Expression Of Coat Protein Gene Research Articles

Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody

Grapevine berry inner necrosis virus (GINV) and grapevine Pinot gris virus (GPGV) are prevalent viral diseases affecting viticulture, posing significant threats in grape-producing regions of China. Previous studies have emphasized the harmful effects of grape viruses on the grape industry all over the world. However, few reports have focused specifically on GINV. In wild grapevines, GINV infection frequently leads to grapevine fanleaf degeneration disease (GFDD). GINV often co-occurs with other grape viruses, exacerbating its harmful effects on the grapevine industry in China. In this study, we collected grapevine samples from Taizhou city, Jiangsu Province, where GINV infection was confirmed. Based on the GINV coat protein (CP) gene, we developed a high-throughput and high-sensitivity direct antigen-coated ELISA and Dot blot assay for field diagnosis of GINV CP in grape samples. The CP gene was cloned from GINV-infected grape samples, and the GINV CP was expressed using the pET30(a) vector. Specific polyclonal antiserum CPGINV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPGINV antiserum, we developed a rapid, precise, and scalable diagnostic method for GINV in the grapevine industry. The established ELISA and Dot blot assays successfully detected GINV-infected grapevine samples. The occurrence of GINV is relatively common in China, which poses a risk of transmission and threatens the healthy development of the grape industry. Therefore, this study prepared CPGINV antiserum and established an efficient, rapid, sensitive, accurate, and high-throughput diagnostic method, providing a foundational approach for the prevention and control of vitis viral diseases.

In silico identification and validation of microRNAs from the genome of Solanum lycopersicum targeting Groundnut bud necrosis orthotospovirus

Tomato, an extensively cultivated edible crop is prone to many viral pathogens. Among them, bud necrosis incited by Groundnut bud necrosis orthotospovirus (GBNV) causes huge yield loss (80–100%). Out of all known strategies for viral disease management in plants, RNA interference (RNAi) mediated approaches have greater potential in managing viral diseases. Small RNAs especially microRNA (miRNA) play a major role in RNAi-mediated defense in plants. Using in silico analysis, we have identified the possible tomato encoded miRNA that have the potential to interact with GBNV-mRNA. The results revealed that, sly-MIR9479-3p, sly-MIR10529, sly-MIR397-5p, sly-MIR10528, sly-MIR477-3p, and sly-MIR6024 predicted by the computational algorithms viz., miRanda, psRNA target, RNA22 and RNA hybrid have the potential to interact with different GBNV-ORFs. Conserved complementary sequences of sly-MIR10529 and sly-MIR6024, targeting GBNV genome regions (ORF2 and ORF5), were confirmed in multiple GBNV isolates. The expression of Sly-miR-10529 (1.72-fold increase),Sly-miR-6024 (2.83-fold decrease) and their corresponding glycoprotein and coat protein gene expression were quantified through q-RT-PCR. These predicted miRNAs can offer useful information for the production of antiviral agents/RNAi mediated transgenic crop plants against GBNV in tomato.

Down Regulation of Potato Virus Y (PVY) Coat Protein (CP) Expression by Iberis gibraltarica Protein Extract

Potato virus Y (PVY) is among the most destructive potato viruses and a solemn threat to efficient seed production worldwide. Almost 70% potato yield losses are due to early PVY infection. Coat protein (CP) gene has a significant function in host adaptation in most of the plant viruses. CP has become more frequent and extensively studied in PVY and other potyviruses to generate resistance against the potyviruses in different crops. In this study, we also targeted the CP gene and evaluate the in vitro potential of Iberis gibraltarica total protein extract against the CP gene expression. Total protein of Iberis gibraltarica, was isolated and quantified. The full length CP gene was amplified from PVY infected potato plant leaves and cloned into mammalian expression vector. It was later transfected into mammalian cell line to obtain the transient expression and mRNA expression of the CP gene against the total protein of Iberis gibraltarica was analyzed through the real-time PCR. Results show that total protein of Iberis gibraltarica down regulates the mRNA expression more than 90% in vitro studies.

