Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots

Smigocki, Ann C.; Puthoff, David P.; Zuzga, Sabina; Ivic-Haymes, Snezana D.

doi:10.1007/s11240-009-9512-3

Cited by 11 publications

(5 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gels were incubated with gentle shaking in 25% (v/v) 2-propanol, 10 mM Tris–HCl pH 7.4 for 30 minutes to remove SDS followed by 10 mM Tris–HCl pH 8.0 for another 30 minutes to renature the proteins [24], [38], [42], [46], [47], [50]. Gels were then soaked with 40 µg/ml bovine trypsin (Sigma) in 50 mM Tris–HCl pH 8.0, 50 mM CaCl 2 for 40 minutes and transferred to a freshly prepared substrate-dye solution consisting of 2.5 mg/ml N-acetyl-DL-phenylalanine ß-naphthyl ester (Sigma) resuspended in dimethylformamide and 0.5 mg/ml tetrazotized O-dianisidine (Sigma) resuspended in 50 mM Tris–HCl pH 8.0 with 50 mM CaCl 2 , for 30 minutes at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene

Smigocki

Ivic-Haymes

et al. 2013

PLoS ONE

View full text Add to dashboard Cite

Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

show abstract

Section: Methodsmentioning

confidence: 99%

Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene

Smigocki

Ivic-Haymes

et al. 2013

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Over 150 sugar beet root genes were identified in a moderately resistant F1016 or a susceptible F1010 sugar beet line (Puthoff & Smigocki, 2007). Several of these genes were characterized as to their functions in resistance mechanisms (Smigocki et al, 2008(Smigocki et al, , 2009Savić & Smigocki, 2012;Smigocki et al, 2013). In a reciprocal study, a PCR-select suppressive subtractive hybridization (SSH) was used to generate SBRM cDNA libraries enriched for larval genes modulated by the interaction of the pest with a moderately resistant or a susceptible sugar beet host .…”

Section: Introductionmentioning

confidence: 99%

Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host

Smigocki

2016

Insect Science

View full text Add to dashboard Cite

Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.

show abstract

“…[ 16 , 17 ] The greatest advantage of hairy roots is their greater biosynthetic capacity for secondary metabolite production than that of their parent plants. [ 18 , 19 ] Even in cases where a particular secondary metabolite accumulates only in the aerial part of an intact plant, hairy roots have been shown to accumulate the same metabolite. [ 20 , 21 ] Hairy roots are also known to produce a spectrum of secondary metabolites that are not present in the parent plant.…”

Section: Introductionmentioning

confidence: 99%

Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents

et al. 2014

View full text Add to dashboard Cite

Background:Polygonum multiflorum is an important medicinal plant. Hairy roots systems obtained by transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes can produce valuable biological active substances, which have immense potential in the pharmaceutical industry.Objective:To optimize the inductive and cultural conditions of P. multiflorum hairy roots and to identify the major active secondary metabolites in hairy roots.Materials and Methods:P. multiflorum hairy root were mediated with A. rhizogenes R1601 to induce hairy roots. Four combinations, including Murashige–Skoog (MS), 1/2 MS, B5, and White, were investigated to optimize the culture medium. MS medium was selected for the growth measurement. The qualitative and quantitative determinations of free anthraquinone in hairy roots were compared with the calli and aseptic plantlets using high-performance liquid chromatography.Results:The inductive rates of hairy roots by leaves were higher than for any other explants. The presence of agropine in the P. multiflorum hairy roots confirmed that they were indeed transgenic. MS medium was the most suitable of the four media for hairy root growth. Meanwhile, the growth kinetics and nutrient consumption results showed that the hairy roots displayed a sigmoidal growth curve and that their optimal inoculation time was 18-21 days. The determination of the anthraquinone constituents indicated that the rhein content of the hairy roots reached 2.495 μg g−1 and was 2.55-fold higher than that of natural plants.Conclusion:Transgenic hairy roots of P. multiflorum could be one of the most potent materials for industrial-scale production of bioactive anthraquinone constituents.

show abstract

Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots

Cited by 11 publications

References 49 publications

Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene

Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene

Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host

Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents

Contact Info

Product

Resources

About