Expression of Hemagglutinin\u2013Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin\u2013Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum ) plant by Agrobacterium -mediated transformation. Results: Putative transgenic plantswere screened in a selection medium containing 50 mg/L kanamycin and 30mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein

Mineralogy, geochemistry, genesis, and industrial application of silica in Arefi area, south of Mashhad

Introduction Arefi quartz-bearing conglomerate (Middle Jurassic) is situated within Binalud structural zone. The unit is trending NW-SE located 25 km south of Mashhad. More than 97% of the pebbles are quartz as mono-crystalline, poly-crystalline, and minor fragments of chert, quartzite, and mica schist. Less that 3% of the remaining minerals are feldspar, mica, chlorite, hornblende, tourmaline, zircon, sphene, and opaque minerals. The cement is mainly silica. Hashemi (Hashemi, 2004) suggested this unit is orthoquartzitic polymictic conglomerate. In this study, we carried out detailed mineralogical studies, geochemical analyses for SiO2 and troublesome elements, determination of quartz pebbles source using geological observations and fluid inclusion microthermometry, and industrial application studies with new insight for porcelain and ceramic factories as the nearest silica-rich reserve to Mashhad. Material and methods 1. Preparing geologic map in 1:10000 scale in the Arefi area. 2. Petrographic study of 65 samples from the quartz-bearing conglomerate unit. 3. Major elements such as SiO2, TFeO, TiO2, and CaO were analyzed at the Maghsoud Porcelain Factories Group, using a Philips PW1480 X-ray spectrometer. 4. Ore dressing analyses in Danesh Faravaran Engineering Company. 5. Fluid-inclusion studies in 4 samples doubly-polished wafers of quartz crystals were studied using standard techniques (Roedder, 1984) and Linkam THM 600 heating-freezing stage (from –190 to 600ºC) mounted on a Olympus TH4–200 microscope stage at Ferdowsi University of Mashhad, Iran. Salinities and density of fluid inclusions were calculated using the Microsoft Excel spreadsheet HOKIEFLINCS-H2O-NACL (Steele-MacInnis et al., 2012; Lecumberri-Sanchez et al., 2012) Results and Discussion Fluid Inclusion studies of both mono- and poly- crystalline quartz revealed that the inclusions consist of three phases (LVS) with NaCl crystals. Homogenization temperature is between 484 and more than 600ºC with average 559ºC and the salinity is between 49.6 and 72.1 wt% NaCl with average 61.2 wt% NaCl. These data indicate a magmatic origin according to Kesler (Kesler, 2005) diagram. Homogenization temperature of two phases (LV) inclusions in the metamorphosed quartz is between 287 and 365ºC with average 318ºC. The main source of quartz pebbles is quartz veins formed within the top of pegmatite-granite (upper Triassic) of Khajeh-Morad area and quartz veins formed due to regional metamorphism. Based on chemical analysis of 93 samples which were taken from the surface (channel method) and power drilling, the SiO2 content is more than 98%, TFeO is less than 0.42% and TiO2 is less than 0.16%. The proved ore reserve is more than 50 million tonnes. Using dry magnetic method, the TFeO became less that 0.03% and TiO2 less than 0.02%. Arefi silica deposit is a first-class reserve and can be used in different types of ceramic. Acknowledgments The Research Foundation of Ferdowsi University of Mashhad, Iran, supported this study (Project No. 29057.2). We also thank Danesh Faravaran Engineering Company for the ore dressing analyses. Reference Hasemi, S.F., 2004. Petrology and depositional environment of Jurrasic conglomerate in south of Mashhad. Unpublished M.Sc. thesis, Ferdowsi University of Mashhad, Mashhad, Iran, 200 pp. (in Persian) Kesler, E.S., 2005. Fluids in Planetary Systems: ore-Forming Fluids. Elements, 1(1): 13–18. Lecumberri-Sanchez, P., Steel-MacInnis, M. and Bodnar, R.J., 2012. A numerical model to estimate trapping conditions of fluid inclusions that homogenize by halite disappearance. Geochim Cosmochim Acta, 92: 14-22. Roedder, E., 1984. Fluid inclusions. Reviews in Mineralogy 12, 644 pp. Steele-MacInnis, M., Lecumberri-Sanchez, P. and Bodnar, R.J., 2012. HOKIEFLINCS-H2O-NACL: A Microsoft Excel spreadsheet for interpreting microthermometric data from fluid inclusions based on the PVTX properties of H2O–NaCl. Computers Geosciences, 49: 334–337. <br

Evaluation of Genetic Diversity in Collection from Iranian Jujube Ecotypes (Ziziphus spp.) using ISSR-molecular Marker

