解剖学报 ›› 2015, Vol. 46 ›› Issue (2): 196-201.doi: 10.16098/j.issn.0529-1356.2015.02.009

• 细胞和分子生物学 • 上一篇    下一篇

不同红色荧光蛋白在酵母细胞中的表达效果分析

吴倩倩 王劲松 王瑞鲲 陆益新 李湘鸣*   

  1. 扬州大学医学院预防医学教研室,江苏 扬州 225001
  • 收稿日期:2014-03-24 修回日期:2014-11-05 出版日期:2015-04-06 发布日期:2015-04-06
  • 通讯作者: 李湘鸣 E-mail:yzzpt@sina.com
  • 基金资助:

    双信号发光酵母细胞高通量筛选化学诱变原的进一步构建研究

Analysis of expression of the different red fluorescent proteins in yeast cells

WU Qian-qian WANG Jing-song WANG Rui-kun LU Yi-xin LI Xiang-ming*   

  1. Department of Preventive Medicine,Medical College of Yangzhou University, Jangsu Yangzhou 225001,China
  • Received:2014-03-24 Revised:2014-11-05 Online:2015-04-06 Published:2015-04-06
  • Contact: LI Xiang-ming E-mail:yzzpt@sina.com

摘要:

目的 比较不同红色荧光蛋白基因在酵母细胞中的表达效果。 方法 利用分子生物学方法,构建了4种红色荧光蛋白(DsRed)基因酵母报告载体,分别是pGPD-DsRed、pGPD-DsRed-express-2、pGPD-yDsRed和pGPD-yDsRed-express-2,后两者含有酵母细胞偏好性的密码子,将4种DsRed酵母报告载体转入W303-1A酵母细胞,利用倒置荧光显微镜观察DsRed的表达,并用多功能酶标仪测酵母细胞的发光强度。结果 各红色荧光发光强度明显不同,其中DsRed-express-2发光最强,其次是yDsRed-express-2,yDsRed发光强度最弱。 结论 在酵母细胞中红色荧光蛋白的强度与密码子偏好性无关;定量红色荧光蛋白表达强度最好选用DsRed-express-2报告基因。

关键词: 红色荧光蛋白, 密码子偏好性, 酵母细胞, 聚合酶链反应

Abstract:

Objective To compare the expression of fluorescent protein gene in yeast cells. Methods Four of red fluorescent protein (DsRed) yeast expression vectors, pGPD-DsRed, pGPD-DsRed-express-2, pGPD-yDsred and pGPD-yDsred-express-2, were constructed using the molecular biology method. pGPD-yDsRed and pGPD-yDsRed-express-2 contained the yeast cell preference codon. These 4 vectors were transformed into W303-1A yeast cells and DsRed expression was observed under the fluorescence microscope. Multifunctional microplate reader was used to measure the luminous intensity of yeast cells. Results The fluorescence intensities of the 4 the DsRed were significantly different. DsRed-express-2 emission was the strongest, followed by yDsRed-Express-2, and the weakest luminous intensity was yDsRed. Conclusion Red fluorescent protein strength has nothing to do with the codon preference in yeast cells. The best way is to select reporter gene of DsRed-express-2 to quantify the expression intensity of red fluorescent protein.

Key words: Red fluorescent protein, Codon preference, Yeast cells, RT-PCR