Md. Shahidul Islam
Bangladesh Jute Research Institute, Dhaka, Bangladesh
Muhammad Shafiul Azam
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Sazia Sharmin
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Abu Ashfaqur Sajib
Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh
Md. Maksudul Alam
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Md. Shamim Reza
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Rajib Ahmed
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Haseena Khan
Molecular Biology Lab, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh
Jute, Salt tolerance, KatE, Fiber crop, Transgenic
Variety and Species
Construction of entry and destination vectors: The katE gene was amplified from E. coli (K-12) genomic DNA in a GeneAmpR PCR System 9700 (Applied Biosystems) using gene-specific primers after the following thermal cycling profile: initial denaturation at 95°C for 4 min, 35 cycles of denaturation at 95°C for 50s, annealing at 60°C for 1 min, extension at 72°C for 1 min 30 s, and a final elongation step at 72°C for 10 min. The amplified fragment was inserted in a pCR/ GW/TOPO TA cloning vector (Invitrogen) following the supplier’s instructions and subsequently introduced into chemocompetent E. coli (DH5α) cells. The presence of the insert in the transformed E. coli was confirmed by nested PCR with NestF and NestR primers (35 cycles, 95°C for 50 s, 52°C for 30s, and 72°C for 1 min). To check the orientation of the inserted gene, the plasmid was amplified with 2 primer pairs, NesF and NesR, and sequenced. This entry vector was recombined with the pH7WGF2 binary vector using LR clonase (Invitrogen) according to the instructions of the supplier (22).
Agrobacterium-mediated transformation of jute plant: A. tumefaciens (LBA4404) carrying the pH7WGF2 vector harboring the katE gene and spectinomycinresistance gene was grown overnight on yeast mannitol broth (YMB) medium containing spectinomycin as the selective agent at 28°C. Bacterial concentration was determined by a spectrophotometer at a wavelength of 600 nm. Jute (C. olitorius var O-72) transformation was performed following a protocol established in our lab (23). Seeds from the mature plants (T1 jute seeds) were grown on MS medium containing hygromycin (25 mg/L) as the selective agent. For comparison, seeds from nontransgenic control plants from the same species were grown on the same medium. These died within a week after germination.
PCR analysis: Genomic DNA was isolated from control (non-infected) and T2 transgenic plants by the CTAB method (24). The presence of the newly introduced sequences in the genomic DNA was confirmed by PCR with Hyg For and Hyg Rev primer pairs (Table) using a thermal cycling profile of 35 cycles of 95 °C for 40 s (denaturation), 60 °C for 50 s (annealing), and 72 °C for 50 s (extension).
Reverse transcription PCR (RT-PCR) analysis: Total RNA was extracted from the T2 transgenic plants and the untransformed control plants by the acid guanidinium thiocyanate-phenol-chloroform extraction method (25), and first-strand cDNA was synthesized using reverse transcriptase (Superscript II; GIBCO BRL) and oligo-dT primers. Expression of the transgenes was checked by RTPCR using primers for catalase (Catalase For and NesR) and hygromycin (Hyg For and Hyg Rev).
Southern blot analysis: In order to verify the presence of the katE gene and to determine its copy number in the T2 plant genomes, 15 μg of gDNA was digested with EcoRI and detected by Southern blot analysis using an immunologic detection protocol (DIG Nucleic Acid Detection Kit, Roche). A 602 bps product of the hygromycin (hyg) resistance gene was amplified, labeled with digoxigenin-dUTP using a DIG DNA labeling Kit (Roche), and used as a probe.
Leaf disk assay: Leaf disk assay was carried out to evaluate the sensitivity of the transformed and untransformed jute plants to sodium chloride (NaCl) stress as described by Fan et al. (1997). Fully developed healthy leaves of wild-type and transgenic T2 plants (of similar age, about 50 days old) were washed with distilled deionized water. Leaf disks (~1 cm diameter) were excised and floated on 7 mL of 250 mM NaCl solution and sterile distilled water (used as an experimental control) for 10 days. The same were kept under continuous white light at 25°C. The effect of salt treatment on leaf disks was observed by monitoring phenotypic changes.
Salinity stress and transgenic plants: Seeds from previously selected T1 transgenic plants (selection was carried out in hygromycin selection medium) were transferred to earthen pots and grown in a greenhouse. Two weeks after transfer to soil, plants were watered with a 150 mM NaCl solution once every 3 days. The relative growth of the seedlings in the presence of continuous salt stress was monitored until the control plants died.
Turkish Journal of Biology, (2013) 37: 206-211
Journal