Tissue specific expression of human calcitonin gene in potato tubers by an organ specific promoter
Hamideh
Ofoghi
Biotechnology Department, Iranian Research Organization for Science and Technology, P.O. Box 15815/3538,
Tehran, I.R. Iran.
author
Fereshteh
Yavari
Biotechnology Department, Iranian Research Organization for Science and Technology, P.O. Box 15815/3538,
Tehran, I.R. Iran.
author
Farhad
Nazarain
Faculty of Agriculture, University of Lorestan, Khorramabad, I.R. Iran.
author
text
article
2012
eng
To increase the production level of heterologous proteins in plants, strategies such as choice of strongerpromoters, optimization of codon usage and specific localization of foreign proteins are of major concern.Calcitonin (CT), a 32 amino acid polypeptide is a powerful and specific inhibitor of bone resorption and isused to treat several human diseases. Calcitonin activity is not species-specific which make it possible toproduce in various animal sources, however, antibody formation in the prolonged application of animal CTleads to gradual decrease or loss of activity. That is why the long term treatment of human patients with CTrequires homologous calcitonin. In this study, a human calcitonin (hCT) gene, driven by two different promoters (granule bound starch synthase I and Cauliflower mosaic virus 35S) was expressed in potato plants, using Agrobacterium-mediated transformation. Molecular analysis, including PCR, RT-PCR, Northerndot blot hybridizations showed that hCT could be successfully transcribed in transgenic potato plants. Theimmunoassay results showed that tissue specific expression in potato, led to almost five-fold more hCTaccumulation when compared to the constitutively expression in all plant tissues.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
79
86
https://www.ijbiotech.com/article_7178_c1cd08e0a484d9ef3790634e64f8f528.pdf
Heterologous expression of the Secale cereal thaumatinlike protein in transgenic canola plants enhances resistance to stem rot disease
Akram
Zamani
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran
author
Mostafa
Motallebi
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
author
Parisa
Jonoubi
Department of Biology, Kharazmi University, P.O. Box 31979-37551, Tehran, I.R. Iran.
author
Nayere Sadat
Ghafarian-Nia
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
author
Mohammad Reza
Zamani
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
author
text
article
2012
eng
Canola (Brassica napus L.) is an important oilseed crop. A serious problem in cultivation of this crop andyield loss, are due to fungal disease stem rot caused by Sclerotinia sclerotiorum. The pathogenesis-related(PR) proteins have the potential for enhancing resistance against fungal pathogen. Thaumatin-like proteins(TLPs) have been shown to have antifungal activity on various fungal pathogens. In this study, the tlp geneisolated from cereal rye (Secale cereal L.) was introduced into canola plants. The amplified DNA fragment(about 500 bp) was analyzed and confirmed by restriction pattern and cloned into pUC19 and designated aspUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains nointron. The tlp gene was predicted to encode a protein of 173 amino acids with an estimated molecular massof 17.7 kDa. The deduced amino acid sequence of TLP showed a significant sequence identity with TLPfrom S.cereal and other plants. We used a transgenic over-expression approach in order to investigate antifungal activity of expressed TLP on Sclerotinia sclerotiorum. TLP was overexpressed under the CaMV35Sconstitutive promoter in (Brassica napus, R line Hyola 308). Transformation of cotyledonary petioles wasachieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerasechain reaction (PCR) and genomic DNA dot blotting. Antifungal activity was detected in transgeniccanola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves oftransgenic canola was significantly retarded when compared to that detected in non-transgenic plants.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
87
95
https://www.ijbiotech.com/article_7173_9de422adcc953788ef26811e48b321ff.pdf
Comparative proteomics analysis of a novel γ-radiationresistant bacterium wild-type Bacillus megaterium strain WHO DQ973298 recovering from 5 KGy γ-irradiation
Mahnaz
Yazdani
Biology Department, Faculty of Science, University of Zanjan, P.O. Box 313, Zanjan, I.R. Iran.
author
Hossein
Naderi-Manesh
Departments of Biophysics and Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14155-111, Tehran, I.R. Iran.
author
text
article
2012
eng
In order to examine radiation-induced proteins in an extremely radio-resistant bacterium, it became possibleto perform comparative proteomic analysis on radio-resistance Bacillus megaterium WHO as a wildtypestrain for the first time. Variation in cellular proteins profiles of the Bacillus megaterium WHO after 5KGy γ-irradiation were analyzed by two-dimensional poly acryl amide gel electrophoresis and silver staining.Although many spots were decreased in density, our primary focus was on the induced spots. Theexpression level of 48 protein spots showed significant increase under radiation stress. Of these spots, 45were identified with MALDI TOF-TOF (peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry) after tryptic in-gel digestion. These proteins exhibitedvarious interesting cellular functions including: (i) transcription (ii) translation (iii) signal transduction (iv) carbohydrate transport and metabolism (v) energy production and conversion (vi) nucleotide transport andmetabolism (vii) posttranslational modification, protein turnover and chaperones (viii) DNA replication, recombination and repair (ix) bacterial general stress response and (iix) different and some still unknownfunctions. The appearance of four spots (24, 27, 30 and 36) in response to γ-irradiation was the distinctresult of present study. These proteins appear to mediate processes related to ionizing radiation resistanceand clearly demonstrate that Bacillus megaterium WHO, significantly has mechanisms contribute to theionizing radiation resistance.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
96
105
https://www.ijbiotech.com/article_7181_e527a452620c921d14fa68ea64b54815.pdf
Construction of a synthetic vector for preparation of a 100 base pair DNA ladder
Mohammad
Rashno
School of Veterinary Medicine, Shahid Chamran University, P.O. Box 61355-145, Ahvaz, I.R. Iran.
