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In human, the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) is critical for tumor angiogenesis. This is a vital process for cancer tumor growth and metastasis. Blocking VEGF/VEGFR2 conjugation by antibodies inhibits the neovascularization and tumor metastasis. This investigation designed to use a transient expression platform for production of recombinant anti-VEGFR2 nanobody in tobacco plants. At first, anti-VEGFR2-specific nanobody gene was cloned in a Turnip mosaic virus (TuMV)-based vector, and then, it was expressed in Nicotiana benthamiana and Nicotiana tabacum cv. Xanthi transiently. The expression of nanobody in tobacco plants were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), dot blot, enzyme-linked immunosorbent assays (ELISA), and Western blot analysis. It was shown that tobacco plants could accumulate nanobody up to level 0.45% of total soluble protein (8.3 µg/100 mg of fresh leaf). This is the first report of the successful expression of the camelied anti-VEFGR2 nanobody gene in tobacco plants using a plant viral vector. This system provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in tobacco plants.

Angiogenesis plays an important role in tumor growth and metastasis of cancer. Vascular endothelial growth factor is the key regulator in stimulating angiogenesis. The VEGF activity is mediated by binding to its cell-surface receptors, mainly VEGFR2. Therefore, inhibition of the VEGF/VEGFR2 interaction by antibodies is investigated as a therapeutic strategy in cancer therapy. Here, we describe transient expression of an anti-VEGFR2 nanobody (3VGR19) by a viral vector based on Turnip mosaic virus in lettuce (Lactuca sativa L.). RT-PCR analysis demonstrated the 3VGR19 transcript expression. Western blot analysis showed the 3VGR19 protein expression with an expected molecular mass of ~15 kDa and based on the ELISA results, the expression level of 3VGR19 was 8 μg/g of leaf fresh weight. Taken together, recombinant 3VGR19 could be efficiently expressed in lettuce leaves and TuMV-based expression system would be an appropriate platform for production of recombinant proteins in lettuce plants.

Salinity influences the agricultural production all over the world. This constrain, similar to others biotic and abiotic stresses generate the reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals. In the evolution process of halophyte plants the mechanisms to detoxify ROS, such as antioxidant enzymes, have been developed. Aeluropus littoralis is a special halophyte that selected to our research, so the plants treated with NaCl at different salt concentration (0, 250, 450 and 650 mM) for a period 45 days. Leaves and roots (separately) collected and their proteins extracted for superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) activity assay. Meanwhile the electrolyte leakage of leaves analyzed and increased at 450 and 650 mM of NaCl concentrations. Superoxide dismutase and catalase showed same pattern for changing in enzymatic activities (increasing activity by salt stress in roots and decreasing in shoot at 450 and 650 mM stress), also peroxidase and ascorbate peroxidase activity almost increased in all stress conditions.

In Arabidopsis thaliana expression of two Desiccation-Responsive rd29 genes are intensity induced under drought, salinity and temperature (heat and cold) conditions. These two genes called, rd29A and rd29B are located in 8-9 Kb of Arabidopsis genome. Also, transgenic plants containing these genes have more tolerant toward environmental stresses. Since rd29A has an important role in various non-biological stresses, in this study genetic and promoter analysis of this gene was performed, rd29A nucleotide sequence was obtained from NCBI gene bank, for bioinformatic analysis used the Map viewer, Plant care and Genevestigator software. In Arabidopsis rd29A has 8048 nucleotides and encode a linear mRNA with 2133bp. It located at AT5G52310 locus on chromosome 5 between nucleotides 21241898 and 21242457 and contains 4 exons and 3 introns. Promoter region analysis to determine the gene regulatory elements performed with using 1500 bp of the 5 'UTR sequence and the results indicated that the existence of diverse regulatory elements such as ABRE, DRE, Box I, C-repeat/DRE and TCA-element which represent involvement of this gene in different physiological pathways in plants. In addition the TATA-Box was identified as Core promoter in -30 position. Also, the pattern of gene expression was examined by Microarrays data and it was found that the expression levels were variable in different developmental stages, so that the lowest and highest expressions were in germination and mature stages. It seems that using of this gene promoter in expression cassettes can raise tolerance of transgenic plants to environmental stresses.

