Flower bud dipping or vacuum infiltration—two methods of Arabidopsis thaliana transformation

Wiktorek-Smagur, Aneta; Hnatuszko-Konka, Katarzyna; Kononowicz, Andrzej K.

doi:10.1134/s1021443709040177

Cited by 11 publications

(7 citation statements)

References 22 publications

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“…After reaching OD 600 = (1.2-1.8), bacteria were harvested using 20-min centrifugation at 1,957 g and suspended in a 5% (w/v) sucrose solution supplemented with 400 ll Sillwet L-77 (Lehle Seeds, the United States). Plants were transformed with the pCAMBIA 1304sak plasmid using the modified Clough and Bent's in planta transformation method (Wiktorek-Smagur et al 2009). …”

Section: Growth Conditions Of Arabidopsis Thaliana Plantsmentioning

confidence: 99%

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Wiktorek-Smagur

Hnatuszko-Konka

Geszberg

et al. 2010

World J Microbiol Biotechnol

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A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta Agrobacterium tumefaciens-mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated plants. The presence of the fusion protein (119 kDa) was confirmed by SDS-PAGE and Western blot analysis in protein extracts from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of 45 transgenic lines of A. thaliana plants.

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Section: Growth Conditions Of Arabidopsis Thaliana Plantsmentioning

confidence: 99%

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Wiktorek-Smagur

Hnatuszko-Konka

Geszberg

et al. 2010

World J Microbiol Biotechnol

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“…The first flowering shoot was removed to stimulate the secondary buds formation. Secondary shoots were subjected to the flower dip Agrobacterium -mediated transformation with recombinant Agrobacterium carrying the shuttle vector pATAG5- sak (Wiktorek-Smagur et al 2009 ). The Arabidopsis shoots were dipped into the Agrobacterium suspension (OD 600 = 0.8–0.9) supplemented with 300–350 µL Sillwet-77 (LEHLE SEEDS) for 2 min.…”

Section: Experimental Protocolsmentioning

confidence: 99%

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants

Hnatuszko-Konka

Luchniak

Wiktorek-Smagur³

et al. 2016

World J Microbiol Biotechnol

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Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.

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“…An overexpression (OE) plasmid pCXSN-FdC1 was constructed, in which the expression of the full-length coding sequence of FdC1 was driven by a CaMV35S promoter ( Chen et al, 2009 ). The construct was transformed into the Agrobacterium tumefaciens strain GV3101 and then into WT Arabidopsis using the floral dip method ( Wiktorek-Smagur et al, 2009 ). The overexpression lines were selected on MS plates containing 30 μg/mL hygromycin.…”

Section: Methodsmentioning

confidence: 99%

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors

et al. 2018

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Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP+ oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

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Flower bud dipping or vacuum infiltration—two methods of Arabidopsis thaliana transformation

Cited by 11 publications

References 22 publications

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors

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