scholarly journals Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants

2012 ◽  
Vol 3 (4) ◽  
pp. 296-300 ◽  
Author(s):  
Nancy Podevin ◽  
Patrick du Jardin
Keyword(s):  
Transgenic Plants ◽  
Plant Transformation ◽  
Camv 35S Promoter ◽  
Viral Gene ◽  
35S Promoter ◽  
Promoter Regions ◽  
Camv 35S ◽  
Transformation Vectors

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.

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.

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.

scholarly journals Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1520
Author(s):  
Dmitry Miroshnichenko ◽  
Aleksey Firsov ◽  
Vadim Timerbaev ◽  
Oleg Kozlov ◽  
Anna Klementyeva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Activity ◽  
Expression Patterns ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Promoter Strength ◽  
Tissue Specific ◽  
Camv 35S

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

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.

scholarly journals Correction: Native Phytoremediation Potential of Urtica dioica for Removal of PCBs and Heavy Metals Can Be Improved by Genetic Manipulations Using Constitutive CaMV 35S Promoter

PLoS ONE ◽  
2017 ◽  
Vol 12 (10) ◽  
pp. e0187053 ◽  
Author(s):  
Jitka Viktorova ◽  
Zuzana Jandova ◽  
Michaela Madlenakova ◽  
Petra Prouzova ◽  
Vilem Bartunek ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Urtica Dioica ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Genetic Manipulations
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