Monitoring Circadian Rhythms in Arabidopsis thaliana Using Luciferase Reporter Genes

2007 ◽  
pp. 143-152
Author(s):  
Anthony Hall ◽  
Paul Brown
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Rhythms ◽  
Reporter Genes ◽  
Luciferase Reporter

scholarly journals Time Lag Between Light and Heat Diurnal Cycles Modulates CIRCADIAN CLOCK ASSOCIATION 1 Rhythm and Growth in Arabidopsis thaliana

2021 ◽  
Vol 11 ◽  
Author(s):  
Kosaku Masuda ◽  
Tatsuya Yamada ◽  
Yuya Kagawa ◽  
Hirokazu Fukuda
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Rhythms ◽  
Plant Growth ◽  
Circadian Clock ◽  
Time Lag ◽  
Luciferase Reporter ◽  
Time Lags ◽  
Light Cycle ◽  
Growth Responses ◽  
Diurnal Cycles

Plant growth responses to cues such as light, temperature, and humidity enable the entrainment of the circadian rhythms with diurnal cycles. For example, the temperature variations between day and night affect plant growth and accompany the time lag to light cycle. Despite its importance, there has been no systematic investigation into time lags, and the mechanisms behind the entrainment of the circadian rhythms with multiple cycles remain unknown. Here, we investigated systemically the effects of the time lag on the circadian rhythm and growth in Arabidopsis thaliana. To investigate the entrainment status of the circadian clock, the rhythm of the clock gene CIRCADIAN CLOCK ASSOCIATION 1 (CCA1) was measured with a luciferase reporter assay. As a result, the rhythm was significantly modulated by the time lag with +10°C heating for 4 h every day but not −10°C cooling. A model based on coupled cellular oscillators successfully described these rhythm modulations. In addition, seedling growth depended on the time lag of the heating cycle but not that of the cooling cycle. Based on the relationship between the CCA1 rhythms and growth, we established an estimation method for the effects of the time lag. Our results found that plant growth relates to the CCA1 rhythm and provides a method by which to estimate the appropriate combination of light–dark and temperature cycles.

scholarly journals Hierarchical Action of Mulberry miR156 in the Vegetative Phase Transition

2021 ◽  
Vol 22 (11) ◽  
pp. 5550
Author(s):  
Hongshun Li ◽  
Yiwei Luo ◽  
Bi Ma ◽  
Jianqiong Hu ◽  
Zhiyuan Lv ◽  
...  
Keyword(s):  
Phase Transition ◽  
Arabidopsis Thaliana ◽  
Woody Plants ◽  
Luciferase Reporter ◽  
Vegetative Phase ◽  
Promoter Sequences ◽  
Over Expression ◽  
Degradome Sequencing ◽  
Pathway Expression ◽  
Reporter Assays

The vegetative phase transition is a prerequisite for flowering in angiosperm plants. Mulberry miR156 has been confirmed to be a crucial factor in the vegetative phase transition in Arabidopsis thaliana. The over-expression of miR156 in transgenic Populus × canadensis dramatically prolongs the juvenile phase. Here, we find that the expression of mno-miR156 decreases with age in all tissues in mulberry, which led us to study the hierarchical action of miR156 in mulberry. Utilizing degradome sequencing and dual-luciferase reporter assays, nine MnSPLs were shown to be directly regulated by miR156. The results of yeast one-hybrid and dual-luciferase reporter assays also revealed that six MnSPLs could recognize the promoter sequences of mno-miR172 and activate its expression. Our results demonstrate that mno-miR156 performs its role by repressing MnSPL/mno-miR172 pathway expression in mulberry. This work uncovered a miR156/SPLs/miR172 regulation pathway in the development of mulberry and fills a gap in our knowledge about the molecular mechanism of vegetative phase transition in perennial woody plants.

Abstract P466: Circadian Rhythm Disruptions in a Novel Diabetic db/db-mPer2 Luc Mouse Model

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Tianfei Hou ◽  
Wen Su ◽  
Ming C Gong ◽  
Zhenheng Guo
Keyword(s):  
Blood Pressure ◽  
Circadian Rhythms ◽  
Real Time ◽  
Clock Gene ◽  
Ex Vivo ◽  
Phase Advance ◽  
Luciferase Reporter ◽  
High Time Resolution ◽  
Peripheral Tissues

