Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

2004 ◽  
Vol 31 (6) ◽  
pp. 573 ◽  
Author(s):  
Somrutai Winichayakul ◽  
Richard L. Moyle ◽  
Dacey J. Ryan ◽  
Kevin J. F. Farnden ◽  
Kevin M. Davies ◽  
...  
Keyword(s):  
Transgenic Arabidopsis ◽  
Specific Activity ◽  
Signal Pathway ◽  
Gus Activity ◽  
Asparagine Synthetase ◽  
Asparagus Officinalis ◽  
Brassica Napus L ◽  
Related Sequence ◽  
Gus Reporter ◽  
Responsive Element

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the –1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in –1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than –405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least –640 bp of the AS promoter but not those with –523 bp or smaller promoter fragments. Fusion of the –640 to –523 bp region to a –381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

The harvest-responsive region of the Asparagus officinalis sparagine synthetase promoter reveals complexity in the regulation of the harvest response

2008 ◽  
Vol 35 (12) ◽  
pp. 1212 ◽  
Author(s):  
Donald A. Hunter ◽  
Lyn M. Watson
Keyword(s):  
Promoter Region ◽  
Constitutive Expression ◽  
Wound Response ◽  
Asparagus Officinalis ◽  
Minimal Promoter ◽  
Nucleotide Substitutions ◽  
Cis Acting ◽  
Gus Reporter ◽  
Highly Active ◽  
Responsive Element

The activity of a 1915-bp asparagine synthetase (AS) promoter of Asparagus officinalis L. was induced in mature leaves of transgenic Arabidopsis thaliana (L.) Heynh. plants when the leaves were detached and held in water for 24 h. To understand this induction by harvest, variants of the AS promoter were linked to the β-glucuronidase GUS reporter gene. Harvest induction in the leaves required detachment and was not simply a wound response. Two regions in the AS promoter (Region A, –640 to –523; Region B, –524 to –383) were independently able to confer harvest response to the otherwise unresponsive –383AS (minimal) promoter. Region A was studied in further detail. Various truncations, deletions, or nucleotide substitutions of Region A affected activity and fold induction of the minimal promoter. However, no harvest-inducible cis-acting element within Region A was identified. Although the minimal promoter contained a dehydration-responsive element and ACGT elements similar to ABA-responsive regulatory motifs these were not needed by the upstream regulatory regions for directing harvest response. When four copies of Region A were linked to the minimal promoter it became highly active in leaves before harvest. Deletions within Region A showed that it required its complete 117 bp for driving harvest response, yet the region cannot simply be thought of as a harvest-responsive module, since its concatemerisation led to constitutive expression.

Analysis of the asparagus (Asparagus officinalis) asparagine synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate Suc-repression

2004 ◽  
Vol 31 (1) ◽  
pp. 63 ◽  
Author(s):  
Somrutai Winichayakul ◽  
Richard L. Moyle ◽  
Simon A. Coupe ◽  
Kevin M. Davies ◽  
Kevin J. F. Farnden
Keyword(s):  
Reporter Gene ◽  
Specific Interaction ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Asparagus Officinalis ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Gus Reporter ◽  
Foliar Senescence ◽  
Responsive Element

