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.

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.

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.

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)

scholarly journals Expression of thaumatin, a new type of alternative sweetener in rice

2020 ◽  
Vol 48 (3) ◽  
pp. 1276-1291
Author(s):  
Shahina AKTER ◽  
Md. Amdadul HUQ ◽  
Yu-Jin JUNG ◽  
Kwon-Kyoo KANG
Keyword(s):  
Transgenic Rice ◽  
Oryza Sativa L ◽  
Plasmid Vector ◽  
Single Copy ◽  
Cauliflower Mosaic Virus ◽  
Pcr Analysis ◽  
35S Promoter ◽  
Alternative Sweetener ◽  
Sweet Proteins ◽  
Transgenic Lines

  Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

Multiplex, Construct-Specific, and Real-Time PCR-Based Analytical Methods for Bt Rice with cry1Ac Gene

2012 ◽  
Vol 95 (1) ◽  
pp. 186-194 ◽  
Author(s):  
Gurinder Jit Randhawa ◽  
Monika Singh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Analytical Methods ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Pcr Assay ◽  
Cry1ac Gene ◽  
Camv 35S ◽  
Bt Rice ◽  
Pcr Assays

Abstract Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) marker gene, and an endogenous α-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise.

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