Modares Journal of Biotechnology

Abstract Aims: The production of recombinant proteins in transgenic plants (molecular farming) is considered a functional aspect of genetic engineering. Unlike animal and bacterial cell-based production systems, proteins produced by plants are very safe and have low production costs due to the absence of common pathogens in humans and animals. The aim of this study was the transient expression of recombinant PARS II endonuclease enzyme using agroinfiltration in tobacco.
Materials and Methods: In this experimental study, the possibility of producing a recombinant form of PARS II endonuclease was investigated, using transient expression system via Agrobacterium. The pBI-Pars expression construct (based on the binary vector pBI121) containing the full sequence of the PARS II encoder, upstream kozak, and a downstream 8x-His tag sequence, was infiltrated into Nicotiana tabacum leaves with Agroinfiltration method. After 72 hours, the expression of PARS II gene in agroinfiltrated leave samples was confirmed through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and protein Dot-blot, using Anti-His antibody at the levels of mRNA and protein, respectively.
Findings: The accuracy of the constructed expression construct was confirmed, and the results of Dot-blot by Anti-His antibody confirmed the expression of the recombinant PARS II protein, while no recombinant protein expression was observed in agroinfiltrated control plants with pBI121 construct. Significant amounts of recombinant PARS II nucleases were produced in tobacco leaves.
Conclusion: Agroinfiltration is an effective and short-term method for mass production of pure recombinant PARS II nucleases in tobacco.

Abstract Aims: In recent years, according the benefits of chloroplast transformation, the cultivation of transplastomic plants and their products have been increased. Due to their biosafety concerns, their identification and labeling have become more widely considered. The aim of this study was to present an optimal method based on polymerase chain reaction (PCR) and nanobiosensor for detection of transplastomic tobacco plants and compare their sensitivity.
Materials and Methods: In the present experimental research, aadA gene as a chloroplast selectable marker was considered to design specific primer and probe. In PCR method, after optimization of aadA gene amplification, its sensitivity was evaluated with different percentages of transplastomic DNA. In nanobiosensor method at first, the labeled aadA probe was immobilized on graphene oxide (GO) and, then, hybridization reaction was optimized to identify target DNA sequence.
Findings: The amplification of 800 bp DNA related to aadA gene was observed. The PCR reaction allowed up to 5% DNA transplostomy tobacco to reproduce the aadA gene. In results of nanobiosensor after immobilization of aadA probe on GO, fluorescence emission was quenched and by adding the trasplastomic tobacco, DNA was observed again. In this method, up to 1% transplastomic tobacco DNA, fluorescence emission was significant in comparison with control tobacco plant.
Conclusion: The PCR method can detect a transplastomic tobacco plant with 5% DNA sensitivity and detect biomarker sensitivity with 1% DNA sensitivity.
The PCR method can detect a transplastomic tobacco plant with 5% DNA sensitivity and nanobiosensor can detect with 1% DNA sensitivity. Therefore, nanobiosensor method is not only a reliable diagnostic method, in addition to the PCR method for detecting transplastomic plants, but also has a higher sensitivity.

Abstract One of the newest methods in plant breeding programs is mapping quantitative trait loci (QTL) with molecular markers. In order to identify QTL associated with some chemical traits such as chlorine, nicotine, sugar concentrations and ash in oriental tobacco, a population of 55 recombinant inbred lines coming from the cross Basma seres 31 × SPT406 were evaluated for above mentioned traits. QTL mapping was performed using linkage map developed on 103 recombinant inbred lines by 64 molecular markers including 14 SSR, 24 ISSR and 26 retrotransposone. The linkage map is composed of 7 linkage groups (LGs). Composite interval mapping revealed 5 QTLs associated with studied traits. Phenotypic variation explained by identified QTLs varied between 0.34 and 0.70. Any QTL was not detected for sugar concentration in tobacco leaves. Common markers between some of studied traits can be due to linkage or pleiotropic effects. The common markers lead to increase the efficiency of marker-assisted selection in plant breeding programs via simultaneously selection for several traits.