scholarly journals The Floral Repressor GmFLC-like Is Involved in Regulating Flowering Time Mediated by Low Temperature in Soybean

2020 ◽  
Vol 21 (4) ◽  
pp. 1322 ◽  
Author(s):  
Jing Lyu ◽  
Zhandong Cai ◽  
Yonghong Li ◽  
Haicui Suo ◽  
Rong Yi ◽  
...  
Keyword(s):  
Low Temperature ◽  
Flowering Time ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Protective Mechanisms ◽  
Floral Repressor ◽  
Mobility Shift Assay ◽  
Delayed Flowering ◽  
Soybean Leaves

Soybean is an important crop that is grown worldwide. Flowering time is a critical agricultural trait determining successful reproduction and yields. For plants, light and temperature are important environmental factors that regulate flowering time. Soybean is a typical short-day (SD) plant, and many studies have elucidated the fine-scale mechanisms of how soybean responds to photoperiod. Low temperature can delay the flowering time of soybean, but little is known about the detailed mechanism of how temperature affects soybean flowering. In this study, we isolated GmFLC-like from soybean, which belongs to the FLOWERING LOCUS C clade of the MADS-box family and is intensely expressed in soybean leaves. Heterologous expression of GmFLC-like results in a delayed-flowering phenotype in Arabidopsis. Additional experiments revealed that GmFLC-like is involved in long-term low temperature-triggered late flowering by inhibiting FT gene expression. In addition, yeast one-hybrid, dual-luciferase reporter assay, and electrophoretic mobility shift assay revealed that the GmFLC-like protein could directly repress the expression of FT2a by physically interacting with its promoter region. Taken together, our results revealed that GmFLC-like functions as a floral repressor involved in flowering time during treatments with various low temperature durations. As the only the FLC gene in soybean, GmFLC-like was meaningfully retained in the soybean genome over the course of evolution, and this gene may play an important role in delaying flowering time and providing protective mechanisms against sporadic and extremely low temperatures.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

scholarly journals TLR4 promoter rs1927914 variant contributes to the susceptibility of esophageal squamous cell carcinoma in the Chinese population

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10754
Author(s):  
Jiaying Li ◽  
Hongjiao Wu ◽  
Hui Gao ◽  
Ruihuan Kou ◽  
Yuning Xie ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Statistical Significance ◽  
Nuclear Extract ◽  
Taqman Probe ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Link Type ◽  
Pcr Rflp ◽  
Dna Protein Interaction ◽  
Mobility Shift Assay

Background Toll-like receptor 4 (TLR4), as a key regulator of both innate and acquired immunity, has been linked with the development of various cancers, including esophageal cancer. This study aims to analyze the association of potential functional genetic polymorphisms in TLR4 with the risk of esophageal cancer. Methods This case-control study involved in 480 ESCC patients and 480 health controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype TLR4 rs1927914 polymorphism. Taqman probe method was used to determine the genotypes of TLR4 rs11536891 and rs7873784 variants. The relationship between TLR4 genetic variation and ESCC risk was analyzed by Logistic regression model by calculating the odds ratio (OR) and 95% confidence interval (95% CI). Results Compared with TLR4 rs1927914 AA genotype carriers, GG carriers had a lower ESCC risk (OR = 0.59, 95% CI [0.38–0.93], P = 0.023). Stratification analysis by age showed that TLR4 rs1927914 GG could affect the risk of ESCC in elderly people (OR = 0.59, 95% CI [0.36–0.97]). Smoking stratification analysis indicated that rs1927914 GG carriers were related to ESCC susceptibility among non-smokers (OR = 0.36, 95% CI [0.18–0.73]). Dual luciferase reporter assay suggested that rs1927914 G-containing TLR4 promoter displayed a 1.76-fold higher luciferase activity than rs1927914 A-containing counterpart in KYSE30 cells. Electrophoretic mobility shift assay (EMSA) showed the KYSE30 cell nuclear extract was able to bind the probe with rs1927914 G allele and this DNA-protein interaction could be eliminated by competition assays with unlabeled rs1927914 G probe, which indicating that the binding is sequence-specific. Our results also showed that TLR4 rs7873784 (G>C) and rs11536891 (T>C) conformed to complete genetic linkage. The genotype distributions of TLR4 rs11536891 variant among ESCC patients and normal controls have no statistical significance. Conclusion The TLR4 rs1927914 variant contributes to the ESCC risk by effecting the promoter activity.

scholarly journals CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2784-2795 ◽  
Author(s):  
Arati Khanna-Gupta ◽  
Theresa Zibello ◽  
Sarah Kolla ◽  
Ellis J. Neufeld ◽  
Nancy Berliner
Keyword(s):  
Luciferase Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Ccaat Displacement Protein ◽  
Lactoferrin Gene ◽  
Mobility Shift Assay ◽  
Silencer Element

