scholarly journals Molecular Cloning and Functional Characterization of Bisabolene Synthetase (SaBS) Promoter from Santalum album

Forests ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 85
Author(s):  
Haifeng Yan ◽  
Yuping Xiong ◽  
Jaime A. Teixeira da Silva ◽  
Jinhui Pang ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Transgenic Arabidopsis ◽  
Core Promoter ◽  
Functional Characterization ◽  
Promoter Sequence ◽  
Inducible Promoter ◽  
Gus Activity ◽  
Regulation Mechanism ◽  
Santalum Album ◽  
Transcriptional Initiation ◽  
Key Enzyme

Bisabolene-type sesquiterpenoids, which have multiple bioactivities, including anticancer activity, are one of the main groups of compounds in the essential oil extracted from Santalum album L. and other Santalum species. Bisabolene synthetase (SaBS) is a key enzyme for the synthesis of bisabolene in S. album, but the regulation of the SaBS gene’s expression is poorly understood. In this study, a 1390-bp promoter sequence of the SaBS gene was isolated from the leaves of six-year-old S. album. A bioinformatics analysis showed that certain environment stresses and phytohormone-activated cis-acting elements were distributed in different regions of the SaBS promoter (PSaBS). Transgenic Arabidopsis carrying full-length PSaBS had significantly higher β-glucuronidase (GUS) activity than the untreated control after treatment with salicylic acid (SA), suggesting that PSaBS is a SA-inducible promoter. Histochemical GUS staining and GUS fluorometric assays of transgenic Arabidopsis showed that the GUS activity directed by PSaBS was mainly expressed in stem tissue, followed by leaves and flowers. Moreover, different regions of PSaBS showed significantly different GUS activity. A 171-bp fragment upstream of the transcriptional initiation codon (ATG) is the core promoter region of PSaBS. Our results provide insight into and a greater understanding of the transcriptional regulation mechanism of the SaBS gene, which could serve as an alternative inducible promoter for transgenic plant breeding.

scholarly journals Analysis of Promoter Elements Involved in the Transcriptional Initiation of RpoS-Dependent Borrelia burgdorferi Genes

2004 ◽  
Vol 186 (21) ◽  
pp. 7390-7402 ◽  
Author(s):  
Christian H. Eggers ◽  
Melissa J. Caimano ◽  
Justin D. Radolf
Keyword(s):  
Borrelia Burgdorferi ◽  
Sigma Factor ◽  
Fluorescent Protein ◽  
Core Promoter ◽  
Expression Patterns ◽  
Inducible Promoter ◽  
Transcriptional Initiation ◽  
E Coli ◽  
Promoter Recognition ◽  
Maximal Expression

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoSBb) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoSBb recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoSBb dependent, while the ospE and flaB reporters were RpoSBb independent. To compare promoter recognition by RpoSBb with that of the prototype RpoS (RpoSEc), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoSEc the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoSBb under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoSEc and RpoSBb each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(−14) in the presumptive −10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoSBb as it is for RpoSEc; (ii) the nucleotide at the −15 position determines RpoS or σ70 selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoSEc- or RpoSBb-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.

scholarly journals Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 418
Author(s):  
Yanbo Huo ◽  
Bin Zhang ◽  
Ling Chen ◽  
Jing Zhang ◽  
Xing Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Interaction Analysis ◽  
Functional Characterization ◽  
Gus Activity ◽  
Core Region ◽  
Regulation Mechanism ◽  
Tripterygium Wilfordii ◽  
Potential Core ◽  
Responsive Element

Miltiradiene synthase (MS) genes, TwTPS27a and TwTPS27b, are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in Tripterygium wilfordii. However, the mechanism underlying the regulation of key genes TwTPS27a/b in triptolide biosynthesis remains unclear. In this study, the promoters of TwTPS27a (1496 bp) and TwTPS27b (1862 bp) were isolated and analyzed. Some hormone-/stress-responsive elements and transcription factor (TF) binding sites were predicted in both promoters, which might be responsible for the regulation mechanism of TwTPS27a/b. The β-glucuronidase (GUS) activity analysis in promoter deletion assays under normal and methyl jasmonate (MeJA) conditions showed that the sequence of −921 to −391 bp is the potential core region of the TwTPS27b promoter. And the TGACG-motif, a MeJA-responsive element found in this core region, might be responsible for MeJA-mediated stress induction of GUS activity. Moreover, the TGACG-motif is also known as the TGA TF-binding site. Yeast one-hybrid and GUS transactivation assays confirmed the interaction between the TwTPS27a/b promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in T. wilfordii), indicating that TwTPS27a/b are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, TwTPS27a and TwTPS27b, as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.