DETECTION AND MOLECULAR CHARACTERIZATION OF ODONTOGLOSSUM RINGSPOT VIRUS (ORSV) JAVA AND BALI ISOLATES

<p>Orchids are one of the most important ornamental plants and cultivated in tropical countries, including in Indonesia. Virus infections become an important limiting factor in orchids cultivation because it causes significant losses of the plants. Odontoglossum ringspot virus (ORSV) is one of the most reported viruses infecting orchids and spread widely in the world. The common symptoms are mosaic with line pattern and necrotic ringspot on leaf surface, and also color breaking on flowers. The purpose of this research was to find out the occurrence of ORSV infecting orchids in Java and Bali, genetic relationship among ORSV isolates based on similarities of coat protein (CP) gene, and to analyze the pathogenicity test of ORSV Java and Bali isolates. Survey and samples collection were conducted in seven locations cultivating orchids in Java and Bali. Detection with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that ORSV was infected three leaf samples of Phalaenopsis sp., called ORSV BOC, ORSV KRB and ORSV TNBB isolates. The results showed 474 bp-amplified DNA band as the expression of ORSV CP gene. Phylogenetic analysis based on nucleotide sequences of CP gene showed that ORSV BOC have similarity close to ORSV Germany, whereas ORSV KRB and ORSV TNBB leads to speciation that possible to be a new strain. Pathogenicity test using various healthy plants showed that ORSV BOC may infected and cause systemic symptoms on Chenopodium amaranticolor, Nicotiana tabacum, Dendrobium sp., Cymbidium sp., Chattleya sp., Phalaenopsis sp., Liparis sp., Spatthoglotis sp. and Pectelis sussanae (L.) Raf.</p><p><br /><strong>Keywords:</strong> ORSV, orchids, coat protein, RT-PCR</p>

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

Background: Cucumber mosaic virus (CMV) has isometric particles with a diameter of about 28 - 29 nm. Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost. Objectives: To this end, our aim was to express the CMV CP gene in E. coli to be used as the antigen for antibody production in the future. Materials and Methods: Coat Protein (CP) gene cDNA from an isolate (B13) of Cucumber Mosaic Virus (CMV) was subcloned from pTZ57RCMVCP to pET21a expression vector and transformed to E. coli strain Rosetta. Expression of CMV CP was successful and confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), wherein a ~30- kDa protein band was revealed. Induction by Isopropyl-Thiogalactoside (IPTG) at final concentrations of 0.5 to 2 mM appeared to produce similar results as to the amount of the expressed protein, which was judged by intensity of the band on SDS-PAGE. Results: The identity of the expressed protein was confirmed by immunoassays such as western blot, Dot-Immunobinding Assay (DIBA) and Enzyme-Linked Immunosorbent Assay (ELISA) by the use of anti-CMV antibody. Conclusions: This is the first report of expression of CMV CP gene in Iran, which is important for the preparation of anti-CMV antibody and paving the way for the use of the virus coat protein as a nanomaterial.

Expression of coat protein gene of Cucumber mosaic virus (CMV-subgroup IA) Gladiolus isolate in Nicotiana tabacum

The coat protein (CP)-mediated resistance against Cucumber mosaic virus (CMV) subgroup IA was developed in transgenic lines of Nicotiana tabacum cv. Petit Havana using Agrobacterium tumefaciens-mediated transformation. Ten independently transformed lines have developed, four of which were tested for resistance against CMV using virus challenge inoculations. The transgenic lines exhibiting complete resistance remained healthy and symptomless in their life span and showed reduced or no virus accumulation in their systemic leaves after virus challenge inoculation. These transgenic lines also showed resistance against CMV strains which are not closely related to CMV-Gladiolus strains. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IA member isolated from India showing resistance to all CMV strains occurring in the same vicinity.

GM plants with RNAi - golden mosaic resistant bean

GM plants with RNAi - golden mosaic resistant bean

Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation.

The absence of resistance genes against biotic stresses like Tobacco streak virus (TSV) within compatible peanut germplasm necessitates the deployment of genetic engineering strategy to develop transgenic resistance. Transgenic resistance in peanut (Arachis hypogaea L.) to peanut stem necrosis disease caused by TSV was obtained by transferring coat protein (CP) gene of TSV through Agrobacterium-mediated transformation of de-embryonated cotyledons and immature leaves of peanut cultivars Kadiri 6 (K6) and Kadiri 134 (K134). Integration of the transgene in T1, T2 and T3 generations were confirmed by PCR with gene-specific primers. On the basis of segregation analysis of the PCR amplicons, homozygosity was confirmed in progeny from five transgenic lines. Six transgenic plants from three different single copy transgenic lines homozygous for the transgene were selected for challenge inoculation in T3 generations. The transgenic lines remained symptomless throughout and showed traces or no systemic accumulation of virus indicating the tolerance/resistance to the TSV infection. CP gene expression was observed in transgenic lines by RT-PCR, real-time PCR and ELISA. The findings provide an effective strategy for developing peanut with resistance to peanut stem necrosis disease.

