scholarly journals Development of the VIGS System in the Dioecious Plant Silene latifolia

2019 ◽  
Vol 20 (5) ◽  
pp. 1031 ◽  
Author(s):  
Naoko Fujita ◽  
Yusuke Kazama ◽  
Noriko Yamagishi ◽  
Kyoko Watanabe ◽  
Saki Ando ◽  
...  
Keyword(s):  
Sex Determination ◽  
Candidate Genes ◽  
Large Scale ◽  
Viral Vector ◽  
Functional Characterization ◽  
Virus Vector ◽  
Functional Gene ◽  
Silene Latifolia ◽  
Vector System ◽  
Dioecious Plant

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.

scholarly journals Expression of HIV-1 gag and env genes using the vesicular stomatitis virus vector system

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Justine M Baek
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Recombinant Virus ◽  
Viral Vector ◽  
Genetically Modified ◽  
Hiv Vaccine ◽  
Virus Vector ◽  
Vector System ◽  
Temperature Sensitive ◽  
Vesicular Stomatitis ◽  
Hiv 1

Traditional vaccine methods have long been employed to control widespread infectious diseases, but so far, all commercially available vaccine strategies have been inadequate in efforts to develop an effective therapeutic HIV vaccine. However, recent advancements in immunological research have led to the generation of novel vaccine strategies, one of which is the recombinant virus vaccine, a method of particular interest that has shown promise in the clearance of HIV infection within HIV-positive patients who have retained immunocompetence. This study examined the stability of expression of HIV-1 genes, gag and env, through a recombinant virus vector, a recombinant vesicular stomatitis virus (VSV), a temperature-sensitive mutant genetically modified to contain the select HIV-1 genes (VSVInd(GML)HIV-1gag-env). For SDS-PAGE, cells infected with VSVInd(GML) temperature-sensitive mutants were incubated at 31 °C and 37 °C, the permissible and semi-permissible growth conditions respectively. Western blot analyses were used to quantitate levels of protein expression of full VSV proteins, Gag, and Env using a primary rabbit antibody of anti-VSV anti-serum and a secondary anti-IgG from rabbit, a primary antibody of anti-p24 anti-serum and a secondary anti-IgG from rabbit, and a primary goat antibody, anti-gp120, and a secondary anti-IgG from goat, respectively. Results indicated that the VSVInd(GML) vector system allowed for high levels of expression of HIV-1 gag and env genes. It is known that the expression of these genes induce the production of major neutralizing antibodies and the stimulation of cytotoxic T lymphocytes, therefore this finding reveals the potential to use a genetically modified recombinant VSV as a universal vector for the development of recombinant virus vaccines. Specifically, the VSVInd(GML) mutant vector is thus an attractive candidate for the viral vector of a therapeutic HIV vaccine system.

Cytochrome P450 mono-oxygenases in conifer genomes: discovery of members of the terpenoid oxygenase superfamily in spruce and pine

2006 ◽  
Vol 34 (6) ◽  
pp. 1209-1214 ◽  
Author(s):  
B. Hamberger ◽  
J. Bohlmann
Keyword(s):  
Cytochrome P450 ◽  
Loblolly Pine ◽  
Large Scale ◽  
Expressed Sequence Tag ◽  
Resin Acid ◽  
Functional Characterization ◽  
Structural Diversity ◽  
Resin Acids ◽  
Full Length Sequence ◽  
Diterpene Resin Acids

Diterpene resin acids, together with monoterpenes and sesquiterpenes, are the most prominent defence chemicals in conifers. These compounds belong to the large group of structurally diverse terpenoids formed by enzymes known as terpenoid synthases. CYPs (cytochrome P450-dependent mono-oxygenases) can further increase the structural diversity of these terpenoids. While most terpenoids are characterized as specialized or secondary metabolites, some terpenoids, such as the phytohormones GA (gibberellic acid), BRs (brassinosteroids) and ABA (abscisic acid), have essential functions in plant growth and development. To date, very few CYP genes involved in conifer terpenoid metabolism have been functionally characterized and were limited to two systems, yew (Taxus) and loblolly pine (Pinus taeda). The characterized yew CYP genes are involved in taxol diterpene biosynthesis, while the only characterized pine terpenoid CYP gene is part of DRA (diterpene resin acid) biosynthesis. These CYPs from yew and pine are members of two apparently conifer-specific CYP families within the larger CYP85 clan, one of four plant CYP multifamily clans. Other CYP families within the CYP85 clan were characterized from a variety of angiosperms with functions in terpenoid phytohormone metabolism of GA, BR, and ABA. The recent development of EST (expressed sequence tag) and FLcDNA (where FL is full-length) sequence databases and cDNA collections for species of two conifers, spruce (Picea) and pine, allows for the discovery of new terpenoid CYPs in gymnosperms by means of large-scale sequence mining, phylogenetic analysis and functional characterization. Here, we present a snapshot of conifer CYP data mining, discovery of new conifer CYPs in all but one family within the CYP85 clan, and suggestions for their functional characterization. This paper will focus on the discovery of conifer CYPs associated with diterpene metabolism and CYP with possible functions in the formation of GA, BR, and ABA in conifers.

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