Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock
Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock
复制标题
DOI：
10.1128/ec.00257-07
复制
发表时间：
2008-01-01
期刊：
影响因子：
--
通讯作者：
Dunlap, Jay C.
中科院分区：
文献类型：
--
作者：
Gooch, Van D.;Mehra, Arun;Dunlap, Jay C.
我们报道了萤火虫荧光素酶基因的完整重建，该基因完全密码子优化在粗脉孢子虫中表达。与以前的版本相比，这个报告将光输出提高了大约4个对数量级，现在产生的光是肉眼可见的，足以监测许多低表达基因的活动。在FRQ或eas/CCG-2启动子驱动荧光素酶的RACE管中生长的菌株的时间推移照片显示，在生长前沿附近和新形成的分生孢子带附近的荧光素酶活性最高。此外，我们还建立了一种山梨糖培养基菌落试验，这将促进基于荧光素酶的筛选。由FRQ启动子驱动荧光素酶的山梨糖生长的菌株菌落的信号表现出昼夜节律的特性，可以跟踪很多天到几周。这位记者现在可以实时跟踪时钟，即使在菌株或在分生孢子带的昼夜节律没有表现出来的情况下也是如此。这一特性已被用于在高温下发现短的、约15小时的周期节律，在高温下很难在比赛管中观察到带状现象，并产生高分辨率的温度相响应曲线。
We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.