Research note: The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

2002 ◽  
Vol 29 (5) ◽  
pp. 649 ◽  
Author(s):  
Aziz Smouni ◽  
Laurent Laplaze ◽  
Didier Bogusz ◽  
Fathia Guermache ◽  
Florence Auguy ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Casuarina Glauca ◽  
Camv 35S ◽  
Highly Active ◽  
One Year

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

2012 ◽  
Vol 39 (9) ◽  
pp. 764 ◽  
Author(s):  
Gi-Ho Lee ◽  
Seong-Han Sohn ◽  
Eun-Young Park ◽  
Young-Doo Park
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Histone Deacetylase 1 ◽  
Transgenic Lines ◽  
Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

scholarly journals A simple and accurate PCR method for detection of genetically modified rice

2019 ◽  
Vol 17 (2) ◽  
pp. 847-851 ◽  
Author(s):  
Payam Safaei ◽  
Ebrahim Molaee Aghaee ◽  
Gholamreza Jahed Khaniki ◽  
Setareh Agha Kuchak Afshari ◽  
Sassan Rezaie
Keyword(s):  
Genetically Modified ◽  
Sucrose Phosphate Synthase ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Rice Varieties ◽  
Camv 35S ◽  
Nos Terminator ◽  
Gm Rice ◽  
Pcr Method

Abstract Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

The Binding of Nuclear Factors to the as-1 Element in the CaMV 35S Promoter is Affected by Cytosine Methylation in Vitro

Plant Biology ◽  
2007 ◽  
Vol 9 (3) ◽  
pp. 435-441 ◽  
Author(s):  
A. Kanazawa ◽  
M. O'Dell ◽  
R. P. Hellens
Keyword(s):  
Cytosine Methylation ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Nuclear Factors

Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

Nematology ◽  
2013 ◽  
Vol 15 (5) ◽  
pp. 519-528 ◽  
Author(s):  
Tina Kyndt ◽  
Hongli Ji ◽  
Bartel Vanholme ◽  
Godelieve Gheysen
Keyword(s):  
Transcriptional Silencing ◽  
Housekeeping Genes ◽  
Cauliflower Mosaic Virus ◽  
Heterodera Schachtii ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Expression Levels ◽  
Camv 35S ◽  
General Decline ◽  
Transgenic Lines

In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H+ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.

scholarly journals Biolistic-mediated Transformation of Lilium longiflorum cv. Nellie White

HortScience ◽  
2008 ◽  
Vol 43 (6) ◽  
pp. 1864-1869 ◽  
Author(s):  
Kathryn Kamo ◽  
Bong Hee Han
Keyword(s):  
Transgenic Plants ◽  
Genomic Dna ◽  
Fusion Gene ◽  
Lilium Longiflorum ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Ms Medium ◽  
Camv 35S ◽  
Npt Ii

Slow-growing compact calluses were initiated from bulb scales of Lilium longiflorum cv. Nellie White that had been cultured for at least 6 months on Murashige and Skoog (MS) medium with 9 μm dicamba. To develop a reliable selection system, the sensitivity of nontransformed calluses and in vitro plants to different selective agents such as phosphinothricin, kanamycin, geneticin, paromomycin, and hygromycin was tested when grown on MS medium. Nontransformed calluses showed high sensitivity to 0.5 mg·L−1 phosphinothricin, 25 mg·L−1 geneticin, and 5 mg·L−1 hygromycin. Nontransformed plants grown in vitro died on either 2 mg·L−1 phosphinothricin or 75 mg·L−1 hygromycin. Plants did not die when grown on either 200 mg·L−1 kanamycin or 100 mg·L−1 geneticin, and 100 mg·L−1 paromomycin stimulated plant growth. Transformation was achieved using biolistics on callus bombarded with either the bar-uidA fusion gene under control of the CaMV 35S promoter or npt II and uidA under control of the CaMV 35S promoter. One week after biolistic bombardment, callus bombarded with the bar-uidA fusion gene was cultured for 1 month on MS medium supplemented with 9 μm dicamba and 0.1 mg·L−1 phosphinothricin and then transferred to 0.2 mg·L−1 phosphinothricin for 1 month followed by 1.0 mg·L−1 for the next 4 months. Regenerating shoots and well-established plants were cultured on MS medium lacking hormones and with either 0.2 mg·L−1 or 2.0 mg·L−1 phosphinothricin, respectively. Callus bombarded with the npt II gene was cultured on MS medium with 50 mg·L−1 geneticin until shoots regenerated. Regenerated shoots were cultured on MS medium lacking hormones. Under optimal conditions, 10 transgenic plants were selected from seven plates of callus bombarded with the bar-uidA fusion gene using phosphinothricin for selection. Both Southern hybridization of genomic DNA and polymerase chain reaction analysis verified the presence of the transgene in transformed ‘Nellie White’ plants. Transgenic plants were phenotypically normal, and they were crossed with nontransformed plants of L. longiflorum cvs. Sakai, Yin tung, Sakai, and Flavo. The presence of the bar gene in 41% of the T1 progeny plants confirmed stable integration of the transgene into the genomic DNA of transgenic lily plants. β-glucuronidase expression resulting from the uidA gene was demonstrated in leaves and roots of some of the transgenic lily plants by histochemical staining, determination of the specific activity of the β-glucuronidase enzyme, and Northern hybridization.