NIR-assisted orchid virus therapy using urchin bimetallic nanomaterials in phalaenopsis

The use of nanoparticles has drawn special attention, particularly in the treatment of plant diseases. Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV) are the most prevalent and serious diseases that affect the development of the orchid industry. In this study we treated nanoparticles as a strategy for enhancing the resistance of orchids against CymMV and ORSV. After chitosan-modified gold nanoparticles (Au NPs) were injected into Phalaenopsis leaves, the injected leaves were exposed to 980 nm laser for light–heat conversion. To evaluate virus elimination in the treated Phalaenopsis leaves, the transcripts of coat protein genes and the production of viral proteins were assessed by reverse transcription-Polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of coat protein genes for both CymMV and ORSV was significantly lower in the chitosan-modified Au NP-treated Phalaenopsis leaves than in the control. Similarly, the amount of coat proteins for both viruses in the Phalaenopsis leaves was lower than that in the control (without nanoparticle injection). We propose that the temperature increase in the chitosan-modified Au NP-treated Phalaenopsis tissues after laser exposure reduces the viral population, consequently conferring resistance against CymMV and ORSV. Our findings suggest that the application of chitosan-modified Au NPs is a promising new strategy for orchid virus therapy.

Cloning and Prokaryotic Expression of <i>Apple Stem Pitting Virus</i> CP Gene in Ya Pear in <i>E. coli</i>

Abstract In order to clarify evolutionary relationship and genetic diversity of apple stem pitting virus (ASPV), and preparation the corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya (Y) pear which infected by ASPV and used as template for cDNA synthesis. The coat protein (CP) gene of ASPV was cloned and sequenced. The CP gene of Ya pear was cloned into expression vector PET-28a, and transformed into E . coli BL21 (DE3), and SDS-PAGE electrophoresis analysis. The results showed that the complete CP gene consisted of 1 194 nucleotides and encodes a polypeptide of 397 amino acid (aa). Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Phylogenetic tree showed that all isolates of the coat protein (CP) genes of ASPV isolates at AA sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups â…i (except NC_003462) and the Ya pear were clustered into group â…¢. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.

The Arabidopsis PEAPOD2 transcription factor interacts with geminivirus AL2 protein and the coat protein promoter

The TrAP protein of bipartite begomoviruses activates the coat protein (CP) promoter in mesophyll and derepresses the promoter in vascular tissue through two sequences, located 60-125 bp and 1.2-1.5 kbp respectively, upstream of the CP gene. TrAP does not, however, exhibit specific binding to either sequence directly. We have identified a plant-specific DNA-binding protein, Arabidopsis PEAPOD2 (PPD2), that specifically binds sequences mediating activation of the CP promoter of Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus in mesophyll. This protein does not however, bind sequences required for TrAP-mediated derepression in phloem. TGMV TrAP interacts with the PPD2/CP promoter complex in electrophoretic mobility shift assays. PPD2 is associated with the nucleus, as expected for a transcription factor, but is not capable of activating transcription directly. Thus, geminivirus TrAP is likely targeted to the CP promoter through interaction with PPD2, leading to activation of CP gene expression.

Transgenic expression of coat protein gene of Rice tungro bacilliform virus in rice reduces the accumulation of viral DNA in inoculated plants

Rice tungro, a devastating disease of rice in south and southeast Asia, is caused by the joint infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to obtain transgenic resistance against RTBV, indica rice cultivar Pusa Basmati-1 was transformed to express the coat protein (CP) gene of an Indian isolate of RTBV. Rice plants containing the transgene integrated in low copy numbers were obtained, in which the CP was shown to accumulate in the leaf tissue. The progenies representing three independent transformation events were challenged with Indian isolates of RTBV using viruliferous Green leafhoppers, and the viral titers in the inoculated plants were monitored using DNA dot-blot hybridization. As compared to non-transgenic controls, two independent transgenic lines showed significantly low levels of RTBV DNA, especially towards later stages of infection and a concomitant reduction of tungro symptoms.

Ecdysone agonist-inducible expression of a coat protein gene from tobacco mosaic virus confers viral resistance in transgenic Arabidopsis.

Constitutive expression of a gene encoding tobacco mosaic virus (TMV) coat protein (CP) in transgenic plants confers resistance to infection by TMV and related tobamoviruses. Here, we examined resistance to TMV by temporal and quantitative control of TMV Cg CP (CgCP) gene expression using a simple, methoxyfenozide-inducible system in Arabidopsis plants. By soil drenching with a commercial ecdysone agonist (Intrepid-2F/methoxyfenozide), most transgenic lines were induced from undetectable levels of gene expression to protein levels from 0.05 to 0.8% (w/w) of CgCP. This corresponds to up to four times the amount of CP produced by the constitutive cauliflower mosaic virus (CaMV) double 35S promoter. CgCP transcripts were induced by 700-fold, without changing the expression patterns of pathogenesis-related (PR) genes. The high level of accumulation of CgCP was sufficient to produce large amounts of virus-like particles that accumulate in large aggregates throughout the cells. In virus challenge assays, treatment with Intrepid-2F prior to TMV infection resulted in high levels of viral resistance, while no treatment or treatment with the inducer following infection did not confer resistance. This report demonstrates chemically controlled disease resistance and confirms the utility of the ecdysone agonist-inducible system under greenhouse conditions.

