مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال بیست و هشتم شماره 3 (پاییز 1394)

Chitinases are one of the important industrial enzymes which have significant role in different industries such as digestion of chitin and chitoligosaccharides, bioremediation and control of plants pathogenic fungal as well as insects. This enzyme catalyzes the β 1→6 glycoside bond and then hydrolyses chitin polymers. chitinases consists of a (ß/α)8-barrel catalytic domain contain of conserved sequence called DXDXE motif on β4 strand which D, E and residue around of this conserved sequence have essential role in cleavage and hydrolysis of the glycosidic bond. Study of active site adjacent amino acids can help us to understand their function in catalytic properties. In this project are mutated Ser390 and Gly191 for investigating role of this two residue existing in the adjacent of conserved sequence at catalytically process of Serratia marcescens B4A chitinase and after of cloning in expression vector and analysis of expression with SDS-PAGE are investigated effect of two mutations on the enzyme activity.

Agrobacterium rhizogenes causes hairy root disease in plants. The hairy roots produced by A. rhizogenes infection are characterized by high growth rate and genetic stability. Hairy root induction cause changes in plant Secondary metabolites production. Phenolic compounds (Flavonoids, Tannins and Anthocyanins) are important antioxidants in radish. In this work, for induction of hairy roots, leaf explants transformed with two Arhizogenes strains (A4 and GUS). Generated Hairy roots confirmed with PCR. Also, Phenolic compounds content in hairy roots was measured. Amplification of 780bp fragment for rolB and 320bp for GUS in transgenic roots confirmed the production of hairy roots. Difference of Phenolic compounds between transgenic and non-transgenic roots was significant. Our results indicate that the insertion of A. rhizogenes T-DNA in to the plant genome stimulate plant defense responses and increase the production of Phenolic compounds.

Lipase (EC 3.1.1.3) is one of the most important class of industrial enzymes. The non-conventional yeast Yarrowia lipolytica is non-pathogenic to humans and is known as safe for some industrial processes. This yeast can produces different types of lipase. Extracellular lipase production depends on medium composition and environmental conditions. Olive oil as carbon source, yeast extract and tryptone as nitrogen sources and pH in different levels were investigated to optimization of lipase production in the mutant strain Yarrowia lipolytica FDY1390 by Taguchi experiment design method. Qualitek-4 software was used to Taguchi experiment design method. The highest lipase production (340 U/mL) was obtained in medium containing 20 g/L Olive oil, 15 g/L yeast extract and tryptone, pH 4 after 72 h of fermentation process. Study and optimization of lipase production can be reduce the cost of production and so use of this enzyme by various industries.

Porcine pepsin A (EC 3.4.23.1), belongs to the aspartic protease family, and is secreted as a zymogen called pepsinogen. Pepsinogen activation occurs at pH values between 1 and 4. Pepsin consists of a bilobal conformation with two catalytic aspartic residues (Asp32 and Asp215) on either side of the active site, The structure of porcine pepsin is predominantly β-strand and random coil with limited α-helix. Porcine Pepsin is most efficient cleaving peptide bonds between hydrophobic and preferably aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. Because the importance of pepsin enzyme as an industrial enzyme in the food industry and nano particles functions in industry, we investigated Structural stability of pepsin in presence and absence of ZnO and Fe3O4 nanoparticles. This study were performed using glycin-Hcl(0.1M) buffer at pH=2 and temperature range (303 to 363) K. porcine Pepsin stability Was not changed in the presence of Zno and Fe3O4 nanoparticles. Strength of the electrostatic and hydrogen interactions between pepsin enzyme and ZnO , Fe3O4 nanoparticles Were a low level in denaturation of pepsin.

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.

Bacteria are the most abundant and richest groups of organisms that are involved in many important ecosystem processes. Despite their ecological importance, there is little literature about biodiversity, especially in aqueous environments. Iran has a variety of lakes in different locations, each of which have a special climate, with unique ecological features. Bazangan Lake, only natural lake with little salty is located in Khorasan Razavi and so far, there has not been researched on its microorganisms. The present study examined the phylogenetic diversity of cultivable bacteria in the Bazangan Lake. Sampling was conducted in November 2010. The bacterial screening led to isolation of 51 gram-positive and 15 gram-negative. 30 isolates were selected to molecular identification using 16S rRNA, studying Morphology, biochemistry and hydrolase enzymes. Identified strains belonged to beta, Gamma-Proteobacteria, Bacteroidetes and Firmicutes. Variety of Gram-ngative genus was more including Luteibacter, Xanthomonas, Varivorax, Collimonas and Flavobacterium. While Gram-positive bacteria is of less diversity including Bacillus, Fictibacillus, Staphylococcus and Paenibacillus. The maximum frequency is related to the genus Pseudomonas. The significant difference was not observed in gram-positive and gram-negative strains by checking hydrolase enzymes.

