Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more popular in plant synthetic molecular biology. These methods enable routine construction large complex multigene structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set novel α-level plasmids compatible with the GB convention that extend ability to rapidly assemble...

This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant plants and recently also cultures are of increasing interest due to speed, safety scalability process. Currently, studies focussing on design virus-derived achieve higher amounts transiently expressed proteins these systems. Here we designed tested single multi-cassette combine elements enhanced replication hypertranslation, assessed...

Within the last two decades, plant viral vectors have emerged as an excellent tool for expression of foreign peptides and proteins. Virus particles carrying antigenic epitopes present some interesting advantages vaccine design other applications. This review covers recent advances in use typical viruses with helical that heterologous particular emphasis on derived from Potato virus X (PVX) its uses.

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis contains a small open reading frame (ORF) (AtWRNexo) with homology to domain hWRN-p. Expression ORF in Escherichia coli revealed an activity AtWRN-exo-p similarities also some significant differences digests recessed strands duplexes 3' --> 5' direction hardly single-stranded or...

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR usually performed classical two-step procedure: the first step, cDNA synthesized by reverse transcriptase (RT), followed amplification thermostable polymerase separate tube second step. However, one-step kits containing multiple enzymes optimized RT and single can also be used. Here, we describe an single-enzyme assay based on RTX DNA that has both activities. The expression plasmid...

Polyclonal antibodies to recombinant Potato virus X (PVX) coat protein (PVX-CP) were developed and their effectiveness determined in different ELISA protocols. The PVX-CP gene was amplified by reverse transcription-polymerase chain reaction, cloned expressed Escherichia coli. For immunization, the CP fractions from bacterial lysate purified either simple fractionation or excision sodium dodecyl sulphate gels. injected into rabbits for antibody production. reacted an indirect plate trapped...

Vector pMPM-A4Ω and vectors pQE-30 pET-45b(+) containing the 6x His-tag sequence were used for expression of Potato leafroll virus (PLRV) structural non-structural proteins in Escherichia coli. Coat protein (CP) RNA-dependent RNA polymerase (RdRp)–fragments RdRp43-616 RdRp304-537 chosen expression. A high level CP was obtained only an system using vector E. coli Rosetta-gami 2(DE3) cells. After purification, His-tagged PLRV immunization rabbits.

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest damage, expressed by tail moment value, was observed and decreased 1st 4th systemic leaves. increased time after inoculation (from day 3 21) higher a part of leaf at petiole compared tip. A Pearson correlation (r = 0.94) between PVX titres ELISA absorbance values observed....

The Potato virus X (PVX)-based vector was used for the construction of N- and C-terminally modified PVX coat protein (XCP) chimeras. N-terminal XCP modifications do not influence viral life cycle, whereas simple C-terminal fusion impedes replication. We designed several chimeras tested their viabilities in various Nicotiana benthamiana genotypes. Our results showed negative impact 3'-terminal modification on chimera's cycle. To ensure chimeric constructs stability, second copy last 60...

Abstract The genes encoding the coat protein (CP) and triple gene block 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) expressed in E. coli Rosetta gami‐2(DE3). purified recombinant antigens used for raising polyclonal antibodies. antibodies against CP successfully Western blot analysis, plate‐trapped ELISA DAS‐ELISA as a coating PVM detection infected potato leaf samples. non‐structural detected TGBp1 only analysis. This is first...

Abstract The sequences of the 3′‐terminal region four Czech Potato virus M isolates VIRUBRA 4/007, 4/009, 4/016 and 4/035 were determined compared with PVM available in GenBank. Among isolates, 4/007 as well showed highest nucleotide identity (93%). Isolates most similar to PV 0273 isolate from Germany wild Russia. Interestingly, 4/009 significantly differed other three was only European that American isolates. Moreover, Republic their host range. Phylogenetic analysis based on ORF 5 coding...

Abstract Potato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly for transient expression of desired peptides fused to the N-terminus its capsid protein (CP). Here we describe testing/investigation new positions based on seven putative surface loops PVX CP. We performed bacterial fourteen different CPs modified by insertion epitope derived from E7 oncoprotein (E7 epitope) Human papillomavirus type 16 with His tag. The vector pMPM-A4Ω Escherichia...

We describe the optimized storage conditions of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV-16). Epitope derived minor capsid L2 was expressed as N-terminal fusion with ACP while an epitope E7 oncoprotein fused to its C-termini. The construct cloned into X potexvirus (PVX) based vector and transiently in plants using Agrobacterium tumefaciens mediated inoculation. effect on serological activity L2ACPE7 studied by ELISA...

We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This consists of three domestication vectors carrying GB parts-the cauliflower mosaic (CaMV) 35S promoter PVX upstream second subgenomic coat protein (PVX CP SGP), nopaline synthase (NOS) terminator downstream first SGP and gene interest (GOI). The full-length clone sequence...

Biologia plantarum, an international journal for experimental botany founded in 1959 by Professor Bohumil Němec. Covers all branches of ranging from molecular biology and biotechnology to whole-plant stand functioning.