scholarly journals Expression Analysis of Hairpin RNA CarryingSugarcane mosaic virus(SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Sehrish Akbar ◽  
Muhammad Tahir ◽  
Ming-Bo Wang ◽  
Qing Liu
Keyword(s):  
Mosaic Virus ◽  
Particle Bombardment ◽  
Rice Plant ◽  
35S Promoter ◽  
Small Interfering Rnas ◽  
Transgenic Resistance ◽  
Hairpin Rna ◽  
Rice Callus ◽  
Transgenic Expression ◽  
Callus Tissues

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research.Sugarcane mosaic virus(SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneouslyCP(Coat Protein) andHc-Pro(helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMVCPandHc-Progenes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with theβ-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

scholarly journals Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA

2020 ◽  
Vol 33 (1) ◽  
pp. 87-97
Author(s):  
Yuh Tzean ◽  
Ho-Hsiung Chang ◽  
Tsui-Chin Tu ◽  
Bo-Han Hou ◽  
Ho-Ming Chen ◽  
...  
Keyword(s):  
Chlorophyll Synthesis ◽  
Tomato Yellow Leaf ◽  
Cauliflower Mosaic Virus ◽  
Rice Tungro Bacilliform Virus ◽  
35S Promoter ◽  
Yellow Leaf ◽  
Hairpin Rna ◽  
Leaf Curl Disease ◽  
Inoculation Assay ◽  
Leaf Curl

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.

scholarly journals Quantification of the 35S Promoter in DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction and Specificity Assessment on Various Genetically Modified Organisms, Part I: Operating Procedure

2005 ◽  
Vol 88 (2) ◽  
pp. 547-557 ◽  
Author(s):  
Sophie Fernandez ◽  
Chrystèle Charles-Delobel ◽  
Angèle Geldreich ◽  
Georges Berthier ◽  
Francine Boyer ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Collaborative Study ◽  
Cauliflower Mosaic Virus ◽  
Performance Criteria ◽  
35S Promoter ◽  
Conserved Sequence

Abstract A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism® 7700 Sequence Detection System and TaqMan® chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

scholarly journals Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading to stable non-coding RNA required for long-distance movement

2012 ◽  
Vol 93 (5) ◽  
pp. 1093-1102 ◽  
Author(s):  
Claire Peltier ◽  
Elodie Klein ◽  
Kamal Hleibieh ◽  
Massimiliano D’Alonzo ◽  
Philippe Hammann ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Cleavage Product ◽  
Yellow Vein ◽  
35S Promoter ◽  
Long Distance ◽  
Non Coding Rna ◽  
Long Distance Movement

Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3′-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3′-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5′ RACE revealed that the truncated forms had identical 5′ ends. The 5′ termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m7Gppp at the 5′ end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5′-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.

PARTICLE BOMBARDMENT-MEDIATED TRANSFORMATION OF ORNITHOGALUM DUBIUM FOR ORNITHOGALUM MOSAIC VIRUS RESISTANCE

2005 ◽  
pp. 183-190 ◽  
Author(s):  
A. Cohen ◽  
A. Lipsky ◽  
T. Arazi ◽  
A. Ion ◽  
R. Stav ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Particle Bombardment ◽  
Virus Resistance

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

Biologia ◽  
2014 ◽  
Vol 69 (7) ◽  
Author(s):  
Fatemeh Mahdavi ◽  
Maziah Mahmood ◽  
Normah Noor
Keyword(s):  
Particle Bombardment ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Vacuum Pressure ◽  
35S Promoter ◽  
Helium Pressure ◽  
Gfp Expression ◽  
Gfp Gene ◽  
Male Flowers ◽  
Green Fluorescent

AbstractThe possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.

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