scholarly journals A 1232 bp upstream sequence of glutamine synthetase 1b from Eichhornia crassipes is a root-preferential promoter sequence

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanshan Zhong ◽  
Xiaodan Lu ◽  
Zhiwei Deng ◽  
Ziqing Lu ◽  
Minghui Fu
Keyword(s):  
Glutamine Synthetase ◽  
Eichhornia Crassipes ◽  
Invasive Plant ◽  
Promoter Sequence ◽  
Regulatory Mechanisms ◽  
Pcr Analysis ◽  
Regulation Mechanism ◽  
Upstream Sequence ◽  
Specific Expression ◽  
N Metabolism

Abstract Background Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism. Results A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5′-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of β-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves. Conclusions EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

Molecular cloning and functional characterization of CvLCYE, a key enzyme in lutein synthesis pathway in Chlorella vulgaris

2021 ◽  
Vol 55 ◽  
pp. 102246
Author(s):  
Sulin Lou ◽  
Xin Lin ◽  
Chenglong Liu ◽  
Muhammad Anwar ◽  
Hui Li ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Chlorella Vulgaris ◽  
Functional Characterization ◽  
Key Enzyme

Functional characterization of an Indian sandalwood (Santalum album L.) dual-localized bifunctional nerolidol/linalool synthase gene involved in stress response

2021 ◽  
Vol 183 ◽  
pp. 112610
Author(s):  
Xinhua Zhang ◽  
Jaime A. Teixeira da Silva ◽  
Meiyun Niu ◽  
Ting Zhang ◽  
Huanfang Liu ◽  
...  
Keyword(s):  
Stress Response ◽  
Functional Characterization ◽  
Santalum Album ◽  
Santalum Album L ◽  
Linalool Synthase

scholarly journals Identification and Functional Characterization of a Cold-Related Protein, BcHHP5, in Pak-Choi (Brassica rapa ssp. chinensis)

2018 ◽  
Vol 20 (1) ◽  
pp. 93
Author(s):  
Jin Wang ◽  
Feiyi Huang ◽  
Xiong You ◽  
Xilin Hou
Keyword(s):  
Brassica Rapa ◽  
Abiotic Stresses ◽  
Quantitative Polymerase Chain Reaction ◽  
Functional Characterization ◽  
Regulation Mechanism ◽  
Pak Choi ◽  
Negative Effect ◽  
Polymerase Chain ◽  
Related Proteins

In plants, heptahelical proteins (HHPs) have been shown to respond to a variety of abiotic stresses, including cold stress. Up to the present, the regulation mechanism of HHP5 under low temperature stress remains unclear. In this study, BcHHP5 was isolated from Pak-choi (Brassica rapa ssp. chinensis cv. Suzhouqing). Sequence analysis and phylogenetic analysis indicated that BcHHP5 in Pak-choi is similar to AtHHP5 in Arabidopsis thaliana. Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative. Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that BcHHP5 was induced to express by cold and other abiotic stresses. In Pak-choi, BcHHP5-silenced assay, inhibiting the action of endogenous BcHHP5, indicated that BcHHP5-silenced might have a negative effect on cold tolerance, which was further confirmed. All of these results indicate that BcHHP5 might play a role in abiotic response. This work can serve as a reference for the functional analysis of other cold-related proteins from Pak-choi in the future.

scholarly journals An NADH-Dependent Reductase from Eubacterium ramulus Catalyzes the Stereospecific Heteroring Cleavage of Flavanones and Flavanonols

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Annett Braune ◽  
Michael Gütschow ◽  
Michael Blaut
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Intestinal Bacteria ◽  
Cyclic Ether ◽  
Content Type ◽  
Oxygen Sensitive ◽  
Key Enzyme ◽  
Catalyzed Reactions ◽  
Dietary Flavonoids

ABSTRACT The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (2S,3S)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

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