Short communication

Expression of a CaMV::sak-gusA-mgfp gene in tobacco plants

Anna Dobrowolska* and Andrzej K. Kononowicz*Department of Cytogenetics and Plant Molecular Biology, University of Lodz, 12/16 Banacha Street, 90-237 Lodz,Poland*Author for correspondence: Tel.:+48-42-635-4425, E-mails: annadobrow@interia.pl, akononow@biol.uni.lodz.pl

Received 18 November 2002; accepted 24 November 2003

Keywords: Nicotiana tabacum, recombinant proteins, staphylokinase, transgenic plants

Summary

A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vectorpCAMBIA1303. The presence of a CaMV::sak-gusA-mgfp gene in Agrobacterium was confirmed by polymerasechain reaction PCR. Tobacco seedlings were used as explants for Agrobacterium tumefaciens-mediated transfor-mation with the pCAMBIA1303sak vector carrying the fusion gene construct CaMV::sak-gusA-mgfp and theexpression of the fusion gene was identified in Nicotiana tabacum plants by b-glucuronidase assay.

Introduction

Staphylokinase is one of the thrombolytic agents, whichinduces fibrin-specific thrombolysis in patients withthrombolytic disorders and therefore it holds promisefor application in thrombolytic therapy (Collen 1998). Itis a plasminogen activator produced by certain strains ofStaphylococcus aureus. It forms a complex with humanplasmin, which in turn activates other plasminogenmolecules to plasmin (Vanderschueren et al. 1995). It ishighly fibrin-selective, as revealed by virtually unalteredlevels of plasma fibrinogen and plasminogen. It isobtainable in high yield by cytosolic expression inEscherichia coli (Schlott et al. 1994).With the advent of biotechnology and gene transfer

technology, different expression systems are beingdeveloped to express recombinant proteins of industrialand pharmaceutical value (Giddings 2001). Molecularfarming in plants can be achieved by stable or transientexpression of recombinant proteins.In the past decade, plants have been actively consid-

ered as an important expression system for a number ofrecombinant proteins such as hepatitis B (HbsAg)(Kapusta et al. 1999) or rabies virus glycoprotein (Gprotein) (Arntzen 1997). Plant expression systems maybe useful for producing pharmaceuticals, as largeamount of protein can be produced at a relatively lowcost. In addition, plants are capable of complex post-translational modification like that in yeast, insect andanimal cell lines. Preliminary clinical trials using potato-produced E. coli enterotoxin showed encouraging resultsagainst diarrhoea in infants (Ma et al. 1998).Based on the observations listed above, our study was

aimed at constructing and identifying the expression of

CaMV::sak-gusA-mgfp gene inNicotiana tabacum. Origi-nally, the gene encoding staphylokinase has been iso-lated from genomic DNA of a wild strain ofStaphylococcus aureus and cloned into the pTrxFusbacterial expression vector (Invitrogen), which enabledexpression of thioredoxin-staphylokinase fusion proteinin E. coli (J. Szemraj & T. Pietrucha, personal commu-nication). We cloned a gene encoding staphylokinaseinto the plant transformation binary vector pCAM-BIA1303. The constructed gene was used to transformtobacco by using an Agrobacterium tumefaciens-medi-ated system and transiently expressed in tobacco seed-lings and regenerated plantlets.The staphylokinase gene was amplified by polymerase

chain reaction (PCR) from plasmid pTrxFussak (kindlyprovided by the Laboratory of Medical Biotechnologyof the Medical University of Lodz) as a template andtwo primers (SIGMA), the forward:(5¢-GCCCATGGATGCTCAAAAAGAAGT-3) and the

reverse one:(5¢-GCAGATCTTTTCTTTTCTATAAC-3), with NcoI

and BglII sites at 5¢ ends, respectively.The amplified fragment of 500 bp size was cloned

into a binary plasmid pCAMBIA1303 (CAMBIA,Australia), which was verified by PCR and NcoIand BglII restriction analysis. The staphylokinase genewas placed under control of the 35S promoter ofcauliflower mosaic virus (Figure 1). The binary plasmidcarries the hygromycin resistance gene. It also carriesfusion of two reporter genes: gusA and mgfp.Recombinant vector pCAMBIA1303sak was trans-formed into nopaline A. tumefaciens strain AGL1 whichwas confirmed by restriction analysis, PCR, and GUSactivity assay.

