DOI: 10.3724/SP.J.1264.2011.00014

Chinese Journal of Multiple Organ Diseases in the Elderly (中华老年多器官疾病杂志) 2011/10:4 PP.340-343

Induced expression and activity analysis of human aldose reductase gene in Saccharomyces cerevisiae

Objective To construct the Saccharomyces cerevisiae cell strains expressing aldose reductase(AR) and AR-green fluorescent protein(AR-GFP) fusion protein, and to detect the target gene expression and the AR enzyme activity. Methods The yeast expression plasmids pYEX-BX inserted with AR and AR::GFP fusion gene were transformed into the yeast strain INVSc1, which were named as XAR and XAG strains respectively. The blank pYEX-BX strain was used as the normal control. The growth curves and the fluorescence were determined in all strains. Northern blot, Western blot and the fluorescent method were used to detect the AR mRNA transcription, AR protein expression, and the AR activity respectively. Results There was no significant difference in the growth rates among three strains. There was a linear relationship between relative fluorescence of the XAG and the growth time. The mRNA transcription and protein expression of AR and AR::GFP were sustainable and stable in XAR and XAG strains. The AR activities in the two strains were both proved by the fluorescent method. Conclusion The yeast expression strain of AR was constructed successfully, which lays basis for its application in the researches on pathogenic mechanism of AR and preliminary screening of new AR inhibitors.

Key words:aldose reductase,yeast,green fluorescent protein,gene expression

ReleaseDate:2014-07-21 15:56:22

[1] Varma SD, Mizuno A, Kinoshita JH. Diabetic cataracts and flavonoids[J]. Science, 1977, 195(4274):205-206.

[2] Bhatnagar A, Srivastava SK. Aldose reductase: congenial and injurious profile of an enigmatic enzyme[J]. Biochem Med Metab Biol, 1992, 48(2): 91-121.

[3] Alexiou P, Pegklidou K, Chatzopoulou M, et al. Aldose reductase enzyme and its implication to major health problems of the 21st Century[J]. Curr Med Chem, 2009, 16(6): 734-752.

[4] Ramana KV, Srivastava SK. Aldose reductase: a novel therapeutic target for inflammatory pathologies[J]. Int J Biochem Cell Biol, 2010, 42(1): 17-20.

[5] 陈思妘, 萧熙佩. 酵母生物化学[M]. 山东科学技术出版社, 1990: 106-260.

[6] 刘 静, 叶 玲, 刘建伟. 糖尿病并发症相关基因醛糖还原酶酵母细胞模型的建立[J]. 中国老年学杂志, 2005, 11(25): 1367-1369.

[7] Schmitt ME, Brown TA, Trumpower BL. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae [J]. Nucleic Acids Res, 1990, 18 (10): 3091-3092.

[8] Sambrook J, David W. 分子克隆实验指南. 黄培堂等译.第3版. 科学出版社, 2002: 76-78.

[9] Burke D, Dawson D, Stearns T. Methods in yeast genetics. A cold spring harbor laboratory course manual (2000 Edition) [M]. 北京: 清华大学出版社, 2002: 115-116.

[10] Bradgord MM. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254.

[11] Lowry OH, Robert NR, Kapphahn JI. The flurometric measurement of pyridine nucleotides[J]. J Biol Chem, 1957, 224(2): 1047-1064.

[12] Trueblood N, Ramasamy R. Aldose reductase inhibition improves altered glucose metabolism of isolated diabetic rat hearts[J]. Am J Physiol, 1998, 275 (1): H75-83.

[13] Mervi HT, Laura S, Laura R, et al. Endogenous xylose pathway in Saccharomyces Cerevisiae[J]. Appl Environ Microbiol, 2004, 70(6): 3681-3686.