Introduction Jujube (Zizyphus jujuba Mill.) as a valuable medicinal plant and adapted to different climatic conditions is widespread in many parts of Iran. Nowadays, beside the export of its fruit, jujube is also used as an herbal medicine to treat the diseases, so it has a high economic value. Study on genetic diversity is the first step to identify and preservation of germplasm. It is also considered as the basic principles of plant breeding. DNA markers seem to be the best way in determination of the genetic diversity. Inter simple sequence repeats (ISSR) markers are highly polymorphic and combine most benefits of Simple Sequence Repeats (SSRs) and amplified fragment length polymorphism (AFLP) to the generality of random amplified polymorphic DNA (RAPD). Materials and Methods In order to study of the genetic diversity among 31 ecotypes collected from eight Jujube-rich provinces, including South Khorasan, Razavi Khorasan, Mazandaran, Golestan, Qom, Isfahan, Lorestan and Fars. Genomic DNA was extracted by CTAB method and polymerase chain reaction (PCR) was performed with 13 ISSR primers in which six most efficient primers were selected. Cluster analysis based on Dice similarity coefficient and Unweighed Pair Group Method with Arithmetic Mean (UPGMA) was carried out and POPGENe.3.2 software was used to determine the similarity of populations with each other. Results and Discussion 84 loci were amplified and 70 of them (83%) revealed a proper polymorphism with the size between 200 and 2000 base pair. The average number of amplified and polymorphic bands per primer was 14 and 11.6 respectively. Primers with di-nucleotide repeats produced more polymorphic bands than ones with tri-nucleotide repeats. It seems that this is due to a higher frequency of sequences containing di-nucleotide repeats in intergenic regions and higher possibility of mutation revealed in more diversity in comparison to gene coding regions. Anchored primers with 1 or 2 nucleotides at the 5’ end make sure annealing only to the ends of SSRs in template DNA, so avoiding internal priming and smear formation. In addition, the anchor lets only a subset of the microsatellites to serve as priming sites. Primers (AC)8YT and (GA)8A with the higher percentage of polymorphism is recommended for further analysis. According to the cluster analysis, the ecotypes could be classified into seven main groups at the 0.85 level of genetic similarity. The most genetic similarity (0.95) was observed between ecotypes from Kalaleh and Doroh and also Noghab and Dustiran and the least genetic similarity (0.48) observed between Kangan and Borzaderan. POPGENe.3.2 software data indicated that populations of Isfahan and South Khorasan had the slightest difference while populations of Isfahan and Razavi Khorasan showed the most difference. Conclusions In general results demonstrated that the total diversity of jujube ecotypes in Iran is summarized in the area of South Khorasan province. Given data showed that South Khorasan has been an original place of cultivation of this medicinal plant, this area could be considered as one of the important centers of jujube diversity. In addition, significant levels of diversity were observed among ecotypes belonging to Isfahan and Mazandaran provinces

Physiological and molecular changes of onion (Allium cepa L.) seeds under different aging conditions

Abstract Background Onion seeds have limited storage capacity compared to other vegetable seeds. It is crucial to identify the mechanisms that induce tolerance to storage conditions and reduce seed deterioration. To address this goal, an experiment was conducted to evaluate changes in germination, biochemical, physiological, and molecular characteristics of onion seed landraces (Horand, Kazerun landraces and Zargan cultivar) at different aging levels (control, three-days and six-days accelerated aging, and natural aging for one year). Results The findings suggest that there was an increase in glucose, fructose, total sugar, and electrolyte leakage in the Horand (HOR), Kazerun (KAZ) landraces, and Zarghan (ZAR) cultivar, with Kazerun exhibiting the greatest increase. The percentage and rate of germination of Kazerun decreased by 54% and 33%, respectively, in six-day accelerated aging compared to the control, while it decreased by 12% and 14%, respectively, in Horand. Protein content decreased with increasing levels of aging, with a decrease of 26% in Kazerun landrace at six days of aging, while it was 16% in Horand landrace. The antioxidant activities of catalase, superoxide dismutase, and glutathione peroxidase decreased more intensively in Kazerun. The expression of AMY1, BMY1, CTR1, and NPR1 genes were lower in Kazerun landraces than in Horand and Zargan at different aging levels. Conclusions The AMY1, BMY1, CTR1, and NPR1 genes play a pivotal role in onion seed germination, and their downregulation under stressful conditions has been shown to decrease germination rates. In addition, the activity of CAT, SOD, and GPx enzymes decreased by seed aging, and the amount of glucose, fructose, total sugar and electrolyte leakage increased, which ultimately led to seed deterioration. Based on the results of this experiment, it is recommended to conduct further studies into the molecular aspects involved in onion seed deterioration. More research on the genes related to this process is suggested, as well as investigating the impact of different priming treatments on the genes expression involved in the onion seed aging process

Transcriptome analysis identified aberrant gene expression in pollen developmental pathways leading to CGMS in cotton (Gossypium hirsutum L.).