In order to cytogenetic study were examined six genotypes of Achillea including, A. millefolium and A. Santolina. In this study were done karyotype determination and determination of the traits contribution in variation. To provide appropriate examined samples, after gathering roots, 0.5 cm root tip was removed and then was carried out pre-treatment and fixation, respectively. After hydrolysis and squash were done imaging and finally were prepared karyotypes. In all genotypes basic chromosome number was x = 9. Three genotypes Aligoodarz, Ardabil and Meshkinshar were diploid and Arak, Ilam and Estahban were tetraploid. Based on Stebbins method, Arak and Ilam were classified in Class 1A, Ardabil and Meshkinshar in Class 2A and Aligoodarz and Estahban in Class 1B, respectively. Mean comparisons revealed that the Estahban and Aligoodarz had the lowest and highest the value of the S, L and T respectively. In factor analysis, two factors were explained more than %90.92 of the variance. The first factor was explained %46.65 of variance and was correlated positively with the L/S and the L-S was correlated negatively with %F and the ratio S/L. Therefore, this factor was named as the arm chromosome ratio arm and Huziwara index. The second factor explained %44.27 of variance and was correlated positively with the S, L, T and %RL. Therefore, this factor was named as the chromosome or genome size. Cluster analysis was classified genotypes into two clusters. In statistical analysis employed software's including, excel (2007), Photoshop (Adobe Photoshop CS2) and SPSS (PASW Statistics18).

Cyanide is a hazardous compound for all living things. Cyanide compounds are commonly used in various industries and cause environmental pollution. Biodegradation is the best method for cyanide elimination in mines and industrial wastewater. The aims of this study were isolation cyanide degrading bacteria in alkaline condition from contaminated soil and investigation of their ability for cyanide degradation. Contaminated soil samples were enriched in minimal salts medium supplemented with 4.3 mM potassium cyanide. Then cyanide degradation and ammonium production was determined in growth medium using picric acid and Nessler’s regent methods. In addition the ability of the isolated bacterium to utilize different cyanide compounds was investigated. A bacterium with ability to degrade cyanide in alkaline condition was isolated and named MF3. The isolate MF3 degraded cyanide in growth medium under alkaline condition after 36 hours. The results showed that there was a direct relation between decreasing of cyanide concentration, increasing of ammonia concentration and growth of MF3. In addition, the isolated bacterium was demonstrated the ability to utilize different cyanide compounds as a sole carbon and nitrogen source. The 16S rDNA sequencing and phylogenetic analysis was exhibited that MF3 strain was similar to Bacillus safensis with 99% homology. The results of current study were demonstrated that MF3 is a suitable candidate for degradation of cyanide in alkaline condition. This isolate could introduce for elimination of cyanide from industrial wastewater and contaminated sites.

Producing tender meat that consumer's desire is one of the major problems facing the sheep industry. Therefore, study of the biochemical mechanisms for muscle degradation is essential at the molecular level. Calpastatin is an endogenous inhibitor and plays an essential role in regulation of calpain activity in cells. Sanjabi sheep is an important animal producing meat in Kermanshah province that until now has not been studied by DNA markers, especially in the case of calpastatin gene. Therefore, present study was conducted to determine the genetic diversity of calpastatin gene in Sanjabi sheep and to compare this breed with other sheep breeds. A 622 bp fragment from this gene was amplified by polymerase chain reaction (PCR) from DNA samples of 142 Sanjabi sheep. PCR products were characterized by the restriction fragment length polymorphism (RFLP) method using two restriction enzymes, MspI, and NcoI, yielding 3 genotypes, MM, MN and NN. Genotype frequencies of MM, MN and NN were 0.67, 0.25 and 0.08, respectively. Gene frequencies of M and N were 0.79 and 0.21, respectively and mean of Nei's and Shanon's indexes were 0.51 and 0.33, respectively. Population was not in Hardy-Weinberg equilibrium for this locus. The results of this experiment indicated that this population is highly polymorphic. Furthermore, in the most studied Iranian sheep breeds, all 3 genotypes of this gene have not been detected whereas in this breed all 3 genotypes were observed.

