حمید رجبی معماری

The use of Spirulina platensis has been expanded in various fields. The main goal of this research is to optimize the growth conditions of this microalgae to reduce costs and increase the benefits of its mass production in seawater. It is also feasible to lessen environmental pollution by producing more spirulina by discovering the best growing conditions for it through the enrichment of Persian Gulf water.

First, the growth conditions of Spirulina platensis were optimized based on three factors: temperature, light, and pH. The temperature factor included four treatments, the light factor included four treatments, and the PH factor included five treatments. Also, Spirulina platensis microalgae were cultured in optimal growth conditions in Persian Gulf water, seawater enriched with 5% Zarrouk, 10% Zarrouk, with urea, and pure Zarrouk culture medium.

The best temperature range for the growth of Spirulina platensis was 27–32 degrees Celsius. Also, the best growth was achieved in 16 hours of light and 8 hours of darkness; for the PH factor, the most appropriate value was determined between 10.72 and 11.47. Also, in the cultivation of Spirulina platensis, optimal conditions for the growth of Spirulina microalgae were obtained in the medium of Zarrouk, urea, 10% Zarrouk, 5% Zarrouk, and seawater, respectively.

Therefore, the growth of Spirulina platensis in Persian Gulf water is slow and shows little performance, and by enriching this water with the mentioned factors, the microalgae enter the logarithmic phase faster and show better performance.

Background and Objectives:

 Khuzestan is the only province with sugarcane cultivation. One of the limiting factors in the production of this crop is the occurrence of frost damages in some years. Since no comprehensive research has yet been conducted on detection and evaluation of bacteria with the potential to form nuclear ice in frostbite, this study was carried out to evaluate this subject.

 Materials and Methods:

 This study was carried out in the laboratory of Institute for Research and Training of Sugar and Auxiliary Industries. Endophytic and epiphytic strains of bacteria were isolated from sugarcane. To test the freezing potential of representative strains, four tests including, "determination of Ice nucleation activity by freezing in the tube", "freezing droplet test at -20 °C", "Ice nucleation test on sugarcane cut leaves in vitro" and "Ice nucleation test on sugarcane plant in greenhouse conditions" were performed. Also the genes of the nucleus were detected by 3076f / 3463r and 3308f / 3463r specific primers. 

Results:

 Results show that bacteria species B. gladioli, B. fungorum, B. cantaminans, M. huakuii, O. ciceri, M. proteolyticum, M. Foliorum, R. solanacearum , R. picketii and X. campestris had different degrees of symptoms. In addition, the genes of the nucleus were detected by 3076f / 3463r and 3308f / 3463r specific primers. For all of above mentioned bacterial strains, specifically strain X. campestris, no references have been reported of ice nucleation activity. To assess susceptibility or resistance of sugarcane, cultivar CP69-1062 were the most susceptible and highly damaged in the tests including "Ice nucleation test on sugarcane cut leaves in vitro" and "Ice nucleation test on sugarcane plant in greenhouse conditions". In addition, CP57-614 and CP48-103 cultivars showed minimum variation in frosting in compression to CP69-1062 cultivar. Two cultivars including CP73-21 and SP70-1143 exhibited a high degree of resistance to frostbite.

 Discussion:

 Due to the high variation among Ice-Nucleating bacteria and the sensitivity of commercial cultivars, our finding can be a suitable option for producers and encourage them to use resistant cultivars in the field.