Male sterility (induced or natural) is a potential tool for commercial hybrid seed production in different crops. Despite numerous endeavors to understand the physiological, hereditary, and molecular cascade of events governing CMS in cotton, the exact biological process controlling sterility and fertility reconstruction remains obscure. During current study, RNA-Seq using Ion Torrent S5 platform is carried out to identify 'molecular portraits' in floral buds among the Cytoplasmic Genic Male Sterility (CGMS) line, its near-isogenic maintainer, and restorer lines. A total of 300, 438 and 455 genes were differentially expressed in CGMS, Maintainer, and Restorer lines respectively. The functional analysis using AgriGo revealed suppression in the pathways involved in biogenesis and metabolism of secondary metabolites which play an important role in pollen and anther maturation. Enrichment analysis showed dearth related to pollen and anther's development in sterile line, including anomalous expression of genes and transcription factors that have a role in the development of the reproductive organ, abnormal cytoskeleton formation, defects in cell wall formation. The current study found aberrant expression of DYT1, AMS and cytochrome P450 genes involved in tapetum formation, pollen development, pollen exine and anther cuticle formation associated to male sterility as well as fertility restoration of CGMS. In the current study, more numbers of DEGs were found on Chromosome D05 and A05 as compared to other chromosomes. Expression pattern analysis of fourteen randomly selected genes using qRT-PCR showed high concurrence with gene expression profile of RNA-Seq analysis accompanied by a strong correlation of 0.82. The present study provides an important support for future studies in identifying interaction between cyto-nuclear molecular portraits, to accelerate functional genomics and molecular breeding related to cytoplasmic male sterility studies in cotton

Characterization of Aspergillus section Flavi from pistachio soils in Iran

During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Kerman Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, northeastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predominant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p < 0.05). The percentage of sclerotium-producing isolates of A. flavus from Rafsanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2) ranged from 274 to 553 ppb (393±17.11) and 394 to 3745 ppb, respectively, while AFB1, and AFB1 + AFB2 ranged from 257 to 392 ppb (285±13.18) and 415 to 1658 ppb, respectively, in Damghan. We found 16 and 20 vegetative compatibility groups (VCGs) for 41 and 37 nit mutant producing isolates of A. flavus from Rafsanjan and Damghan, respectively. From Damghan the VCG diversity for A. flavus isolates was greater (54%) than from Rafsanjan (39%). Because there were a few number of sclerotium-producing isolates of A. flavus, we did not determine the relationships between sclerotium production with VCGs and/or geographical distribution in the three pistachio production regions. This study was the first to determine the strain and VCG diversity of A. flavus soil isolates from Iranian pistachio orchards

Expression of Hemagglutinin–Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin–Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco (Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein

Characterization of Aspergillus section Flavi from pistachio soils in Iran

Abstract: During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Ker-man Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, north-eastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predomi-nant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p &lt; 0.05). The percentage of sclerotium-producing isolates of A. flavus from Raf-sanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2

An Efficient Protocol for Embryonic Carcinoma Cells P19 Differentiation to Cardiomyocytes Using Oxytocin as Inducer

Background: The capability of embryonic carcinoma cells P19 in differentiation to Cardiomyocyte was examined through inducing effects of Oxytocin (OT) and 5-Azacytidin (5Az) individually and compared with each other in laboratory condition. Materials and Methods: P19 Embryoid Bodies (EBs) was formed through hanging drops method. Then, EBs were treated with (5Az) or (OT) and the EB medium (Ctrl) until 12 days. Morphology and beating number per minute were recorded every two days. Viability was carried out every three days. The expression of several cardiomyocyte-associated genes was assessed by RT-PCR. Results: The beating area percentage of EBs in OT treatment groups was more than that of the 5Az group in all days of experiment. However, only in final stage, a significant increase was observed in beating area of OT group. There was no significant difference in viability and morphological changes. OT induction expressed three more specific proteins in cell culture than 5Az. Conclusion: Statistical analysis revealed that response to OT inducer was more excessive than 5Az in all treatment groups. The Oxytocin was found to be effective inducer of cardiomyocytes differentiation from embryonic carcinoma cells P19 than 5-azacytidine