In asparagus (Asparagus officinalis L.), increased levels of asparagine (Asn) and Asn synthetase (AS) transcript are detected during foliar senescence and in harvested spears, possibly triggered by signals from a reduced supply of carbohydrate. To identify cis-elements mediating this regulation, the asparagus AS gene promoter was isolated and analysed by DNA sequencing, followed by expression of AS::GUS (β-glucuronidase) reporter-gene constructs in transgenic tissue, and electrophoretic mobility shift assays (EMSA). The 1958-base pair (bp) region of the AS promoter upstream of the translation initiation ATG (–1958 bp region) was sufficient to confer sucrose (Suc)-regulated expression on the GUS reporter gene in asparagus callus and protoplasts, which were transformed by particle bombardment and electroporation, respectively. Removal of Suc from callus or protoplast media resulted in the induction of GUS activity. Deletion analysis of this 1958-bp fragment identified elements in the –640 to –266�bp region as important for both high GUS levels and mediating the Suc response. This was supported by EMSA results, which showed the formation of three nuclear protein–DNA complexes with the –558 to –284 bp fragment of the promoter. A 20-bp oligonucleotide, designed to match the sequence from –423 to –404 bp, was able to out-compete formation of one of these protein-DNA complexes, suggesting a specific interaction with this sequence. This region of the promoter, overlapping with the 20-bp oligonucleotide sequence, contains a 10-bp stretch identical to a sequence previously shown to mediate low Suc induction of an Oryza sativa (rice) α-amylase gene, and may thus represent a conserved Suc-responsive element.

scholarly journals Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

2010 ◽  
Vol 23 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Miin-Huey Lee ◽  
Chiu-Min Chiu ◽  
Tatiana Roubtsova ◽  
Chien-Ming Chou ◽  
Richard M. Bostock
Keyword(s):  
Gene Expression ◽  
Redox Regulation ◽  
Brown Rot ◽  
Gus Expression ◽  
Apple Fruit ◽  
Gus Activity ◽  
Monilinia Fructicola ◽  
Gus Reporter ◽  
Flanking Regions ◽  
Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

Isolation and evaluation of three novel native promoters in Brassica napus

Botany ◽  
2013 ◽  
Vol 91 (6) ◽  
pp. 414-419 ◽  
Author(s):  
Limin Wu ◽  
Aliaa El-Mezawy ◽  
Saleh Shah
Keyword(s):  
Brassica Napus ◽  
Reporter Gene ◽  
Genetic Modification ◽  
Gus Activity ◽  
The Other ◽  
Genome Walking ◽  
Constitutive Promoter ◽  
Brassica Napus L ◽  
Transgenic Canola ◽  
Uida Reporter Gene

To provide effective and specific native promoters for canola (Brassica napus L.) genetic modification, three promoters were isolated by genome walking from this species. These three promoters were fused to the uidA reporter gene (GUS) and were independently used to generate populations of transgenic canola plants. Plants transformed with BnPGPro-GUS (B. napus putative germin promoter) exhibited GUS activity in all the tissues tested at a level comparable to those transformed with CaMV35 S promoter. This indicates that BnPGPro may serve as a native constitutive promoter for canola. The other two promoters, BnPro3-GUS and BnPro5-GUS (B. napus, promoter 3 and 5), exhibited GUS activity in various tissues. None of these two promoters expressed in embryo, however. These novel Brassica native promoters can be used to modify canola genes for various purposes.

scholarly journals EFFICIENT REGENERATION AND TRANSFORMATION PROTOCOLS FOR SAFFLOWER (CARTHAMUS TINCT0RIUS L.)

HortScience ◽  
1994 ◽  
Vol 29 (7) ◽  
pp. 744
Author(s):  
Charleen M. Baker ◽  
William E. Dyer
Keyword(s):  
Gus Activity ◽  
Direct Regeneration ◽  
Leaf Spot Disease ◽  
Hypocotyl Explants ◽  
Gus Reporter Gene ◽  
Gus Reporter ◽  
Ti Plasmids ◽  
Efficient Regeneration ◽  
Esterase Gene ◽  
Whole Plants

Our goal was to develop efficient regeneration protocols for safflower that could be used in conjunction with Agrobacterium tumefaciens -mediated transformation to introduce genes conferring economically important traits. Direct regeneration of whole plants has been achieved from cotyledon and hypocotyl explants of 30-day-old `Centennial' and `Montola' seedlings. Explants transformed with Ti plasmids containing NPTII and the β-glucuronidase (GUS) reporter gene produced kanamycin-resistant calli and shoots testing positive for GUS activity. Current work is incorporating the bar gene into appropriate Ti plasmids that will be used to confer glufosinate herbicide resistance to elite safflower cultivars. An esterase gene from Bacillus subtilis will be introduced to confer resistance to Alternaria carthami leaf spot disease.

