Abstract：
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 vectorissuccessfullyconstructed andthe 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 hypertonictreatment contribute to maturation of the red fluorescent protein.
收稿日期: 2011-12-12
基金资助:
国家自然科学基金（81072673）；
中央级公益性科研院所基本科研专项（2010ZD04）
通讯作者:
孔建强
E-mail: jianqiangk@imm.ac.cn
引用本文:
史彦薇, 王志芳, 王伟, 安建梅, 孔建强. 红色荧光蛋白在酿酒酵母中的表达特性研究[J]. 《中国医药生物技术》杂志, 2012, 7(2): 93-99.
SHI Yan-Wei, WANG Zhi-Fang, WANG Wei, AN Jian-Mei, KONG Jian-Qiang. Rapid cloning and expression of Dsred gene in Saccharomyces cerevisiae. , 2012, 7(2): 93-99.
2012, Vol. 7 
