红色荧光蛋白在酿酒酵母中的表达特性研究

目的研究红色荧光蛋白编码基因Dsred在酿酒酵母菌中的快速克隆与表达。方法根据已发表基因序列设计引物,采用连续重叠PCR方法快速克隆获得全长Dsred基因,将其与pMD-18T载体连接后进行测序鉴定。通过In-fusion方法将鉴定正确的pMD-Dsred重组载体与酿酒酵母表达载体pYeDP60进行连接,测序后利用LiAc方法将鉴定正确的pYeDP60-Dsred重组表达载体转化至酿酒酵母菌W303-1B中,PCR扩增筛选阳性克隆,获得的W303B[pYeDP60-Dsred]工程菌经诱导培养后进行SDS-PAGE电泳分析和绿光激发荧光成像检测。将工程菌分别接种至YPD、YPG、SCG和SCD培养基,培养48、72、96、120和144h后分别测定吸光度(A600)值。取诱导表达后的工程菌(菌液组)进行离心(菌体组)、离心后加甘油(高渗组)处理,分别置于–70、–20、4、28和37℃条件培养,荧光显微镜观察其表达特性。结果连续重叠PCR扩增和测序鉴定结果表明,扩增获得的Dsred基因长为678bp,其序列与已发表的基因序列完全一致;Dsred基因已成功插入pYeDP60-Dsred重组表达载体,且受酵母诱导型启动子GAL10-CYC1调控表达。PCR扩增筛选和SDS-PAGE分析显示,pYeDP60-Dsred重组表达载体已成功导入酿酒酵母菌中,诱导表达产物相对分子质量与预期相符,且诱导后工程菌可在绿光激发下发出红色荧光。4种培养基内工程菌的菌体生长情况无明显差别,重组Dsred红色荧光蛋白表达不会抑制酿酒酵母菌体生长。荧光显微镜观察显示,工程菌经不同处理后,高渗组红色荧光蛋白成熟时间最短,菌液组时间最长;红色荧光蛋白在37℃条件下培养时成熟最早,但降解速度较快。结论成功构建了pYeDP60-Dsred重组酿酒酵母表达载体,实现了Dsred基因在酿酒酵母菌体内的异源表达。红色荧光蛋白表达对酿酒酵母菌体生长无明显影响,且离心保留菌体和加入甘油等缺水高渗处理都有利于红色荧光蛋白的成熟。

Rapid cloning and expression of Dsred gene in Saccharomyces cerevisiae

Objective To study rapid cloning and expression of Dsred gene in Saccharomyces cerevisiae. Methods The primers were designed on the published gene sequence. The full-length Dsred gene was amplified by successive overlap PCR, and then the amplification product was cloned into vector pMD-18T. After DNA sequencing, the identified recombinant vector pMD-Dsred was cloned into Saccharomyces cerevisiae expression vector pYeDP60 by In-fusion method. After DNA sequencing, the identified recombinant expression vector pYeDP60-Dsred was transfected into Saccharomyces cerevisiae W303-1B by LiAc method. The positive clones of the engineered strain W303B[pYeDP60-Dsred] were selected by PCR. After that, the induced recombinant expression products were detected by SDS-PAGE method and green excitation fluorescence imaging. W303B[pYeDP60-Dsred] were inoculated into YPD, YPG, SCG, SCD medium respectively, and absorbance value were measured after cultured for 48, 72, 96, 120, 144 h respectively. The induced W303B[pYeDP60-Dsred] (bacteria solution group) were treated with centrifuging (bacterial group), adding glycerine after centrifuging (hypertonic group) respectively, and then were cultured at –70, –20, 4, 28, 37 ℃ respectively to observe the maturation time and stability of protein. Results The length of Dsred gene obtained by PCR amplification was 678 bp and its sequence was consistent with the published gene sequence. Dsred gene was inserted into recombinant expression vector pYeDP60-Dsred successfully, and its expression was regulated by cerevisiae inducible promoter GAL10-CYC1. The molecular mass of expression product of the engineered strain W303B [pYeDP60-Dsred] transformed with recombinant expression vector pYeDP60-Dsred was consistent with expectation, and the engineered strain W303B [pYeDP60-Dsred] excited by green fluorescence showed red fluorescent. The engineered strain W303B [pYeDP60-Dsred] growth in four kinds of medium had no significant difference. The recombinant Dsred red fluorescent protein expression didn’t inhibit the growth of Saccharomyces cerevisiae. After different treatments, the time of red fluorescent protein maturation from the engineered strain in hypertonic group was shortest, and that in bacteria solution group was longest. The time of red fluorescent protein maturation was shortest at 37 ℃, but its degradation rate was fast. Conclusion The recombinant Saccharomyces cerevisiae expression vector is successfully constructed and the heterologous expression of Dsred gene in Saccharomyces cerevisiae is achieved. There is no significant effect on growth of Saccharomyces cerevisiae by the red fluorescent protein. Water-limited and hypertonic treatment contribute to maturation of the red fluorescentprotein.