Db/db mouse, which lacks functional leptin receptor, is an extensively used model of obesity and type 2 diabetes. We and others have demonstrated that db/db mouse has disruptions in circadian rhythms of behavior, physiology and some clock genes. However, systemic investigations of the alterations in clock gene oscillations in multiple systems with high time resolution in this model are impeded by the impractical demand for large number of animals. To overcome this limitation, we cross bred the db/db mouse with mPer2 Luc mouse in which the clock gene Period2 is fused with a luciferase reporter thus allow real-time monitoring of the clock gene Per2 oscillations. The generated db/db-mPer2 Luc mice had the typical diabetic mellitus including obesity, hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance. In addition, the db/db-mPer2 Luc mice also exhibited disruptions in circadian rhythms in behavior (locomotor activity), physiology (blood pressure) and metabolism (respiratory exchange ratio and energy expenditure). Using the LumiCycle system, we monitored in real-time of the Per2 oscillations in both the SCN central clock and multiple peripheral tissues ex vivo . The results showed no difference in the phase of the central SCN Per2 oscillation. However, the peripheral tissues that related to metabolism, such as liver and white adipose clocks, displayed 3.28±0.86 and 4.64±1.06 hours of phase advance respectively. Aorta, mesentery artery and kidney, organs play important role in blood pressure homeostasis, showed 0.99±0.37, and 2.12±0.4, and 2.21±0.5 hours phase advance respectively. Interestingly, no difference was observed in the lung and adrenal gland. We then investigated the Per2 oscillation in vivo by using the IVIS imaging system. Consistent with the ex vivo results, the liver Per2 oscillation were phase advanced in vivo. Our findings demonstrated that clock gene Per2 oscillations were disrupted in multiple peripheral tissues but not in central SCN. Moreover, the extent of phase advance in peripheral tissue varies largely. Our results suggest dyssynchrony of the clock oscillations among various peripheral systems likely contribute to the multiple disruptions in physiology and metabolism in diabetic db/db mice.

Bioluminescence and chemiluminescence literature—Luciferase reporter genes—Lux andLuc

1993 ◽  
Vol 8 (5) ◽  
pp. 267-291 ◽  
Author(s):  
P. J. Hill ◽  
G. S. A. B. Stewart ◽  
P. E. Stanley
Keyword(s):  
Reporter Genes ◽  
Luciferase Reporter

FIGalpha, a germ cell specific transcription factor involved in the coordinate expression of the zona pellucida genes

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 4939-4947 ◽  
Author(s):  
L. Liang ◽  
S.M. Soyal ◽  
J. Dean
Keyword(s):  
Transcription Factor ◽  
Zona Pellucida ◽  
Reporter Genes ◽  
Single Copy ◽  
Luciferase Reporter ◽  
Embryonic Fibroblasts ◽  
Transcription Start Sites ◽  
Helix Loop Helix ◽  
E Box ◽  
Dna Complex

The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3, encoded by single-copy genes whose expression is temporally and spatially restricted to oocytes. All three proteins are required for the formation of the extracellular zona matrix and female mice with a single disrupted zona gene lack a zona and are infertile. An E-box (CANNTG), located approximately 200 bp upstream of the transcription start sites of Zp1, Zp2 and Zp3, forms a protein-DNA complex present in oocytes and, to a much lesser extent, in testes. It has been previously shown that the integrity of this E-box in Zp2 and Zp3 promoters is required for expression of luciferase reporter genes microinjected into growing oocytes. The presence of the ubiquitous transcription factor E12 in the complex was used to identify a novel basic helix-loop-helix protein, FIGalpha (Factor In the Germline alpha) whose expression was limited to oocytes within the ovary. The ability of FIGalpha to transactivate reporter genes coupled to each of the three mouse zona promoters in heterologous 10T(1/2) embryonic fibroblasts suggests a role in coordinating the expression of the three zona pellucida genes during oogenesis.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Evangelia Skarpidi ◽  
George Vassilopoulos ◽  
Qiliang Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Luciferase Activity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Gene Activity ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Highly Sensitive

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

scholarly journals Analysis of Promoter Function in Aspergillus fumigatus

2012 ◽  
Vol 11 (9) ◽  
pp. 1167-1177 ◽  
Author(s):  
Sanjoy Paul ◽  
J. Stacey Klutts ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Reporter Gene ◽  
Reporter Genes ◽  
Antifungal Drugs ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Gene System ◽  
Reporter Gene System

ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

scholarly journals The Paeonia qiui R2R3-MYB Transcription Factor PqMYB113 Positively Regulates Anthocyanin Accumulation in Arabidopsis thaliana and Tobacco

2022 ◽  
Vol 12 ◽  
Author(s):  
Xiaokun Liu ◽  
Jingjing Duan ◽  
Dan Huo ◽  
Qinqin Li ◽  
Qiaoyun Wang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Myb Transcription Factor ◽  
Anthocyanin Synthesis ◽  
Regulation Mechanism ◽  
Reporter Assay ◽  
Anthocyanin Accumulation ◽  
Tree Peony ◽  
R2r3 Myb

Paeonia qiui is a wild species of tree peony native to China. Its leaves are purplish red from the bud germination to the flowering stage, and anthocyanin is the main pigment in purplish red leaves. However, the anthocyanin synthesis regulation mechanism in tree peony leaves remains unclear. In this study, an R2R3-MYB, PqMYB113 was identified from the leaves of P. qiui. Phylogenetic analysis revealed that PqMYB113 clustered with Liquidambar LfMYB113 and grape VvMYBA6. Subcellular location analysis showed that PqMYB113 was located in the cell nucleus. The transient reporter assay suggested that PqMYB113 was a transcriptional activator. The overexpression of PqMYB113 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) resulted in increased anthocyanin accumulation and the upregulation of CHS, F3H, F3’H, DFR, and ANS. The dual luciferase reporter assay showed that PqMYB113 could activate the promoters of PqDFR and PqANS. Bimolecular fluorescence complementation assays and yeast two-hybrid assays suggested that PqMYB113 could form a ternary MBW complex with PqbHLH1 and PqWD40 cofactors. These results provide insight into the regulation of anthocyanin biosynthesis in tree peony leaves.

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