Sugarcane Mosaic Virus (SCMV) is one of the important diseases of the sugarcane. In this study, the infection of sugarcane mosaic virus in imported and existing cultivars was screened based on nucleic acid-based molecular methods. During 2014–15, samples showing disease symptoms were collected from 90 different cultivars grown in six sugarcane agro-industries and their partial blades were separated. The samples were freeze-dried and powdered in liquid nitrogen. Suitable primers were designed to amplify 1040 bp from the nuclear inclusion B and coat protein gene sequences using the RT-PCR method. Ten out of 90 surveyed samples were SCMV-infected and produced expected-size fragments. These samples belonged to the cultivars of IRC99-06, V58-4, and Q58 from Sugarcane Research and Training Institute for the Development of Industries in Khuzestan, IRC99-09, IRC00-21, and 4380-3 from Salman Farsi agro-industry, CP80-1557 and V68-74 from Imam Khomeini agro-industry and two uncertain cultivars. Four out of 10 detected samples were directly sequenced and deposited in GenBank. A BLASTn search of the four detected isolates, including Kh10, Kh41, Kh44, and Kh28, showed the highest identity to isolates from Iran and Argentina. Phylogenetic tree constructed using the maximum likelihood algorithm showed that the Iranian isolates were probably originated from Argentina and China.

Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans. An effective way to treat and prevent the disease is vaccination. Since production of conventional HBV vaccines is very expensive, use of transgenic plants as an alternative bioreactor has recently become of interest to many researchers. In this study, the HBsAg gene has been transferred to pepper plants (Nicotiana tabacum) through the leaf disk technique. The recombinant plant expression vector, pCAMBIA containing HBsAg was cloned into E. coli strain JM107 and was then introduced into Agrobacterium tumefaciens strain LBA4404. Young tobacco leaves were used as explants and co-cultivated with A. tumefaciens. The transformants were regenerated on selection medium containing 1mg.l-1 BAP, 0/1mg.l-1 NAA, 500 mg.l-1 cephotaxim and 15 mg.l-1 hygromycin. After the growth of plantlets (about 15 cm), genomic DNA was extracted from putatively regenerated plants by the CTAB method. The presence of HBsAg gene in transgenic plants was detected using PCR analysis. Finally expression of HBsAg gene was tested via RT-PCR analysis.

RNA extraction is considered a crucial step in the molecular studies and the results of subsequent work such as, PCR, Real-TIME PCR and RACE PCR depend on the quality and quantity of the extracted RNA. Some standard methods are available in total RNA extraction of plant tissue, which are effective for the ordinary plants. But it is difficult to extract the total RNA from the plants with high levels of carbohydrates and secondary metabolites such as phenolic compounds. Yarrow is one of the valuable medicinal and industrial species of the pasture of Iran that contains high levels of secondary metabolites. So far, there is no report on a suitable method for extraction of total RNA from it.
Material and Total RNA extraction was done through three methods including RNXTM(-Plus), Lithium Chloride and Phenol-Chloroform. Then the quality and quantity of extraction RNA was evaluated.
The results showed that among the three methods, extracted RNA with phenol-chloroform had high quantity and quality as the A 260/280 ratio was well within the accepted range of 1.8-2.
Using phenol-chloroform method tends to remove polyphenols and polysaccharides from plant tissue cell contents and causes more efficient RNA extraction. Given of this, the phenol-chloroform method is recommended for total RNA extraction of valuable plant Yarrow in future studies.

RNA extraction from hard wood tissues, especially in coniferous family trees, is very hard because they have high concentrations of phenolic compounds, polysaccharides, secondary metabolites and lignin. Moreover, up to now, no commercial kit is made for the mentioned purpose. In this study, the problems were resolved and a rapid, simple, and economic protocol is presented for RNA extraction from bark tissue of Cedrus deodara and Abies grandis, which belong to conifer family. Ratios of optical density of 260/280 nm and 260/230 nm were respectively obtained as 1.88 and 1.1 for Cedrus deodara and 1.8 and 0.9 for Abies grandis. Modifications for optimization of RNA extraction were preheating treatment of extraction buffer, and decreasing centrifuge rounds to eliminate DNA contaminations and DEPC. Synthesis of cDNA was performed using extracted RNA. RT-PCR successfully produced expected bands. Therefore, it seems that the presented protocol is suitable for RNA extraction from woody tissues.

