Improved GFP Variants to Study Gene Expression in Haloarchaea
Improved GFP Variants to Study Gene Expression in Haloarchaea
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DOI：
10.3389/fmicb.2019.01200
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发表时间：
2019-05-29
影响因子：
5.2
通讯作者：
Pfeifer, Felicitas
中科院分区：
文献类型：
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作者：
Born, Johannes;Pfeifer, Felicitas
盐古菌启动子活性的研究是专门利用酶作为报告者进行的。另一种报告基因是编码绿色荧光蛋白（GFP）的基因，这是一种简单快速的研究启动子强度的工具。然而，用于分析盐古菌中蛋白质稳定性的GFP变体smRS-GFP，由于荧光信号过低，不适合量化弱启动子活性。通过引入10个氨基酸取代，我们将smRS-GFP的荧光增强了3.3倍，得到了mGFP6。以mGFP6为报告子，研究了6种具有不同启动子强度的卤古菌启动子。其中，铁氧还蛋白基因的清洁启动子P-fdx和核糖体16S rRNA基因的P2活性最强。在盐盐杆菌PHH1中，p-vac区驱动气体囊泡蛋白（gvp）基因表达的启动子活性低得多。基础启动子强度依次为P-pA、P-pO、P-pF、P-pD。所有启动子均表现出生长依赖的活性模式。gpe诱导的P-pA和P-pD活性较高，但低于P-fdx或P2启动子活性。mGFP6报告基因也被用于研究三种不同gvp mrna的5'-非翻译区（5'-UTRs）的调控作用。5'-UTR的缺失总是导致表达增加，这意味着5'-UTR对翻译有负面影响。实验证实mGFP6是研究盐古菌基因表达的简单、快速、敏感的报告基因。
The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters P-fdx of the ferredoxin gene and P2 of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (gvp) genes in Halobacterium salinarum PHH1. The basal promoter strength dropped in the order P-pA, P-pO > P-pF, P-pD. All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of P-pA and P-pD were high, but lower compared to the P-fdx or P2 promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5'-untranslated regions (5'-UTRs) of three different gvp mRNAs. A deletion of the 5'-UTR always resulted in an increased expression, implying a negative effect of the 5'-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.