scholarly journals Analysis of the Promoter of Emb5 from Zea mays Identifies a Region of 523 bp Responsible for Its Embryo-Specific Activity

2020 ◽  
Author(s):  
Yang Li ◽  
Liping Yu ◽  
Qiushuang Wang ◽  
Xiangyu Zhao ◽  
Xinzheng Li ◽  
...  
Keyword(s):  
Transgenic Arabidopsis ◽  
Specific Activity ◽  
Initiation Site ◽  
Pharmaceutical Products ◽  
Full Length ◽  
Pathway Engineering ◽  
Specific Expression ◽  
Transcriptional Initiation ◽  
Arabidopsis Seed ◽  
Metabolic Pathway Engineering

Abstract The maize Emb5 is an abscisic acid–responsive gene which is specifically expressed in the late embryo during seed maturity. To further dissect and identify the elements specific for its embryo expression pattern, we investigated the activity of the − 1653 bp upstream of the “full-length” promoter region of this gene in transgenic Arabidopsis plants. We first confirmed that the “full-length” promoter could indeed drive the expression of β-glucuronidase reporter gene (GUS) in the transgenic Arabidopsis seed embryo. Subsequently, DNA fragments of ~ 500 bp in length were generated after a series of progressive deletions from positions − 1653 bp to − 1 bp relative to the transcriptional initiation site. These fragments were fused with GUS and introduced into Arabidopsis. Measurement of the GUS activity in the immature seeds isolated from the transgenic plants revealed that the region between positions − 523 bp and − 1 bp, namely ProEm-D, is absolutely required and sufficient for the temporal and embryo-specific expression of GUS with an activity comparable with the full-length Emb5 promoter in Arabidopsis. Therefore, our results clearly demonstrated that the 523 bp ProEm-D can replace the − 1653 bp Emb5 promoter to drive embryo-specific expression in Arabidopsis seed. Because of its small size and strong embryo-specific activity, it could become the promoter of choice in metabolic pathway engineering to transfer multiple genes for the production of valuable pharmaceutical products in seeds, such as polyunsaturated fatty acids found in fish oils, or pro-vitamin A where at least three transgenes are required to assemble the entire metabolic pathways.

Analysis of a promoter for the NADH - glutamate synthase gene in rice (Oryza sativa): cell type-specific expression in developing organs of transgenic rice plants

2000 ◽  
Vol 27 (9) ◽  
pp. 787 ◽  
Author(s):  
Soichi Kojima ◽  
Michiko Kimura ◽  
Yukine Nozaki ◽  
Tomoyuki Yamaya
Keyword(s):  
Oryza Sativa ◽  
Oryza Sativa L ◽  
Glutamate Synthase ◽  
Gus Activity ◽  
Start Codon ◽  
Assimilate Transport ◽  
Upstream Region ◽  
Vascular Bundles ◽  
Specific Expression ◽  
Gus Reporter

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 The entire 3.7 kbp 5´-upstream region (–2840 to +886) from the translational start codon of NADH–glutamate synthase (NADH–GOGAT, EC 1.4.1.14) gene from rice (Oryza sativa L.) or the region sequentially deleted from the 5´-end was fused with the β−glucuronidase (GUS) reporter gene. The chimeric gene was introduced into calli derived from rice scutellum via Agrobacterium tumefaciens-mediated transformation and tissue-specific GUS activity determined in T0 generations. When the entire region was fused, GUS activity was detected in vascular bundles of the developing leaf blade and in dorsal and lateral vascular bundles of developing grains. This corresponds with our previous immunodetection of NADH–GOGAT protein (Hayakawa et al., Planta 193, 455–460, 1994). A series of deletion experiments showed that a 149-nucleotide region between –142 and +7 was essential for promoter activity in the NADH–GOGAT gene.

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