Abstract Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

scholarly journals A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

2021 ◽  
Author(s):  
NGUYEN HOAI NGUYEN
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Chromatin Immunoprecipitation ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Mobility Shift Assay

Abstract To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

scholarly journals TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hualiang Qiao ◽  
Yongwei Liu ◽  
Lingling Cheng ◽  
Xuelin Gu ◽  
Pengcheng Yin ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Leaf Senescence ◽  
Brachypodium Distachyon ◽  
Wrky Transcription Factor ◽  
Luciferase Reporter ◽  
Virus Induced Gene Silencing ◽  
Reporter System ◽  
Mobility Shift ◽  
Yield And Quality ◽  
Mobility Shift Assay

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

scholarly journals Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Agriculture ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1286
Author(s):  
Joon Ki Hong ◽  
Eun Jung Suh ◽  
Sang Ryeol Park ◽  
Jihee Park ◽  
Yeon-Hee Lee
Keyword(s):  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Crop Improvement ◽  
Sequencing Analysis ◽  
Vrn1 Gene ◽  
Floral Repressor ◽  
Dna Sequencing Analysis ◽  
Delayed Flowering ◽  
Golden Gate Cloning

The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

scholarly journals An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene

2004 ◽  
Vol 32 (3) ◽  
pp. 811-823 ◽  
Author(s):  
A Blondet ◽  
M Doghman ◽  
P Durand ◽  
M Begeot ◽  
D Naville
Keyword(s):  
Granulosa Cells ◽  
Gene Promoter ◽  
Cell Types ◽  
Melanocortin Receptor ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
E Box ◽  
Mobility Shift Assay

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.

scholarly journals Transcription of the Sox30 Gene Is Positively Regulated by Dmrt1 in Nile Tilapia

2019 ◽  
Vol 20 (21) ◽  
pp. 5487 ◽  
Author(s):  
Yaohao Tang ◽  
Xiaoyan Li ◽  
Hesheng Xiao ◽  
Minghui Li ◽  
Yueqin Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nile Tilapia ◽  
Regulatory Element ◽  
Transcriptional Profiling ◽  
Sex Differentiation ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Related Transcription Factor ◽  
Mobility Shift Assay ◽  
Male Specific

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.

scholarly journals Drought and Darkness during Long-Term Simulated Shipping Delay Post-Shipping Flowering of Phalaenopsis Sogo Yukidian ‘V3’

Horticulturae ◽  
2021 ◽  
Vol 7 (11) ◽  
pp. 483
Author(s):  
Ju Hui Jeong ◽  
Wook Oh
Keyword(s):  
Low Temperature ◽  
Chlorophyll Content ◽  
Continuous Light ◽  
Growth Chamber ◽  
Control Group ◽  
Treatment Groups ◽  
Psii Photochemistry ◽  
Flower Quality ◽  
Delayed Flowering

We investigated the relationship between simulated shipping (SS) without watering or light and post-shipping growth and flowering of Phalaenopsis Sogo Yukidian ‘V3’. Two experimental environments were created: a low-temperature chamber for simulated shipping and a growth chamber for simulated finishing at the destination. Plants from both the control and treatment groups were moved from the low-temperature chamber to the growth chamber after the end of the simulated shipping. Control plants received continuous light and regular irrigation; plants in the treatment group were placed in the low-temperature chamber under light (LSS) or dark (DSS) conditions for 10, 20, 30, 40, or 50 days, without irrigation. Once DSS duration exceeded 40 days, the leaf-yellowing rate increased rapidly. Chlorophyll content decreased from day 10 to 30 of DSS and slightly increased in LSS and DSS over 40 days. The photochemical reflectance index decreased with the SS duration. The maximum quantum yield PSII photochemistry (Fv/Fm) values sharply decreased after the end of SS; after 40 days, neither LSS nor DSS plants recovered to the normal range. In the same SS duration, the number of days to spiking was delayed in the DSS. In addition, the number of days to spiking was delayed, owing to the longer SS duration. LSS for 50 days induced early flowering, as in the control group, but lowered flower quality. The results demonstrate that drought stress from long-term shipping (>40 days) delayed flowering. In particular, DSS delayed flowering more than LSS due to the decrease in chlorophyll content and the reduction in carbohydrates through respiration.

scholarly journals Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences

2012 ◽  
Vol 194 (18) ◽  
pp. 4904-4919 ◽  
Author(s):  
Lara L. Hause ◽  
Kevin S. McIver
Keyword(s):  
Binding Site ◽  
Streptococcus Pyogenes ◽  
Transcriptional Activation ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Content Type ◽  
Promoter Sequences ◽  
Mobility Shift Assay

ABSTRACTThe Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.

Sign in / Sign up

Export Citation Format

Share Document