Studies have shown that aberrant methylation of the tumor suppressor genes could be lead to silencing and reducing of their gene expression, consequently occurring the cancer.The aim of this study was investigation of the aberrantDNAmethylation of p14,p15,p16 genes in Iranian patients with Esophageal Squamous cell carcinoma(ESCC) and its association with demographic and pathological characteristics. Therefore we extracted the DNAs and RNAs of44 tumor tissues and 19 normal tissues from 44 unrelated Iranian patients with ESCC. The methylation specific PCR(MSPCR)was performed for both methylated and unmethylated status of these genes and the expression of these genes have done with RT-PCR.The results showed the 53%,9% and 27% methylation for the p14,p15 and P16 genes respectively in the tumor tissues and no methylation frequency in none of the normal sample. Regarding the gene expression, there was an inverse relationship between methylation status and gene expression(p

Tail and other adipose fat comprise a considerable fraction of carcass composition. This study aimed to assess association between polymorphism in thyroglobulin gene (TG) and carcass traits in Afshari sheep. To do this, a study was conducted using 97 male lambs of Afshari breed. Lambs were at age of 11 month before slaughtering. Primers were designed based on sequences available for cow. To identify different genotypes SSCP procedure were implicated on PCR products. Six different genotypes namely AA, AB, BB, AC, D- and EE were found according to the bands on non-denaturing polyacrylamide gel. There were significant differences between back fat thickness (BFT) among genotypes and BB and D- had the most and least BFT respectively. Among all genotypes, BB had lowest carcass percentage (42.7) and Longissimus dorsi muscle diameter (2.09) and live weight in animals of this genotype was also the lowest. In contrast genotype AB had the highest live weight. These results are indicating that TG can be considered as a candidate gene when improvement of the carcass quality is a goal in breeding planes

Sulphur is one of the waste elements in coal material which is in two forms, organic and inorganic. The amount of sulphur more than acceptable quantity in coal concentration affects the environment and coke characteristics. The quality of steel is depended on the quantity of sulphur in coke. High sulphur in entering materials to the steel furnace causes inferior steel. Also, sulphur in heat coal makes SO2 gas which has negative environmental effects. Different chemical, physical and biological techniques are investigated for removing sulphur from coal. In recent years, new method for desulfurization from coal is expressed with improving biotechnology and microbial mining. In this research, feasibility study of depyritization from Tabas coal using bioflotation method in laboratory scale was considered. In these tests, Pyrite in coal pulp depressed with desulfurization bacteria. To carry out bioflotation tests, a sample from C1 seam (Tabas, Parvadeh Mine, no. 2) was used. After comminuting and screening, the coal material less than 500 micron was used as flotation feed. A Thiobacillus ferrooxidans bacterium was obtained from the biotechnology laboratory at Sarcheshme Copper Mine. Thiobacillus ferrooxidans was cultured in 9K medium. Bioflotation tests were carrying out in Denver flotation cell and the important parameters such as pulp density, particle size, bacterial density and interaction time were optimized. Final results indicated that by using the mentioned method, the pyritic and total sulphur were decreased about %62 and %35, respectively.

In order to identify genetic loci associated with flowering time in oriental-type tobacco, the genetic population comprising 100 F2 individuals from the cross between two oriental-type genotypes Basma Seres 31 (maternal) × SPT 406 (paternal) were evaluated for days to flowering character. In molecular experiment, linkage map with 23 SSR and 29 ISSR markers were prepared which covered 570/8 cM of tobacco genome. Using interval and composite interval mapping procedures, 9 and 2 QTLs were identified for studied character, respectively. In this study, the most identified QTLs were located on linkage group 5. Genetic loci qDF5-3 and qDF2-27, identified via interval mapping, with 0.8 and 36 percent of R2 were the minor and major QTLs, respectively. According to results, the percentage of phenotypic variance (R2) explained by identified QTLs through composite interval mapping (qDF5-1 and qDF5-2), ranged from 1.95 to 15.34. Results revealed the role of both additive and dominance effects in genetic control of days to flowering in oriental-type tobacco.

Chitinolytic enzymes are involved to break down chitin waste products such as chitin shells of crustaceans and cell walls of fungi and production of chitin oligosaccharides. Ideal catalytic properties of these enzymes make them an attractive target for use in industry or as a biocontrol. In this study to amplify and cloning of chit36 gene from native Iranian isolate, Trichoderma atroviride PTCC5220, Specific primers (chit36pf/chi36r) were designed and then intended to produce in periplasmic form, amplified fragment was cloned into pET26b() vector. The new construct was transformed into E. coli BL21-DE3 bacteria. The results of SDS-PAGE showed no evidence of protein expression in any of the conditions of temperature and different levels of IPTG at different cell fractions. So expression of Chit36 protein with the removal of signal peptide (to produce cytoplasmic form) with histidine sequence at the carboxyl end was planned. Bacteria containing recombinant construct were evaluated at different induction conditions. Despite detection of any band in any induction conditions using SDS-PAGE, low expression of proteins in two colonies were confirmed by Western blot assay. In order to optimize the expression condition, recombinant protein expression levels relative to the total bacterial protein in different induction conditions was evaluated using the quantitative measurements of protein bands on SDS-PAGE gels. The results showed that the best condition to induce expression is at OD600 : 0.3 and 1 mM IPTG and incubation time of 6 hours. In these conditions expression level of recombinant protein relative to total protein was 45.57%.