Functional Characterization of Serotonin N-Acetyltransferase Genes (SNAT1/2) in Melatonin Biosynthesis of Hypericum perforatum

Hypericum perforatum is a traditional medicinal plant that contains various secondary metabolites. As an active component in H. perforatum, melatonin plays important role in plant antioxidation, growth, and photoperiod regulation. Serotonin N-acetyltransferase (SNAT) is the key enzyme involved in the last or penultimate step of phytomelatonin biosynthesis. A total of 48 members of SNAT family were screened and analyzed based on the whole genome data of H. perforatum, and two SNAT genes (HpSNAT1 and HpSNAT2) were functionally verified to be involved in the biosynthesis of melatonin. It was found that HpSNAT1 and HpSNAT2 were highly expressed in the leaves and showed obvious responses to high salt and drought treatment. Subcellular localization analysis indicated that these two proteins were both localized in the chloroplasts by the Arabidopsis protoplasts transient transfection. Overexpression of HpSNAT1 and HpSNAT2 in Arabidopsis (SNAT) and H. perforatum (wild-type) resulted in melatonin content 1.9–2.2-fold and 2.5–4.2-fold higher than that in control groups, respectively. Meanwhile, SNAT-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. In this study, the complete transgenic plants of H. perforatum were obtained through Agrobacterium-mediated genetic transformation for the first time, which laid a significant foundation for further research on the function of key genes in H. perforatum.

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Functional characterization of the H+/K+ ATPase in wild type and gastrin knock-out mice

Zeitschrift für Gastroenterologie ◽

10.1055/s-2007-982721 ◽

2007 ◽

Vol 45 (05) ◽

Author(s):

A Schnur ◽

P Hegyi ◽

V Venglovecz ◽

Z Rakonczay ◽

I Ignáth ◽

...

Keyword(s):

Functional Characterization ◽

Wild Type ◽

Knock Out ◽

Knock Out Mice

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

Algal Research ◽

10.1016/j.algal.2021.102246 ◽

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

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Identification and Functional Characterization of the CVOMT and EOMT Genes Promoters from Ocimum Basilicum L

10.21203/rs.3.rs-642688/v1 ◽

2021 ◽

Author(s):

Fatemeh Khakdan ◽

Zahra Shirazi ◽

Mojtaba Ranjbar

Keyword(s):

Water Deficit ◽

Transcriptional Control ◽

Functional Characterization ◽

Pcr Analysis ◽

Water Deficit Stress ◽

Specific Expression ◽

Stress Dependent ◽

First Time ◽

Dependent Mechanism

Abstract Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This indicates these promoters are able to drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

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Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

International Journal of Molecular Sciences ◽

10.3390/ijms20174081 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4081 ◽

Cited By ~ 1

Author(s):

Lin Chen ◽

Xiaohong Liu ◽

Xiaojia Huang ◽

Wei Luo ◽

Yuming Long ◽

...

Keyword(s):

Cell Wall ◽

Fluorescent Protein ◽

Functional Characterization ◽

Protein Fusion ◽

Drought Treatment ◽

Vacuolar Invertase ◽

Maize Leaves ◽

Green Fluorescent ◽

Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

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Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

Visual Neuroscience ◽

10.1017/s0952523812000223 ◽

2012 ◽

Vol 29 (4-5) ◽

pp. 211-217 ◽

Cited By ~ 18

Author(s):

CONSTANZE BICKELMANN ◽

JAMES M. MORROW ◽

JOHANNES MÜLLER ◽

BELINDA S.W. CHANG

Keyword(s):

Half Life ◽

Functional Characterization ◽

Egg Laying ◽

Sensory Transduction ◽

Wild Type ◽

Functional Variation ◽

Bovine Rhodopsin ◽

Tachyglossus Aculeatus

AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

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Functional characterization of wild-type and mutant human sialin

The EMBO Journal ◽

10.1038/sj.emboj.7600464 ◽

2004 ◽

Vol 23 (23) ◽

pp. 4560-4570 ◽

Cited By ~ 62

Author(s):

Pierre Morin ◽

Corinne Sagné ◽

Bruno Gasnier

Keyword(s):

Functional Characterization ◽

Wild Type

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Whole‐genome sequence data of Hypericum perforatum and functional characterization of melatonin biosynthesis by N‐acetylserotonin O‐methyltransferase

Journal of Pineal Research ◽

10.1111/jpi.12709 ◽

2020 ◽

Author(s):

Wen Zhou ◽

Ying Wang ◽

Bin Li ◽

Linda Petijová ◽

Suying Hu ◽

...

Keyword(s):

Genome Sequence ◽

Hypericum Perforatum ◽

Sequence Data ◽

Functional Characterization ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Sequence Data

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2066 Functional characterization of mutant BRCA1

Journal of Clinical and Translational Science ◽

10.1017/cts.2018.77 ◽

2018 ◽

Vol 2 (S1) ◽

pp. 13-13

Author(s):

John Barrows ◽

David Long

Keyword(s):

Functional Characterization ◽

Coiled Coil ◽

Wild Type ◽

Coiled Coil Region ◽

Egg Extracts ◽

Study Population ◽

Xenopus Egg ◽

Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

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