Phycocyanin is a blue pigment in two eukaryote algal genera and in cyanobacteria as Spirulina. This pigment has various biology activities and are utilised in a number of applications in foods, cosmetics and pharmaceuticals. A lot advantage of phycocyanin studied by many researchers but the scale-up of these methods is difficult and expensive while production of recombinant phycocyanin is more convenience and inexpensive to scale up protein desire. The purpose of this study was to isolation and cloning of phycocyanin alpha subunit gene in expression vector and production of recombinant protein in E.coli to provide industrial production of phycocyanin. The genomic DNA of Spirulina platensis was prepared and used for PCR as template. phycocyanin alpha subunit gene amplified by designed specialize primers was cloned in a pET43.1a expression vector, under the control of T7 promoter using NdeI and NotI restriction enzymes. The cloning of phycocyanin alpha subunit gene is confirmed by colony PCR, digestion and DNA sequencing. The constructs were transformed into E.coli strain BL21 (DE3). Expression of phycocyanin alpha subunit gene was examined by 12.5 % SDS-PAGE analysis at 8 hrs after induction by IPTG. The SDS-PAGE analysis showed that alpha subunit phycocyanin was produced in E.coli expression system. Our study provided the production of recombinant phycocyanin. Also overexpression of the synthetic alpha subunit phycocyanin in a bacterial system (E.coli BL-21) showed that E.coli can be used to produce this desire protein in large quantity.

Yarrow (Achillea millefolium) is an herbaceous and perennial plant species which belongs to the Asteraceae family. Yarrow's essential oil has different compounds of monoterpene and sesquiterpens, which its main constituents are pinene and linalool. These compunds have anti-microbial and anti-pest activities and also can be used in the food industry, perfumery and cosmetics.The aim of the present study was to use the degenerate primers approach in order to isolate Pinene synthase and linalool synthase genes from Yarrow plant. Up to date, there is no any report on the availability of these genes in the world gene bank. In this investigation the total RNA was extracted from Yarrow then pinene synthase and linalool synthase genes were isolated, using degenerate primers and Polymerase Chain Reaction (PCR). PCR amplified two bands of 250 bp and 720 bp. The sequence data were compared with NCBI gene bank data. The results of diversity study among varieties and families based on Pis and Lis genes showed most similarity between Achillea and Artemisia plants. Also this similarity was seen between Asteraceae and Lamiaceae families and these families grouped together same group. These results also showed a relatively high similarity of Pis and Lis with some other plants which confirmed sequencing data.

Sugarcane is one of the most important industrial plants. There are many ways to improve the agronomic traits of this plant, such as genetic engineering and gene transfer methods. These modern techniques depend on optimizing the regeneration of plants from callus tissue. In this study, we attempted to optimize all different stages of tissue culture for regenerating the plants from callus tissue derived from terminal roll lives of two commercial sugarcane cultivars, CP48-10 and CP69-1062. Two types of cutting were applied, ring cutting and half-cylinder cutting from roll leaves. Among these cutting types, ring cutting from the roll leaves gives better results. Using solid MS medium with 3 mg per liter 2,4-D could attain the maximum callus production. To produce shoots from callus, the hormone combination of 1 mg per liter BAP and 2 mg per liter Kinetin showed the best response. Seedlings produced in MS medium containing 5 mg per liter IBA, 1 mg per liter Kinetin and 3 mg per liter NAA accompanied by 3 g per liter activated charcoal rendered the most root production. In the treatments with activated charcoal, the regenerated seedlings had superiority for the traits, days to root emergence, root length and number of roots in plants.

The most important composition character within a plant breeding program is to have type and amount of genetic diversity. In addition to using a visual approach and genetic tools to discern plant variation, application of statistical methods can be utilized to screen and classify genetic differences among plants within a plant population. A was study set up asarandomized complete block design with three replications to evaluate the geneticdiversity of ten different dill (Anethuprogrammm graveolens) germplasms. Analysis of variance showed significant differences (α = 0.01) among germplasms regarding the plant characters. Results elucidated the existence of great morphological/phytochemical variation among these germplasms. Application of principle components analysis has led the germplasms into four main classes based on the characters growth performance. Although, germplasms have geographically different growing conditions, some of those classified in a similar group according to their growing and plant diversity. Further analysis could categorize some of germplasms such as Varamin, Hamadan and Dezful into a distinct group having the highest rate of Carvon concentration in theirtissues, especially in the seeds.

