To improve Agrobacterium-mediated transformation of tobacco, factors influencing gene delivery, including genotype of the plant, bacterial strain, and Agrobacterium transformation procedure, were tested via direct somatic embryogenesis. Leaf tissue of three different tobacco genotypes (Nicotiana tabacum L. cvs. Samsun, and Xanthi, and N. benthamiana) were used as explant. Leaf explants were transformed using three Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the binary vector pCAMBIA1304 using three different types of transformation methods as named Agro-inoculation, Agro-infection and Agro-injection. Selection of hygromycin resistant shoots was conducted on MS medium containing 3.0 mgL-1 BAP and 0.2 mgL-1 IAA,

Introduction: Omega 3 desaturase (fatty acid desaturase 3 or delta 15 desaturase) is an important polyunsaturated fatty acid (PUFA) in oilseeds which plays a great role in converting 18:2 to 18:3. Omega 3 fatty acids have a crucial function in human and plants physiological activity due to presence in cell transmembrane. Materials and Methods: In the present study, at first the fatty acid desaturase 3 (FAD3) gene was cloned from Camelina sativa by cloning via T/A cloning vector (pTG19-T plasmid) and sequenced it. Second, some different bioinformatics software were used to characterize the CsFAD3 gene and its protein. Results: Sequencing analysis of the CsFAD3 gene showed that this fragment contains 1164 bp and the start and stop codons were

The plant-based expression systems are extensively used to produce different biopharmaceuticals such as vaccines, antibodies, growth factors, and cytokines. To produce recombinant human interferon-γ (hIFN-γ) protein in tobacco, factors influencing gene delivery were first evaluated for higher efficiency of transient expression by fluorometric measurement of GUS activity. Higher levels of transient expression were observed in leaves of Nicotiana tabacum cv. Samsun infiltrated with GV3101 strain (optical density equal to 1.0 at 600 nm) under treatment of 200 μM AS at 4 days post agroinfiltration (dpa). The Samsun cv. Proved to be amenable with 1.4- and 1.5-fold higher levels of transient expression than Xanthi and N. benthamiana, res

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The EL

ResultsThe results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 ?g. g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively.

This study was carried out to analyze the performance of natural resources cooperatives in Paveh County, Iran. Cochran’s formula was used for determining the population sample size among members of active cooperatives in the study area. Data was collected using a fully structured questionnaire whose validity confirmed by both faculty members of university and cooperative organization experts. Cronbach’s alpha coefficient estimated through a pilot study was used for measuring the reliability level of questionnaires. Correlation analysis and stepwise multiple linear regression were used to analyze the data. Pairwise comparisons indicated that social, economic and environmental factors were respectively the most important criteria contribu

Chloroplast genetic engineering enhances capacity of plants as a bioreactor for the production of recombinant proteins through increasing transgenes expression level and protein accumulation. In this feasibility study, the potential of high-yield expression of interferon gamma (IFNg), a valuable therapeutic human protein, is explored for tobacco chloroplasts. The interferon gamma gene (ifnG) and aminoglycoside 3'-adenylyltransferase (aadA)(as a selectable marker gene) were introduced into the inverted repeat (IR) of tobacco plastome as a dicistronic operon using a biolistic-mediated transformation method. The presence and expression of ifnG in the regenerated plants were confirmed in DNA, RNA, and protein levels by polymerase chain reaction

To improve the Agrobacterium-mediated transformation of tobacco, factors influencing gene delivery, including genotype of the plant, bacterial strain, and Agrobacterium transformation procedure, were tested via direct somatic embryogenesis. Leaf tissue of three of different genotypes of Nicotiana tabacum L. cvs. Samsun, and Xanthi, and N. benthamiana were used as explant. Leaf explants were transformed using three Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the binary vector pCAMBIA1304 by using three different types of transformation methods as named Agro-inoculation, Agro-infection and Agro-injection. Selection of hygromycine resistant shoots was conducted on MS medium containing 3.0 mgL-1 BAP and 0.2 mgL-1 I

Nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. It has some benefits such as fast and large quantity production of recombinant proteins with low cost. In this research, the green fluorescent protein (GFP) was transiently expressed in two tobacco species via turnip mosaic virus (TuMV) derived vector, a virus which can infect a wide range of plant species. Florescence microscopy results indicated that TuMV could infect tobacco plants and accumulate GFP protein in plant leaves. In addition, RT-PCR, Dot-Blot and ELISA assays demonstrated the recombinant gene transcription, translation and stability. This is the first report of using TuMV-based viral vectors fo

ResultsDespite the very promising advances in the field of molecular farming, we still face two serious challenges which needs to be taken seriously; insufficient accumulation levels of recombinant proteins and lack of efficient purification methods. To achieve the high levels of production, several factors should be taken into consideration, such as choice of a

In this study, the ELP sequence was fused to human interferon-γ (hIFN-γ) and hIFN-γ-ELP fusion protein accumulated with high levels of yield and purity, compared with the corresponding unfused hIFN-γ protein. The hIFN-γ was exclusively produced in the form of insoluble inclusion bodies while the hIFN-γ was relatively soluble when expressed as an ELP fusion protein. The insoluble inclusion bodies were then solubilized under denaturing conditions, refolded in the presence of arginine and purified by single-step ion-exchange chromatography. The fusion to ELP signidficantly increased the accumulation of hIFN-γ by 10-fold with a stable expression on average of 46.85% of total soluble protein (TSP). Furthermore, three rounds of Inverse Tra

The production of recombinant proteins for medical and industrial uses is widely enhanced in plants and transient expression systems have made much progress in recent years. However, low yield of production and the lack of suitable purification methods for recovering of recombinant proteins has led to develop different fusion protein strategies such as elastin-like polypeptide (ELP) fusions. In this study, the ELP sequence was fused to human interferon-γ (hIFN-γ) and transiently expressed in Nicotiana tabacum plants by Agroinfiltration. The ELP fusion significantly increased the accumulation of hIFN-γ by 2.6-fold with a high expression at an average of 22.48% of total soluble protein (TSP). The analysis of different salt concentrations o

Oilseed crop oils contain a variety of unsaturated fatty acids that are synthesized and regulated by fatty acid desaturases (FADs). In this study, 14 FAD3 (ω3 desaturase) protein sequences from oilseeds are analyzed and presented through the application of several computational tools. The results indicated a close relationship between Brassica napus and Camelina sativa, as well as between Salvia hispanica and Perilla frutescens FAD3s, due to a high similarity in codon preferences in codon usage clusters and the phylogenetic tree. The cis-acting element results reveal that the seed-specific promoter region of BnFAD3 contains the critical conserved boxes such as HSE and ABRE, which are involved in responsiveness to heat stress and abscisic a

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in

Key message This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Abstract Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They c

Antagonistic fungi are well-known as viable alternatives to chemical control of root-knot nematodes. In this paper, serine protease Ac1 as an important pathogenicity factor was used to enhance the antagonistic activity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 (second stage juveniles). Ac1 gene was extracted from A. conoides and cloned in pCAMBIA1304 vector. The recombinant plasmid was then transferred to these fungi using two strains of Agrobacterium tumefaciens (LBA4404 and AGL1). Transgenic isolates were confirmed by PCR amplification of Hygromycin B resistance gene (hph) as selectable marker, protease assay using casein substrate and in vitro bioassay. Transferring of Ac1 gene by homologous recombination

The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference bet