Transgenic cotton (Gossypium hirsutum) over-expressing alcohol dehydrogenase shows increased ethanol fermentation but no increase in tolerance to oxygen deficiency

2000 ◽  
Vol 27 (11) ◽  
pp. 1041 ◽  
Author(s):  
Marc H. Ellis ◽  
Anthony A. Millar ◽  
Danny J. Llewellyn ◽  
W. James Peacock ◽  
Elizabeth S. Dennis
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Fermentation ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Mild Stress ◽  
Waterlogging Tolerance ◽  
Over Expression ◽  
Camv 35S ◽  
Adh Activity

Cotton (Gossypium hirsutumL.) was transformed with constructs for the over-expression of two enzymes involved in ethanol fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), with the goal of increasing waterlogging tolerance. Four independent transgenic lines transformed with the cotton Adh2 cDNA driven by the CaMV 35S promoter showed ectopic expression of this isozyme in leaves and up to 20-fold greater in vitro ADH activity in roots. Under conditions of O2 deficiency, excised roots from these transgenic plants showed up to 80% increase in ethanol evolution compared to untransformed roots. Conversely, one line transformed with a construct containing the Adh2 coding region in the antisense orientation showed a 65% decrease in ADH activity and a 25% decrease in ethanol production from anaerobic roots relative to untransformed cotton. Lines transformed with a rice Pdc1 cDNA driven by the CaMV 35S promoter showed high levels of expression of the transgene-encoded protein in leaves, but only very low levels of protein and no in vitro enzyme activity detectable in the roots of these plants. Roots from plants transformed with the 35S-Pdc construct did not produce more ethanol than roots from untransformed controls. We tested the ability of cotton roots to withstand low O2 treatments under hydroponic conditions. Neither the ‘ADH’ nor the ‘PDC’ transgenics showed more tolerance than the wild-type on the basis of root growth under a mild stress (5% O2), a strong stress (0% O2 with or without a 5% O2 pretreatment), or in recovery growth following these treatments. Our results show that over-expression of ADH can lead to ethanol over-production (even though the activity of this enzyme by far exceeds that of PDC, its precursor in the pathway), but this is not sufficient to increase waterlogging tolerance in cotton.

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

2007 ◽  
Vol 26 (8) ◽  
pp. 1195-1203 ◽  
Author(s):  
Xuelian Zheng ◽  
Wei Deng ◽  
Keming Luo ◽  
Hui Duan ◽  
Yongqin Chen ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Promoter Sequence ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Specific Gene ◽  
Gene Promoters ◽  
Adjacent Tissue ◽  
Camv 35S ◽  
Organ Specific

scholarly journals Modification of Recombination-based GATEWAYTM Binary Destination Vector with Novel Promoter for Agrobacterium-mediated Transformation of Rice

1970 ◽  
Vol 17 (1) ◽  
pp. 47-58
Author(s):  
Md Rakibul Islam ◽  
Richard Malo ◽  
Rumana S Tammi ◽  
Sharmin Jahan ◽  
Lisa Parvin ◽  
...  
Keyword(s):  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
Upstream Region ◽  
35S Promoter ◽  
Destination Vector ◽  
Camv 35S ◽  
Promoter Cloning ◽  
Expression Of Genes ◽  
Efficient Expression ◽  
Rice Leaves

The GATEWAYTM Binary Destination Vector pH7WG2 is available for easy insertion of genes for transformation into plants. The gene of interest integrates downstream of the Cauliflower Mosaic Virus Promoter CaMV 35S by recombination. The CaMV 35S promoter is however not suitable for transformation and expression of genes in monocots like rice. We isolated and cloned a ~1100 bp upstream region from two rice (Pokkali and IR64) Na/H antiporter genes into the GATEWAYTM promoter cloning vector pHGWFS7. The Pokkali promoter expressed the â-glucuronidase or GUS gene ~25-fold more efficiently than the CaMV 35S promoter in rice calli, while that of IR64 was 7-fold more. The IR64 promoter however showed efficient expression in transgenic rice leaves. The promoter from Pokkali Na/H antiporter was used to replace the CaMV 35S sequence in pH7WG2. The CaMV 35S region was cut out and the linear vector fragment blunted and T-tailed. After amplification of the promoter from Pokkali rice DNA, it was A-tailed and ligated to the modified T-vector. The resultant vector, named pH7WG3, following the nomenclature at the gateway site, www.plantgenetics.rug.ac.be/gateway, can now be used for recombination of any genes for efficient rice transformation.Key words: Recombination, Binary vector, Promoter, Transformation, RiceDOI = 10.3329/ptcb.v17i1.1120Plant Tissue Cult. & Biotech. 17(1): 47-58, 2007 (June)

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