The aim of the current study is to amplification the regularity region encompassed the mutations changing the Myostatin gene express and also to determine sequential analysis. The blood samples taken from 12 Najdi Cattles in Najdi Cattle station in Shushtar city، Khuzestan province. Then the DNA extracted to amplification 730bp and 561bp fragments. Sequencing was performed after the precision in PCR products put-upon Agarose gel 1% and Myostatin gene pro-motor was determined. In Myostatin gene’s pro-motor region، three mutations، Insertion، Deletion and Substitution in 561bp fragment and the two mutations، Deletion and Substitution in 730bp were appeared، results pointed out. Reported mutations on boxes and pro-motor components did not influence upon Myostatin gene.

The production of human gamma interferon in eukaryote expression systems refers as a therapeutic recombinant protein which has significant impact in medical studies. Unique com position of gamma interferon makes such protein as a suitable tool against cancer. It is documented that phosphinothricin (PPT) classified as non-selective herbicide group of bialaphos acts an inhibitor for glutamine synthetase. The bar gene is encoding the phosphinothricin-N-acetyltransferase (PAT) enzyme. This enzyme capable of boosting resistance against PPT herbicide¡ thus it can be selected as a selective marker within plant population. Then various colony PCR techniques¡ enzymatic digestion and sequencing were used to confirm the accuracy of fusion of IFNγ-bar gens within expression transporter. Using freezing and thawing method to transfer the pCAMBIA1305.1- IFNγ-bar construction into strain of LBA4404 of agrobacterium¡ then disc leaves was used to integrate into the genomic of tobacco plant. The transgenic plants were selected under selector condition which possess 30 mg/l of hygromycin. After the developed roots were transferred into soil¡ and PCR technique was used to confirm the presence of IFNγ-bar in the genomic of plants. Dot blot analysis was applied to detect IFNγ-bar protein in transgenic of to tobacco plants.

Interferons are part of the body''s defense system against viruses. These proteins are generated in the body، and then they trigger the cells around them to produce inhibitor proteins against virus replication. The purpose of this study was cloning and expression of the human interferon alpha-2bgene in preplasmic space of Escherichia coli. Subjects and

In this research، Alpha 2b interferon gene was cloned in a pET-26b (+) expression vector، under the control of T7 promoter and pelB periplasmic signal peptide sequence using NcoI and XhoI restriction enzymes. The expression vector was transformed into Escherichia coli strain BL21 (DE3). Cloning of alpha interferon gene confirmed using colony PCR، digestion and DNA sequencing. Gene expression of alpha interferon was examined by SDS-PAGE and Dot blot analysis. Also the possibility of periplasmic targeting was determined using SDS-PAGE and dot blot analysis at 15 hrs after induction.

The results showed that Alpha 2b interferon was produced in bacterial expression system and targeted to periplasmic space.

Our study showed that the production of pharmaceutical recombinant protein in preplasmic space of bacterial expression system is fissible and can be ustilized as a cheaper source and more efficient than conventional expression systems.

Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression، biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band، which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

