌株和非激活形式基因（Hac1u）原位插入过表达菌株，并初步观察Hac1基因的2种不同编码蛋白在菌丝内部的运动情况，为进一步研究转录调控蛋白Hac1的功能奠定基础。【方法】分别克隆ptrA筛选标记、gpdA启动子、Hac1i编码区、eGFP编码区及终止子，利用融合PCR融合克隆片段，构建Hac1i随机插入表达盒；再分别克隆Hac1上游臂及编码区、eGFP编码区及终止子、ptrA筛选标记、Hac1下游臂序列，融合克隆片段，构建Hac1u原位插入表达盒。将2种表达盒分别转化斜卧青霉原生质体，通过吡啶硫胺素筛选标记、PCR扩增等检测获得阳性转化子，荧光显微镜观察融合蛋白的表达及运动情况。【结果】成功构建了Hac1i基因随机插入表达盒和Hac1u基因原位插入表达盒，利用原生质体转化将2种表达盒转入宿主斜卧青霉菌株，经PCR初步验证，得到了阳性转化子。阳性转化子在麸皮培养基上培养2～4 d后，可在菌丝内部清晰观察到强烈的绿色荧光信号，表明融合蛋白在菌丝体内得到了正确表达。【结论】成功构建了Hac1i基因随机插入和Hac1u基因原位插入菌株。 |
| 关键词: 斜卧青霉 荧光蛋白 转录调控蛋白Hac1 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（31200058） |
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| Construction of over expression strain of green fluorescent protein labeled transcription regulation protein Hac1 |
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MA Xiaojuan,WEI Xiaomin,LAI Hangxian,et al
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| Abstract: |
| 【Objective】This paper constructedover expression strain by randomly inserting activated transcription regulation protein gene Hac1(Hac1i) and stain in-situ inserted by non-activated transcription regulation protein gene Hac1(Hac1u) of Penicillium decumbens with enhanced green fluore scent protein as a reporter gene.The motion of these two encoded proteins in the strains was also observed.This paper would provide basis for further investigating the function of transcription regulation protein Hac1.【Method】The random integration expression cassette of Hac1i was constructed by cloning selection marker,gpd Apromoter,Hac1i protein-coding region,eGFP protein-coding region and terminatorand fusing these fragmentsusing PCR fusion.The in-situ integration expression cassette of Hac1u was constructed by cloningup-stream sequence and coding region of Hac1,protein-coding region of eGFP and terminator,selection marker and down-stream sequence of Hac1,and fusing these fragments.Then these two expression cassettes were put into Penicillium decumbens protoplasts,positive trans formants were detected by thiamine pyridine resistance and PCR amplification,and eGFP was observed under microscope.【Result】The random integration of Hac1i and situ integration of Hac1u were success fully cloned by fusion PCR,and they were inserted into host Penicillium decumbens strainusing protoplast transformation.PCR verification indicated that positive trans-formants were success fully obtained.Green fluorescence distribution emerged on hyphae carryingthe expression cassettes Hac1i and Hac1u after 2 to 4 days culture on bran medium.【Conclusion】Strainswithrandom integration Hac1i and situ integration Hac1u were constructed successfully. |
| Key words: Penicillium decumbens fluorescent protein transcription regulation protein Hac1 |