author
Masoud Reza
Seyfi Abad Shapouri
Department of Pathobiology, School of Veterinary Medicine, Shahid Chamran University, P.O. Box 61355-145, Ahvaz, I.R.
Iran.
author
Abbas
Jolodar
Department of Basic Sciences, School of Veterinary Medicine, Shahid Chamran University, P.O. Box 61355-145, Ahvaz, I.R. Iran.
author
text
article
2012
eng
DNA size markers are widely used to estimate the size of DNA samples on agarose or polyacrylamide gelelectrophoresis (PAGE). DNA markers can be prepared by mixing PCR products with definite sizes.Alternatively, they are prepared by restriction enzyme digestion of the genomic DNA of bacteriophages ornatural and synthetic DNA plasmids. The present study describes engineering of a synthetic plasmidwhich produces a 100 bp DNA ladder, a popular DNA size marker, upon digestion with a single restrictionenzyme. Our strategy consisted on sequential cloning of ten PCR products of 100 to 1000 bp in plasmidpTZ57R, using the BamHI and BglII restriction enzymes and releasing the fragments from the recombinantplasmid by enzyme EcoRV. This strategy could be applied to construct various complex synthetic vectorsto produce different DNA ladders.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
106
110
https://www.ijbiotech.com/article_7183_3df1f424069d4cd00bb82afc28a74d4c.pdf
Isolation, purification and characterization of proline dehydrogenase from a Pseudomonas putida POS-F84 isolate
Hamid Shahbaz
Mohammadi
Department of Biochemistry, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, I.R. Iran.
author
Eskandar
Omidinia
Department of Biochemistry, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, I.R. Iran.
author
text
article
2012
eng
The purpose of this study was to isolate and characterize Proline Dehydrogenase (ProDH) enzyme frommicroorganisms isolated from soil in Iran. Isolation and screening of L-proline degradative enzymes from soilsamples was carried out. The isolate was characterized by biochemical markers and 16S rRNA geneanalysis. The target ProDH was purified and the effects of pH and temperature on the activity and stabilitywere tested. Among the 150 isolates recovered from 30 soil samples, only one was identified asPseudomonas putida displayed the highest enzyme activity toward L-proline (2200U/l). The enzyme wasidentified as a ProDH and had Km value of 35 mM for L-proline. The molecular mass of the purified ProDHwas about 40 kDa, and determined to be a monomeric protein. The N-terminal amino acid sequences of thesubunit of P. putida enzyme were determined to be MLTSSLTRIIGKSGE. ProDH exhibited high activity attemperature range of 25 to 35ºC and the highest activity was achieved at 30ºC. It was almost stable at temperatures between 25-30ºC for 2 h. The optimum pH of ProDH activity was determined in pH=8.5 and it was stable in pH range of 8.0-9.0 up to 24 h. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. Briefly, a ProDH flavonzyme was purified and characterized from a P. putida bacterium. Thespecificity of P. putida enzyme toward L-proline is advantageous for the application to the L-prolineanalysis.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
111
119
https://www.ijbiotech.com/article_7184_b1f57d714094e1e1b75c446c35a41f26.pdf
Purification and characterization of a thermostable neutrophilic metalloprotease from Pseudomonas sp. DR89
Ahmad
Asoodeh
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, P.O. Box 9177948974,
Mashhad, I.R. Iran
author
Hossein
Mohammadian Musaabadi
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, P.O. Box 9177948974, Mashhad, I.R. Iran.
author
text
article
2012
eng
A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified ina mineral spring in Iran. The enzyme was purified from the isolate to 21-folds in a three-step procedure involving ammonium sulfate precipitation, Q-Sepharose ionic exchange and Sephadex G-100 gel filtrationchromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresisanalysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity wasdecreased by Zn2+, Mn2+, H2O2 and cetyl trimethylammonium bromide (CTAB), whereas its activity wasincreased by Ca2+, Mg2+, Cu2+ and dimethyl sulfoxide (DMSO). Na+, phenylmethyl sulfonylfluoride (PMSF),β-mercaptoethanol, sodium dodecyl sulfate (SDS), and Triton X-100 did not show a considerable effect onits activity. Casein was a better substrate than bovin serum albumin (BSA) and gelatin for this enzyme. Thekinetic parameters (Km and Vmax) of the purified protease towards caseinolytic activity were also determined.These properties of the enzyme make it suitable for use in food industries.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
120
128
https://www.ijbiotech.com/article_7182_f60c62442c75f030490d7927185764ba.pdf
Association of prolactin and prolactin receptor gene polymorphisms with economic traits in breeder hens of indigenous chickens of Mazandaran province
Hamed
Rashidi
Laboratory for Molecular Genetics and Animal Biotechnology, Department of Animal Science, Faculty of Animal
Science and Fisheries, Sari Agricultural Science and Natural Resources University, P.O. Box 578, Sari, I.R. Iran.