In this study, Fusarium root rot from Tubers in Dezful (Khuzestan, Iran) were identified and genetic diversity of domnnat species was determined. Root Associated fungi were isolated using common isolation technique in laboratory. Collectively 143 isolates of fungi including 110, 27, and 6 at 4 species of Fusarium oxysporum, F. solani, F.equiseti were recovered and identified respectively. Genetic diversity of the population of F.oxysporum was determined using VCG and RAPD techniques. For VCG method, 45 isolates of fungus were selected randomly. Then nit mutant were generated on MMC and Czapeck media each containing 3% KClO3. Phenotypic classes of nit mutants were determined according of the growth types on basal medium containing one of four nitrogen sources (Nitrate, Nitrate, Hypoxanthine, and Ammonium). From recovered nits, 301, 171, and 45 were nit 1, nit 3 and nit M respectively. Complementation test was conducted among different nit mutant of different isolates in all combinations. Results revealed that all isolates were placed into 4 VCG groups, the largest one containing 28 and the rest containing 10, 3 and 4 isolates respectively. In addition, genetic diversity of theses isolates was studied using thirteen primers. Cluster analysis of RAPD data was done using UPGMA, Single and Complete methods. The best results obtained by UPGMA and dice coefficient, which distinguished six main groups at 61% similarity level. In this grouping, group I, III and VI had 2 members each, group II had 12 members. Ten of that belonging to VCG a and groups IV and V had one member each. No close relation was observed between VCG and RAPD method results. Pathogenicity test that was conducted using selected isolates of different VCGs revealed that all were pathogenic to plant. This research is first report of genetic diversity of F. oxysporum on Tuberose in Iran.

There are many problems with most of the available diagnostic tests used to diagnose Legionella pneumonia, including inadequate sensitivity and specificity, and inability to provide a result in a clinically useful time period. Legionella pneumophila PAL protein has been considred as a target for detecting of Legionella infection from urine specimen, because it is conserved sequence and is secreted into the urine. The aim of this study was to optimize expression and purification of L. pneumophila PAL protein. In this experimental study, optimizing of 5 parameters (cell density, induction time, growth temperature, IPTG concentration and type of medium) was performed. After expression, periplasmic extract was prepared and recombinant PAL protein purified using Ni2+-charged resin column. Finally, recombinant PAL protein was verified by Western blotting. In terrific broth medium, the optimum condition of r-PAL protein induction was occurred at an OD600 of 0.6, 1mM IPTG concentration and 15 hours incubation at 25ºC Recombinant periplasmic PAL protein was highly purified (>80%) using Ni-NTA column. Western blotting analysis showed that recombinant PAL protein was also specifically recognized by anti-His6-peroxidase antibody. By purification of recombinant PAL protein in purity greater than 80% it can be used to evaluate its capacity in diagnosis of Legionella infection and preparation of diagnostic kit.

Root and crown rot of corn and rape seed is one of the most important disease attack to these crops at various stages of their growth and cause considerable damage to these crops. For study and identification of the Cylindrocarpon species associated with corn and rape seed plants in Khuzestan province, this study was conducted during growing season of 2006-2007. Samples were collected from different field in different region of Khuzestan In that study 207 isolates of Cylindrocarpon were recovered. isolates belonged to the 5 species which based on there frequency are as follow: C. destructans (96), C. didymum(69), C. hederae(25),), C. obtusisporum(14), C. macrodidymum (3). This is the first report of the association of these species of Cylindrocarpon with corn and rape plants in the World. Cylindrocarpon isolates specific primers ITS4 & ITS5 were used. specific primers amplified a 500-560 bp band which is a confirmation of Cylindrocarpon species. Comparsion of the generated sequences from the mentioned species with the avalaible sequences in the gene Bank, using Blast Browers indicated that all of the generated sequences beloneged to the Cylindrocarpon species. Cylindrocarpon grouping using ITS sequences was corresponding to the classification of this geneus based on morfhological characteristics. This showen that sequences determination of ribosomal its rigon is a valuable tool in the assessment of the phylogenetic relation of Cylindrocarpon species.

Spinach is one of edible leafy vegetable in many countries worldwide and is the most prime and informative plant species for studying plant chloroplast. A main advantage of spinach which makes it remarkable among other plants is that its leaf possesses abundant number of chloroplasts and each chloroplast sustains a very large genomic molecule. This characteristic of spinach makes it a unique crop that can serve as a valuable genetic model for studying chloroplast manipulation at the molecular genetic level. Therefore, implementing a technique which can lead to identify an appropriate way to induce adequate plant regeneration from explants has become significantly important with a great demand. A study was conducted to examine optimization of callogenesis and regeneration of two spinach cultivars; Orai and Viroflay using MS based medium with various combination of plant growth regulators (PGRs); IAA, 2,4-D and GA3 at different concentration rates in order to evaluate regeneration induction of leaf, hypocotyl and cotyledon explants under three separate experimental studies. The results indicated that leaf explants from both cultivars were capable of producing the highest callogenesis on growth media which supplemented with 0.5 mg/l 2,4-D and 2 mg/l Kinetin. When calli was transferred into medium containing concentration of 0.1, 2 and 1 mg/l of 2,4-D, Kinetin and GA3 respectively, only the explants of Orai cultivar was able to induce regeneration. Also, hypocoyl drived calli from Viroflay cultivar showed the best regeneration when transferred on medium containing 2 mg/l IAA and 3.4 mg/l GA3. There was no regeneration from cotyledon explants of both cultivars on all media.

