Iranian Journal of Biotechnology
Volume:10 Issue: 2, Spring 2012

To increase the production level of heterologous proteins in plants, strategies such as choice of stronger promoters, 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 is used to treat several human diseases. Calcitonin activity is not species-specific which make it possible to produce in various animal sources, however, antibody formation in the prolonged application of animal CT leads to gradual decrease or loss of activity. That is why the long term treatment of human patients with CT requires 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, Northern dot blot hybridizations showed that hCT could be successfully transcribed in transgenic potato plants. The immunoassay results showed that tissue specific expression in potato, led to almost five-fold more hCT accumulation when compared to the constitutively expression in all plant tissues.

Canola (Brassica napus L.) is an important oilseed crop. A serious problem in cultivation of this crop and yield 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 gene isolated 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 as pUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains no intron. The tlp gene was predicted to encode a protein of 173 amino acids with an estimated molecular mass of 17.7 kDa. The deduced amino acid sequence of TLP showed a significant sequence identity with TLP from 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 CaMV35S constitutive promoter in (Brassica napus, R line Hyola 308). Transformation of cotyledonary petioles was achieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerase chain reaction (PCR) and genomic DNA dot blotting. Antifungal activity was detected in transgenic canola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves of transgenic canola was significantly retarded when compared to that detected in non-transgenic plants.

In order to examine radiation-induced proteins in an extremely radio-resistant bacterium, it became possible to perform comparative proteomic analysis on radio-resistance Bacillus megaterium WHO as a wild-type strain. Variation in cellular proteins profiles of the Bacillus megaterium WHO after 5 KGy g-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. The expression level of 48 protein spots showed significant increase under radiation stress. Of these spots, 45 were 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 exhibited various interesting cellular functions including: (i) transcription (ii) translation (iii) signal transduction (iv) carbohydrate transport and metabolism (v) energy production and conversion (vi) nucleotide transport and metabolism (vii) posttranslational modification, protein turnover and chaperones (viii) DNA replication, recombination and repair (ix) bacterial general stress response and (iix) different and some still unknown functions. The appearance of four spots (24, 27, 30 and 36) in response to g-irradiation was the distinct result of present study. These proteins appear to mediate processes related to ionizing radiation resistance and clearly demonstrate that Bacillus megaterium WHO, significantly has mechanisms contribute to the ionizing radiation resistance.

DNA size markers are widely used to estimate the size of DNA samples on agarose or polyacrylamide gel electrophoresis (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 or natural and synthetic DNA plasmids. The present study describes engineering of a synthetic plasmid which produces a 100 bp DNA ladder, a popular DNA size marker, upon digestion with a single restriction enzyme. Our strategy consisted on sequential cloning of ten PCR products of 100 to 1000 bp in plasmid pTZ57R, using the BamHI and BglII restriction enzymes and releasing the fragments from the recombinant plasmid by enzyme EcoRV. This strategy could be applied to construct various complex synthetic vectors to produce different DNA ladders.

The purpose of this study was to isolate and characterize Proline Dehydrogenase (ProDH) enzyme from microorganisms isolated from soil in Iran. Isolation and screening of L-proline degradative enzymes from soil samples was carried out. The isolate was characterized by biochemical markers and 16S rRNA gene analysis. The target ProDH was purified and the effects of pH and temperature on the activity and stability were tested. Among the 150 isolates recovered from 30 soil samples, only one was identified as Pseudomonas putida displayed the highest enzyme activity toward L-proline (2200U/l). The enzyme was identified as a ProDH and had Km value of 35 mM for L-proline. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein. The N-terminal amino acid sequences of the subunit of P. putida enzyme were determined to be MLTSSLTRIIGKSGE. ProDH exhibited high activity at temperature 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. The specificity of P. putida enzyme toward L-proline is advantageous for the application to the L-proline analysis.

A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified in a 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 filtration chromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of 5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity was decreased by Zn2+, Mn2+, H2O2 and cetyl trimethylammonium bromide (CTAB), whereas its activity was increased by Ca2+, Mg2+, Cu2+ and dimethyl sulfoxide (DMSO). Na+, phenylmethyl sulfonylfluoride (PMSF), b-mercaptoethanol, sodium dodecyl sulfate (SDS), and Triton X-100 did not show a considerable effect on its activity. Casein was a better substrate than bovin serum albumin (BSA) and gelatin for this enzyme. The kinetic 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.

Polymorphisms in 5’-flanking region of prolactin (PRL), exon 2 and exon 5 of prolactin receptor (PRLR) genes and its association with growth and egg traits were examined in breeder hens of Mazandaran native fowls breeding station. A single nucleotide polymorphism at site C-2402T and a 24 bp nucleotide sequence insertion at situation -382 in 5’-flanking regions of PRL gene were identified. PCR amplification together with Restriction 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 promoter region 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 gene using PCR-Single Strand Conformation Polymorphism (PCR-SSCP) and PCR-RFLP analyses, respectively. A novel mutation consists of a BamHI restriction site found in the exon 5 of PRLR gene. The results showed significant association between SNP in exon 2 with body weight at hatch, age at sexual maturity, and between 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 may influence the production traits in chicken.

The genotypes for calpastatin (CAST) and calpain (CAPN) loci were determined by PCR-RFLP and PCR-SSCP methods. 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 different gene variants. Heterozygosis was low for both loci. Chi-square test confirmed Hardy-Weinberg equilibrium only in CAST locus using SSCP method. Detected polymorphisms and associations of genetic variation with meat production and tenderness may help to find the effective genotypes of Zel sheep for the economic traits.