World Journal of Microbiology & Biotechnology 20: 333–335, 2004. 333� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Two–three week old seedlings of Nicotiana tabacum cvWisconsin 38 were used as explants for transformation.They were infected with AGL1 A. tumefaciens carryingpCAMBIA1303sak binary vector by the vacuum infil-tration method and incubated for 5 min in YEBmedium. Infected explants were transferred on MSmedium and grown for 2 days in the dark. Thenexplants were placed on MS medium containing0.1 mg NAA/l, 1.0 mg BAP/l, 100 mg hygromycin/land 400 mg carbenicillin/l. Petri-plates were sealed withParafilm and incubated at 26 �C under a 16 h photope-riod. After 4 weeks, small shoots were regenerated fromthe callus. After another 2–4 weeks, long, healthy shootswere dissected from the explants and transferred to MSmedium for rooting. After 3–4 weeks, when roots werewell developed, plantlets were transferred to the soil.Throughout the entire regeneration process (except therooting stage), explants were grown under continuousselection pressure: hygromycin (100 mg/l) and carbeni-cillin (400 mg/l) for a thorough plant selection and toeliminate Agrobacterium contamination from the cul-ture.Expression of the gusA gene was examined histo-

chemically in transformed seedlings and plantlets oftobacco plants that survived selection.Eleven days after Agrobacterium infection, trans-

formed and control seedlings were subjected to histo-chemical analysis for b-glucuronidase (GUS) activity.Plant tissue were incubated for 24 h in reaction buffer(5 mg X-Gluc in 50 ll DMSO, 10 ml 0.1 M phosphatebuffer pH 7.0, 70 ll 14 M mercaptoethanol, 200 ll0.5 M EDTA, 100 ll Triton X-100). Then plant tissuewas washed with distilled water and incubated for24–48 h in 70% ethanol to remove chlorophyll. Thesamples were analysed using Analysis software (GeranySoft Imaging System GmbH). The same procedure ofGUS assay was applied to 7–8 week old transformedand control plantlets.The presence and expression of the CaMV::sak-gusA-

mgfp gene in Agrobacterium tumefaciens AGLI Rfr wasconfirmed by PCR amplification (Figure 2) and NcoIand BglII restriction analysis (not shown).Expression of the gene fusion was analysed by

immunoblot assay using mouse polyclonal anti-staphy-lokinase antibody (Kucharczyk T.E. Co.), which indi-cated that the transformed bacteria expressedstaphylokinase protein (the results not shown).An expression cassette consisting of a hygromycin

selection marker and the CaMV::sak-gusA-mgfp fusion

gene was introduced into tobacco seedling cells byAgrobacterium tumefaciens AGLI Rfr carrying thebinary vector pCAMBIA1303sak. One hundred four-teen 2–3 week-old tobacco seedlings were transformed.After 8 days of incubation on MS medium underselection pressure, the transient expression was detectedby GUS assay in 14 transformed tobacco seedlings. Theexpression was observed in all examined seedlings,especially in young leaves and 57% of the roots.Then all seedlings were incubated on MS medium

under selection pressure. Embryogenic callus wasobserved only in 13 explants (14.8%). After 7 weeks ofincubation on MS medium under selection pressure, theexpression of the CaMV::sak-gusA-mgfp gene in Nico-tiana tabacum was detected by GUS assay. Expressionwas found only in 2 out of 76 (2.6%) regeneratedtobacco plants examined (the results not shown).Transient gene expression in plants has advantages

over the generation of stably transformed transgenicplants, and it seems particularly suited for verifying thatthe gene product is functional and stable before movingon to large-scale production in transgenics (Fischeret al. 1999). The results presented in this research showthat the gene construct CaMV::sak-gusA-mgfp detectedby GUS assay works properly in tobacco plants.The production of staphylokinase protein from

microorganisms in edible plants may provide humantherapeutic protein in the future because of the reducedrisk of mammalian viral contamination, the ability to dolarge scale-up at low cost, and the low maintenancerequirements.

Figure 1. Outline of the T-DNA construct of pCAMBIA1303sak plasmid indicating location of sak sequence insert.

Figure 2. PCR analysis of binary vector pCAMBIA1303sak to detect

the presence of sak gene in Agrobacterium. Lane M: 50 bp ladder

(SIGMA): 1031, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100

and 50 bp; Lane 1: pTrxFussak plasmid DNA; Lane 2: PCR without

DNA template; Lanes 3–9: plasmid DNA isolated from 7 Agrobac-

terium clones.

334 A. Dobrowolska and A.K. Kononowicz

Acknowledgement

This work was supported by grant (A.K.K.) from theState Committee for Scientific Research (KBN grant no.6 P04B 003 18), Poland.

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