According to the wide range of monoclonal antibodies applications in distinction and treatment of diseases, their production from safe, permanent and inexpensive sources has a great importance. The camel-based single-domain antibody (VHH) has individual characteristics such as: similarity to human VH fragment, solubility, high affinity and specific attachment to the antigen. So it has preferred to the other kinds of antibodies. In this report the antibody gene against MUC1 was transferred into Canola plants by agrobacterium mediated transformation method. The transformed plants initially selected on kanamycin (10-25 mg l-1). The presence and expression of the transgene was confirmed in transformants by PCR and SDS-PAGE. This study is an attempt for transfering an antibody gene to Canola and preparing the best field for production of other recombinant proteins in this plant.

“Molecular farming” is the use of plants، and potentially animals، as the means to produce therapeutic and industrial valuable proteins using genetic engineering techniques. Firefly luciferase is one of the most important industrial enzymes which has various applications in biotechnology and molecular biology such as microbial contamination detection by ATP amount، preparation of cancer diagnosis kits، drug screening، biosensors، protein folding and protein-protein interaction examinations، pyrosequencing، etc. luc is also used as an ideal reporter gene in order to optimize gene transformation systems in genetic engineering. In this study، Iranian firefly Lampyris turkestanicus luciferase gene (luc) was cloned and introduced into tobacco plants. The gene was isolated by PCR and appropriate primers، designated according to luc termini، restriction sites and a plant highly expression sequence (Kozak sequence)، and then cloned in pCAMBIA1304 vector، under the control of CaMV35S promoter and NOS terminator. The construct was confirmed by several colony PCR، PCR، digestion، sequencing and BLAST analysis. The Agrobacterium–mediated transformation method was used to introduce luc into tobacco genome and putative transgenic plants were selected on the selective medium containing hygromycin and cefotaxim. Finally، transgenic plants were confirmed by PCR analysis on transgenic and control plants’ genomic DNA.

By using molecular farming, plants are used as a bio-reactor for producing low costrecombinant proteins. This technology had been successful in the production of primaryproducts in the commercial field. Other recombinant products are in final steps of production.In the last 10 years, up to 100 different recombinant proteins are produced in transgenicplants. Plants have many advantages in comparison with other expression systems, especially in economic, safety, operations and production aspects. However, there are some problems for using plants as a bio-reactor with the goal of recombinant proteins production that should be considered. Some of these problems are: the quality of final product, extraction and the processing of plant derived pharmaceutical macromolecules and bio-safety. In this study, we will review the global view of molecular farming and some successful cases in Iran. During 8 years of research in the field of molecular farming in Iran, especially in Tarbiat Modarres University, different kinds of recombinant proteins such as VHH single domain antibody in tobacco and canola and tPA protein in tobacco were expressed. The human gamma interferon in canola and tobacco and also the luciferase enzyme in tobacco, African violet and canola were produced. Also, useful projects are executed in the field of transplastomic plants in order to express the mentioned genes in the chloroplast. In transferring the human pro-insulin gene to chloroplast genome, this gene was bombarded onto tobacco leaf ex-plants using a particle delivery system. Leaf ex-plants produced adventitious shoots when cultured onshoot-inducing medium containing spectinomycin. The results of PCR confirmed that thepro-insulin gene was present in the chloroplast genome.