Constructs for expression in tobacco and E. coli
The IpaB coding region from Shigella flexneri for expression in tobacco plants and E. coli was amplified using the following primers, which were designed in this study:
ipaB-tob-F: 5’-aata ctc gag gcc gcc acc atg cat aat gta agc acc aca ac-3’
ipaB-tob-R: 5’-atat ctc gag tca tag ctc atc ttt ctc aga gtg gtg gtg gtg gtg gtg agc agt agt ttg ttg caa att g-3’
ipaB-eco-F: 5’-ata gca cca tgg gac ata atg taa gca cca ca-3’
ipaB-eco-R: 5’-agc tct aga gta gtt tgt tgc aaa att g-3’
Forward primer for cloning the PCR product in pCambia1304 (a gift from Dr. Rajabi Memari; Shahid Chamran University of Ahvaz), contained restriction site for XhoI (bold) and Kozak sequence (underlined). In the reverse primer a restriction site for Xho1 (bold), a KDEL retention signal (underlined), and His-tag sequence (italic) were included. Restriction sites for enzymes NcoI and XbaI were incorporated in forward and reverse primers used for amplification of the gene for E. coli system, respectively.
The chemicals were purchased from Sigma and Merck companies, the enzymes and molecular weight markers were purchased from Fermentas, Lithuania.
The molecular biology techniques were done according to the gene cloning manual (22). The gene for use in both plant and bacterial system was amplified according to the Enzyme manufacturer protocol (Fermentas, Lithuania), briefly in aliquots of 50 µl containing 500 ng of template, 1.5 U High Fidelity Enzyme Mix, 2 mM MgSo4, 200 µM dNTP mixture 1 µM of each primer (Gene Fanavaran, Tehran). The mixture was subjected to an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s and extension 72°C for 4 min and a final extension of 72°C of 10 min. PCR products were visualized by UV and eluted by gel extraction kit (Fermentas, Lithuania) according to manufacturer's instructions. The purified fragments were cloned in pTZ57T according to the supplier's instructions (Fermentas, Lithuania) and sequencing was performed by a commercial facility (Gene Fanavaran, Tehran).
After the sequence verification, the fragment to be cloned in pCambia1304 containing Kanamycin resistance gene was digested with XhoI and ligated with similarly digested vector which had been treated with shrimp alkaline phosphatase in place of hygromycine. Orientation of the cloned gene was determined by digestion with EcoRI.
The vector containing ipaB gene with correct orientation was transformed into competent Agrobacterium tumefaciens strain LB4404 (kindly provided by Dr. Salmanian, National Iranian Genetic Engineering Center) by freeze and thaw method (23). Transformed bacteria were cultured on LB plates containing kanamycin (50 mg/ml) and incubated at 28°C for 2 to 4 days.
Agro infiltration of leaves of tobacco plant N. tabacum var. samsun (kindly provided by Dr. Rajabi Memari, (Shahid Chamran University of Ahvaz) was performed using the method described by D. Aoust et al
(24). Briefly, a single colony of Agrobacterium LBA4404 containing recombinant plasmid was cultured in 5 ml LB medium containing kanamycin (50 mg/ml) at 120 rpm at 28°C for one day. Four hundred µl of the culture (OD=0.8) was centrifuged for 20 min at 4°C (2500 ×g). The pellet was dissolved in 1 ml of resuspension solution (10 Mm MgCl2, 10 mM 4-morpholino ethanesulfonic acid (MES), 20 g/l Sucrose, 100 µL Acetosyringone (100 µM) and 1% Tween 20 in MS media) and incubated for one day. Four hundred l of the culture (OD=0.8) was centrifuged for 20 min at 4°C (2500 ×g). The pellet was dissolved in 1 ml of resuspension solution (10 Mm MgCl2, 10 mM 4-morpholino ethanesulfonic acid (MES), 20 g/l Sucrose, 100 L Acetosyringone (100 M) and 1% Tween 20 in MS media) and incubated for 1 hr at 28°C at 120 rpm. Tobacco leaves were immersed in bacterial suspension and transfered to a glass container attached to a vacuum pump (0.5 mbar) for 4 min. Breaking the pressure in container facilitates entry of bacteria into the leaves. The infiltrated leaves were then rinsed by sterile water and put in transparent containers and incubated at 28°C under continuous illumination for 7 days.
Expression of GFP in the leaves was investigated under UV light (350 nm), Image machine 440 CF (Kodak UK). A week after agro infiltration, the leaves were used for total protein extraction (25). Briefly, tobacco leaves were ground using mortar and pestle and liquid nitrogen and 1 g of leaf-powder was added to 1 ml of protein extraction buffer containing phenylmethylsulfonyl fluoride (2 mM). The plant extract was loaded to a 12% SDS-acrylamide gel (26) and after electrophoresis the proteins were transferred to nitrocellulose membrane (Schleider and Schull, Germany) for Western blotting using semi-dry blotting unit according to manufacturer's instructions. The membranes were blocked with 0.5% skim milk in Tween-PBS and probed with anti-IpaB MAb (1:1000) a kind gift from Dr. Farida Nato, Pasteur Institute of Paris. Goat anti-mouse antibody (Sigma, USA) was used as the secondary antibody (1:8000) and the membranes were developed using DAB (3-3’ Diaminobenzidine Sigma, USA). The HRP conjugated anti-5xHis antibody was purchased from Qiagen and added in 1/10000 dilution.
The sequence of the gene for IpaB amplified with primers containing NcoI and XbaI restriction sites was verified (Gene Fanavaran, Tehran) and after double digestion with these enzymes and gel purification was cloned in similarly digested and purified pBAD/gIIIA (Invitrogen, USA).
Competent E. coli Top 10 F’ (Invitrogen, USA) was transformed with the construct and the transformed bacteria were selected on LB plates containing 50 µl ampicilin. The transformed bacteria were induced with different amounts of L-arabinose according to the supplier's specifications. SDS-PAGE and Western blotting was performed as described above with HRP-conjugated anti-myc (Invitrogen, USA).