Cloning and characterization of the light-inducible Gacab promoter from Gossypium arboreum

2005 ◽  
Vol 2 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Li Wei-Min ◽  
Wang Zhi-Xing ◽  
Pei Xin-Wu ◽  
Jia Shi-Rong
Keyword(s):  
Promoter Sequence ◽  
Inducible Promoter ◽  
Regulatory Elements ◽  
Gus Expression ◽  
Full Length ◽  
Expression Vectors ◽  
35S Promoter ◽  
Gossypium Arboreum ◽  
Gus Histochemical Assay

AbstractA 1009 bp promoter sequence of cab gene, which encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins, was cloned from Gossypium arboreum. Sequence analysis showed that it had no obvious homology with previously published cab promoters. The full-length Gacab promoter and 5′ deletions with length of 197, 504 and 779 bp were fused with gus (uid A) gene, respectively, and plant expression vectors were used for transformation of Nicotiana tabacum cv. NC89. β-Glucuronidase (GUS) histochemical assay of transgenic tobacco plants showed that GUS was expressed specifically in leaves and young green tissues. GUS was not detected in the leaves of transgenic plants grown in the dark for 6 days. However, it was highly expressed in the leaves of these plants after induction with light for another 6 days, demonstrating that the full-length Gacab promoter is a light-inducible promoter. Transient GUS expression in rice calli indicated that the expression level of Gacab504::gus was the highest and stronger than that of the CaMV 35S promoter, while expression was reduced for Gacab197::gus, Gacab779::gus and Gacab1009::gus constructs. This suggests that −197 bp to −1 bp is a basic promoter of Gacab, some positive regulatory elements may exist in −504 bp to −197 bp, and the fragment −1009 bp to −504 bp may contain negative elements.

scholarly journals Isolation, Cloning and Characterization of a Constitutive Plant from Potato Aquaporin Gene

2020 ◽  
Vol 63 (3) ◽  
pp. 169-178
Author(s):  
Hira Mubeen ◽  
Rubab Zahra Naqvi ◽  
Ammara Masood ◽  
Mushtaq A. Saleem ◽  
Aftab Bashir ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Crop Improvement ◽  
Regulatory Elements ◽  
Gus Expression ◽  
Cis Acting ◽  
Transcription Start Sites ◽  
Expression Studies ◽  
Aquaporin Gene ◽  
Highly Expressed Genes

Plasma membrane intrinsic proteins (PIP1) are the most common integral membrane proteins belong to a larger family of intrinsic aquaporin proteins. They are member of aquaporin gene family and have gained importance as highly expressed genes in plants. In this study, the promoter of aquaporin PIP1 gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements, transcription start sites and transcription factor binding sites of selected promoter were identified through different bio-informatics tools. Many light responsive, phytohormone, stress and defense related cis-regulatory elements were detected in PIP1 promoter region indicating its role as a constitutive promoter. The PIP1 promoter was isolated from Solanum tuberosum. It was initially cloned in TA vector (pTZ57R/T) and later transferred to plant expression binary vectors, pGR1 and pGA482 for transient and stable expression studies in tobacco. The GUS expression results of PIP1 promoter in different tobacco tissues showed its functional importance in regulating gene expression in a constitutive manner. Further, it was concluded that the PIP1 aquaporin promoter is constitutively expressed with a strength equivalent to CaMV 2x35S promoter. These findings indicated the significance of isolated promoter for genetic engineering of plants for crop improvement.  

Molecular Characterization of Mouse CREB3 Regulatory Factor in Neuro2a Cells

2021 ◽  
Author(s):  
Kentaro Oh-hashi ◽  
Tomoyuki Hasegawa ◽  
Yoshihisa Naruse ◽  
Yoko Hirata
Keyword(s):  
Luciferase Activity ◽  
Intracellular Localization ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Regulatory Factor ◽  
Neuro2a Cells ◽  
Positive Effect ◽  
Stress Pathway

Abstract We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged full-length CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.

scholarly journals Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

2002 ◽  
Vol 9 (1) ◽  
pp. 46-53 ◽  
Author(s):  
W.-G. Son ◽  
T. A. Graham ◽  
V. P. J. Gannon
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Full Length ◽  
Expression Vectors ◽  
Protein Fusion ◽  
Western Blots ◽  
E Coli ◽  
Specific Pcr ◽  
His Tag

ABSTRACT Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and ε (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.

scholarly journals CLONING OF ProAV PROMOTER ISOLATED FROM TIGER PRAWN Penaeus monodon

2009 ◽  
Vol 4 (1) ◽  
pp. 1
Author(s):  
Andi Parenrengi ◽  
Alimuddin Alimuddin ◽  
Sukenda Sukenda ◽  
Komar Sumantadinata ◽  
Muhammad Yamin ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Penaeus Monodon ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Polymerase Chain Reaction Method ◽  
Viral Gene ◽  
Isolation And Characterization ◽  
Tiger Prawn

Promoter is a specific DNA sequence involved in the transcription of a particular gene. It is usually located in the upstream of the gene they regulate. Isolation and characterization of promoter is essentially needed in order to establish the sequence analysis and transcription factor that are used in the regulation of gene expression. The research was conducted to analyze the characteristics of Penaeus monodon anti viral gene promoter (ProAV) towards generation of auto-transgenic tiger prawn, P. monodon. ProAV promoter was isolated by PCR (Polymerase Chain Reaction) method and the purified DNA fragment was cloned into pGEM-T Easy cloning vector. The promoter sequence was characterized by using BLAST-N and Genetyx version 7 softwares. The results showed the success in isolating a promoter from tiger prawn of 368 bp in length. BLAST-N analysis showed that the sequence of isolated promoter has high similarity (95%-98%) compared to the other promoters in the GeneBank. The study revealed the existence of important transcription factors (TATA box, MRE, TCF-1, and other potential regulatory elements) are identified in the promoter sequence.

scholarly journals Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

2013 ◽  
Vol 142-143 ◽  
pp. 447-457 ◽  
Author(s):  
Afonso C.D. Bainy ◽  
Akira Kubota ◽  
Jared V. Goldstone ◽  
Roger Lille-Langøy ◽  
Sibel I. Karchner ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Characterization ◽  
Pregnane X Receptor ◽  
Full Length ◽  
Receptor Expression

scholarly journals Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

2021 ◽  
Vol 22 (12) ◽  
pp. 6450
Author(s):  
Anita Wiśniewska ◽  
Kamila Wojszko ◽  
Elżbieta Różańska ◽  
Klaudia Lenarczyk ◽  
Karol Kuczerski ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Heterodera Schachtii ◽  
Myb Transcription Factor ◽  
Parasitic Nematodes ◽  
Regulatory Sequences ◽  
Gene Encoding ◽  
Constitutive Overexpression

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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