The role of the antioxidant defense system in salt tolerance of Aeluropus littoralis has not been yet reported; therefore in the present study, the changes of catalase (CAT) activity in this halophyte plant was investigated and CAT gene was isolated. The leaves of treated and control plants were harvested at various times, starting 1 day prior to initiating treatment, then periodically at 72-h intervals for 21 days. The data collected showed that CAT activity increased significantly with time in plants treated with 200, 400, and 600 mM NaCl when compared with the control plants. Maximum enzyme activity was observed between the 6th and 12th day at all NaCl concentrations. CAT gene was isolated and cloned via pTZ57R/T cloning vector in Escherichia coli. CAT gene encoded 494 amino acids and had also high homology of 90, 87, 86, and 86% with CAT genes from Zea mays, Oryza sativa, Triticum aestivum, and Hordeum vulgare, respectively.

High-level reactive oxygen species reduce plant growth and development. Plants use different systems to mitigate such effects. Superoxide dismutase (SOD) is one of the most important antioxidants, which detoxifies superoxide radicals. The expression of SOD enzymes increases under stress conditions in salt- and drought-tolerant plants. Because SOD plays a critical role in plants' tolerance to stressful conditions, we provide molecular characterization of SOD genes. In this study, extraction of RNA and construction of cDNA were done for gene amplification from halophyte plant Aeluropus lagopoides. Two SOD genes were isolated and cloned in Escherichia coli. Then the genes were sequenced. Two novel SOD genes, AlSOD1 and AlSOD2, have been deposited in Genebank under accession # HM467789 and HM536180.1. The result of bioinformatics analysis indicated the genes were complete and open reading frames encoded 152 amino acids in both SOD proteins. Molecular weight and isoelectric points, respectively, were 15.097 an...

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which plays an important role in central carbon metabolism, was studied in Aeluropus lagopoides, which is an important perennial grass found in drought-stricken areas and deserts from Africa to South Asia. Aeluropus lagopoides is an ideal candidate plant system to identify novel or enhanced stress tolerance mechanisms. The GAPDH has stable expression under different conditions and it is a typical housekeeping gene in prokaryotes and eukaryotes. So far, there are no reports about identification of housekeeping genes for gene expression analysis in Aeluropus lagopoides. Thus in the present study, a GAPDH cDNA was cloned and sequenced from this plant. Our results revealed that the open reading frame (ORF) (1014 bp) encoded a polypeptide of 337 amino-acid residues. The deduced amino acid sequence indicated a protein with a molecular weight of 364.95 kDa and estimated isoelectric point value of 7.06. Multiple alignments of putative amino acid sequences indicate...

Reactive oxygen species (ROS) derived from molecular oxygen under biotic and abiotic stress such as salinity which have deleterious effects on cell metabolism. The toxic effect of ROS counteract by enzymatic as well as non-enzymatic antioxidant system. Superoxide dismutase (SOD) has a potential role for elimination of ROS. Halophytes respond to salt stress at different levels and can be a model for increasing salt tolerance in crop plants. Thus salt tolerance gene isolation and cloning of gene as well as subsequent transformation are first step for sensitive crop improvement. Aeluropus littoralis is a halophyte plant from poaceae family can be as a beneficial plant with high potential for creal breeding. There was no report on isolation of SOD gene from A. littoralis and little genomic study of this plant carried out. In this study a novel gene from A. littoralis isolated. This gene amplified by reverse transcription-PCR and cloned in E. coli pTZ57R/T cloning vector. The AlSOD gene sequence contained 456bp and the deduced transcripts encoding 152 amino acids shared a high homology with those putative CuZnSOD of higher plants like Zea mays and Oryza sativa.