author
Ghodrat
Rahimi-Mianji
Laboratory for Molecular Genetics and Animal Biotechnology, Department of Animal Science, Faculty of Animal
Science and Fisheries, Sari Agricultural Science and Natural Resources University, P.O. Box 578, Sari, I.R. Iran.
author
Ayoub
Farhadi
Laboratory for Molecular Genetics and Animal Biotechnology, Department of Animal Science, Faculty of Animal
Science and Fisheries, Sari Agricultural Science and Natural Resources University, P.O. Box 578, Sari, I.R. Iran.
author
Mohsen
Gholizadeh
Laboratory for Molecular Genetics and Animal Biotechnology, Department of Animal Science, Faculty of Animal
Science and Fisheries, Sari Agricultural Science and Natural Resources University, P.O. Box 578, Sari, I.R. Iran.
author
text
article
2012
eng
Polymorphisms in 5’-flanking region of prolactin (PRL), exon 2 and exon 5 of prolactin receptor (PRLR) genesand its association with growth and egg traits were examined in breeder hens of Mazandaran native fowlsbreeding station. A single nucleotide polymorphism at site C-2402T and a 24 bp nucleotide sequence insertionat situation -382 in 5’-flanking regions of PRL gene were identified. PCR amplification together withRestriction Fragment Length Polymorphism (RFLP) and direct agarose gel electrophoresis were used to identify different genotypes at C-2402T and a 24 bp indel (insertion-deletion) at the site of -358 of PRL gene, respectively. Nucleotide substitution of C to T and a 24 nucleotides insertion (I) or deletion (D) in promoterregion of PRL gene resulted in three genotypes with the frequency of CC (0.10), CT (0.84), TT (0.06)and II (0.39), ID (0.40), DD (0.21), respectively. There were no heterozygous females and only two genotypes A/A (0.54), B/B (0.46) and AA (0.72), BB (0.28) were identified in exon 2 and exon 5 of PRLR geneusing PCR-Single Strand Conformation Polymorphism (PCR-SSCP) and PCR-RFLP analyses, respectively. Anovel mutation consists of a BamHI restriction site found in the exon 5 of PRLR gene. The results showedsignificant association between SNP in exon 2 with body weight at hatch, age at sexual maturity, andbetween SNP in exon 5 and egg number. Individuals with AA genotype produced higher eggs than BB genotype (P<0.05). These results showed that the PRLR locus can be considered as a major gene that mayinfluence the production traits in chicken.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
129
135
https://www.ijbiotech.com/article_7185_dcb1ab94e41843501315de5e542d4967.pdf
Genetic variability of calpastatin and calpain genes in Iranian Zel sheep using PCR-RFLP and PCR-SSCP methods
Elena
Dehnavi
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
4913815739, Gorgan, I.R. Iran.
author
Mojtaba
Ahani Azari
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
4913815739, Gorgan, I.R. Iran.
author
Saeed
Hasani
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
4913815739, Gorgan, I.R. Iran.
author
Mohammad Reza
Nassiry
Department of Animal Science, Ferdowsi University of Mashhad, P.O. Box
9177948978, Mashhad, I.R. Iran.
author
Mokhtar
Mohajer
Golestan Agriculture Jahad, P.O. Box 49174, Gorgan, I.R. Iran.
author
Ali Reza
Khan Ahmadi
Department of Animal Science, Faculty of Gonbad, P.O. Box 4971799151, Gonbad, I.R. Iran.
author
text
article
2012
eng
The genotypes for calpastatin (CAST) and calpain (CAPN) loci were determined by PCR-RFLP and PCRSSCPmethods. Blood samples were collected from 200 pure-bred Zel sheep from Shirang’s Zel sheep Breeding Station located in south-west of Golestan, Iran. Extraction of genomic DNA was performed based on the modified salting out method. Based on results, two investigated loci were polymorphic and had differentgene variants. Heterozygosis was low for both loci. Chi-square test confirmed Hardy-Weinberg equilibriumonly in CAST locus using SSCP method. Detected polymorphisms and associations of genetic variationwith meat production and tenderness may help to find the effective genotypes of Zel sheep for the economictraits.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
10
v.
2
no.
2012
136
139
https://www.ijbiotech.com/article_7186_89811a119d0a52c54948579103ebee61.pdf