RNA phage Q beta coat protein as a carrier for foreign epitopes.

The Q beta gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known 'display vectors' on the basis of coat proteins of RNA phage group I, group III phage Q beta-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called A1 protein for insertion of the appropriate immunological epitopes. 'Mosaic' capsids presenting model hepatitis B virus preS1 and HIV-1 gp120 epitopes and formed by Q beta CP together with A1-derived proteins were obtained as a result of (1) suppression of leaky UGA stop codon of the CP gene and (2) simultaneous expression of 'pure' CP and full-length A1-derived genes obtained after the changing of CP-terminating UGA to strong UAA stop codon or sense GGA codon, respectively.

Nucleotide Sequence of the 3'-Terminal Region of Bean Yellow Mosaic Virus RNA and Resistance to Viral Infection in Transgenic Nicotiana benthamiana Expressing Its Coat Protein Gene.

The sequence of 4, 306 nucleotides from the 3'-poly(A) tract of bean yellow mosaic virus (BYMV) RNA has been determined. This sequence contained a single, long open reading frame (ORF), that when compared with amino acid sequences of other potyviruses was expected to encode carboxyl terminus of CI, 6K, NIa, NIb and coat protein (CP). The CP-coding sequence was predicted to be 819 nucleotides in length, encoding a protein of 273 amino acids. To produce transgenic plants resistant to viral infections, the BYMV CP gene was fused to a high expression promoter cassette containing a duplicated enhancer sequence of cauliflower mosaic virus 35S promoter followed by the 5'-untranslated sequence of tobacco mosaic virus RNA. The chimeric gene was introduced into Nicotiana benthamiana by Agrobacterium-mediated gene transfer, and the expression of CP gene was verified by Western blot analysis. Self-fertilized R1 progeny expressing detectable levels of CP showed significant delay in symptom development, as well as an attenuation of the symptoms.

Expression of Coat Protein Gene from Cucumber Mosaic Virus Strain C in Tobacco: Protection Against Infections by CMV Strains Transmitted Mechanically or by Aphids

The coat protein (CP) gene from cucumber mosaic virus (CMV) strain C was engineered for expression in plants and transferred into the genome of tobacco (Nicotiana tabacum Xanthi'). Transfer of the CP gene was confirmed, and plants containing it produced the expected 1,400-nucleotide mRNA and 24-kDa protein products, which were detected by northern and western blots, respectively (...)

Are the PR1 proteins of tobacco involved in genetically engineered resistance to TMV?

Transgenic tobacco plants constitutively expressing the coat protein (CP) of tobacco mosaic virus (TMV) exhibit enhanced resistance to TMV (P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, Science, 232, 738–743,1986; R. S. Nelson, P. Powell Abel, and R. N. Beachy, Virology 158,128–132,1987). To determine if this enhanced resistance might be mediated through the PR1 family of pathogenesis-related (PR) proteins, their synthesis was examined. In transgenic plants derived from NN genotypic tobacco, a high proportion (∼ 80%) of those producing CP also expressed the PR1 genes at low levels. However, this correlation between TMV CP and PR1 gene expression was not observed in similarly transformed nn genotypic tobacco plants. Therefore, it appears unlikely that PR1 proteins play a critical role in genetically engineered resistance in transgenic plants producing TMV CP.

Effects of transposition and deletion upon coat protein gene expression in bacteriophage f1

We have studied expression of the major coat (gene VIII) protein in vivo in cells infected with variant fl phage whose genomes had been restructured in vitro. This gene with its most proximal promoter and terminator behaves as an independent transcription unit: coat protein expression is unaffected by transposition of the gene to the intergenic space (IG) and is independent of orientation. Deletion of the DNA sequence (−35 region) upstream of the Pribnow box of the promoter for the transposed gene eliminates coat protein expression. The position and orientation of the transposed DNA within the IG can, however, have marked effects upon phage DNA replication. In mutants diploid for the coat protein gene, the kinetics of phage production are altered by both the level of DNA replication and the level of coat protein expression. In partial diploid mutant infections virions can be assembled from mixtures of wild-type fl and variant coat protein molecules in which amino acid polymorphisms occur near the amino terminus.