Kavita P, Burma Pradeep Kumar
Department of Genetics, University of Delhi South Campus, New Delhi, India.
J Biosci. 2008 Sep;33(3):337-43. doi: 10.1007/s12038-008-0053-4.
The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, beta-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp ) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.
报告基因的使用极大地促进了启动子活性的评估。然而，通过报告基因评估的活性不仅反映了启动子的强度，还反映了报告基因编码的mRNA和蛋白质的稳定性。虽然稳定的报告基因产物在分析弱启动子的活性时是一个优势，但它却成为理解启动子时间表达模式的主要限制，因为即使启动子活性停止后报告基因产物仍会持续存在。在本研究中，我们对两种报告基因β-葡萄糖醛酸酶（gus）和绿色荧光蛋白（sgfp）进行了比较分析，以研究绒毡层特异性启动子A9（拟南芥）和TA29（烟草）的时间表达模式。无论使用哪种报告基因，通过报告基因（gus或sgfp）的转录谱评估的A9和TA29启动子的活性都是相同的。然而，虽然使用gus推导的启动子活性在时间上超出了启动子的实际活性，但通过sgfp荧光记录的结果与转录谱的相关性更好。因此，我们的结果表明，与gus相比，sgfp是评估启动子时间活性更好的报告基因。尽管早期有几篇报道评论了使用GUS作为报告蛋白推导启动子时间活性时可能存在的误差，但我们通过实验证明了使用gfp等报告基因分析时间表达模式的优势。
