MODERN GENETICS JOURNAL (MGJ)
Year:2014 | Volume:9 | Issue:3 (38)|Papers Count:17

one of the most common diseases in zoonotic is Bovine tuberculosis. It has been more attended because of prevalence and economic loss. Although, many efforts have been done to produce the vaccine, but the research in this area is going on.Mycobacterium bovis (M. bovis) is the most important pathogen in bovine tuberculosis. The ESAT-6 antigen is considered as a protective immunogenic and also its important candidate for DNA vaccine. BCG vaccine makes a variable immunity period and it is not desirable. Immunization with recombinant plasmid encoding protective proteins is a promising vaccination technique Therefore, the aim of this study was cloning and expression of recombinant protein in the mice myoblast cells of BALB /c as the immunogenicity of candidate. The ESAT-6 antigen fromM. bovis was cloned in a eukaryotic expression plasmid pcDNA3.1 (+). The integrity of the constructed plasmid was confirmed by using clone PCR, restriction enzyme mapping and sequencing.Recombinant plasmid pcDNA3.1 (+) /ESAT-6 injected into Myoblast cells. After one week the samples was taken from muscle tissue. The electrophoresis and western blotting confirmed the recombinant protein of ESAT-6 (rESAT-6) in Myoblast cells. Also the structure prediction showed that the recombinant protein antigenicity and hydrophobicity index is high. The current research indicated that rESAT-6 could be used as a research experimental tool in protection assays against bovine tuberculosis.

Basil (Ocimumbasilicum), a medicinal plant of the Lamiaceae family, has ring-shaped compounds, oils, antibacterial and antioxidant characteristics. Chitosan is the main compound of fungal species and could be usedas biotic elicitor to improve secondary metabolites. In the present study, the effect of chitosan on phenylpropanoid compounds, gene expression, and activity of phenyl alanine ammonia lyase (PAL) was evaluated. The plants were treated at pre flowering stage with 2 g/L chitosan and harvested after 1, 2, 3, and 5 days after chitosan. Essential oils analysis showed that methylchavicol and methyleugenol increased under chitosan, so that the most increase was after 1 and 2 days after treatment, respectively. The results of PAL activity and gene expression showed that gene expression and PAL activity increased one day after chitosan and decreased five days after chitosan.Totally, changes in gene expression and PAL activity in different harvest stages are consistent with phenypropanoid compounds changes. Thus, chitosan, as a biotic elicitor, increased phenypropanoidcompounds by increasingPAL gene expression and activity.

Nowadays, study on the abnormal protein aggregation has been interested by researchers in different areas of science especially medicine and biotechnology. The aggregated amyloid fibrils of proteins play the major role in several important diseases including Alzheimer’s’, Parkinson’s’ and type II diabetes. In biotechnology, a major problem during production and purification of the recombinant proteins and peptides is the formation of irreversible aggregates. In the recombinant proteins some tags are usually added in the C-terminal or N-terminal of the genes which can change the physicochemical properties of proteins. So far, no comprehensive study has been carried out on the effect of these sequences on the aggregation of recombinant proteins. In this study, by using AGGRESCAN bioinformatics algorithm, the effect of different tags on propensity of the aggregation of recombinant proteins was investigated. Results demonstrated that the tags can affect the protein aggregation. To determine accuracy ofin silico prediction, the fibrillation of alpha-synuclein with or without His-tag was assessed by circular dichroism, atomic force microscopy, fluorimetry and the amyloid standard tests. Alpha-synuclein is the key protein in the development of neurodegenerative diseases by formation the extensive plaques. The bioinformatics program predicted that His-tag can induce the protein tendency to aggregation. Data obtained from experimental study also confirmed thein silico results and showed that the presence of the tag inspire the fibrillation process in alpha-synuclein. Thus it is suggested in many studies, especially studies related to the pathogenesis of fibrillation or exploring the inhibitors of protein aggregation such tags removed from proteins.

Genetic diversity is a necessary factor for breeding programs and increasing genetic gain of selection in crop plants. Molecular markers have been successfully used for analysis of genetic diversity in various crops. For this purpose the 20 bread wheat cultivars were in a completely randomized design and five physiological traits, total chlorophyll, chlorophyll a, chlorophyll b, carotenoids and their stability was assessed in the cytoplasm membrane. Study of genetic diversity accomplished by 12 SSR marker primers. In order to determine genetic relation between cultivars, cluster analysis in Ward method was performed and cultivars were into four groups. The association between 40 microsatellite bands of 12 primer pairs and 5 physiological traits recorded on 20 wheat bread cultivars.Polymorphic information contents (PIC) of loci were in the range of 0.32 (Xgwm10) to 0.725 (Xgwm33).By multiple regression stepwise relationship of each 5 physiological traits and the 40 polymorphic markers were studied. The result of stepwise regression analysis showed the significant relationship of 2 markers with 5 physiological trait at the contort temperature and also 6 marker with those physiological marker at the severe level of spring cold stress, may facilitate preliminary selection of these traits for breeding purposes.Moreover, our results showed that some of the markers were related to more than one trait, implying close linkage and/or peliotropy of these genes on chromosomes.

Microsatellite markers are important for phylogenetic and gene mapping studies. Several researches developed microsatellite markers (either genomic or transcriptomic) in different Citrus species during recent years. For the first time in this study, Clementine (Citrus clementina) deep transcriptome sequencing data was used for developing Citrus microsatellite markers. Primers were designed for some of these loci and their band patterns were assessed for phylogeny studies in 34 citrus genotypes. Totally 9082 SSRs were identified from 75659 Unigenes obtained from deep transcriptome sequencing of Clementine. Di- and tri- nucleotide repeats had the maximum frequencies respectively. A high number of developed EST-SSRs confirmed Next Generation Sequencing method to be highly efficiencent for molecular marker development. Totally 25 microsatellite loci were selected for primer designing. From these totally 65 alleles obtained with average of 4.25 alleles and 2.53 effective alleles per locus. The average of Polymorphic Information Content (PIC) was 0.48 and the highest PIC observed for ABRII7 and ABRII9. Phylogenetic study for 34 Citrus genotypes was done based on NJ algorithem and Uncorrected P distance. All of genotypes located in 4 groups: Pummelo, Citon, Mandarin and Trifoliate orange. This classification was similar to those obtained by previous studies illustrateed a high efficiency of microsatellite markers developed using deep transcriptome sequencing.

Identification and selection of varieties with improved qualitative and quantitative traits has a particular importance in breeding process of current varieties and developing new cultivars. Chemical mutagens provide a diverse gene pool in plant resources and as a complementary tool are applicable in breeding programs. In this study, ethyl methane sulfonate (EMS) was used to create genetic variability in rice (cv. Neda), and the role of gained mutations was examined in improving salt tolerance. M2 mutant lines were evaluated at germinating and seedling stages under NaCl salinity (-1.2 MPa), that was resulted in identifying 9 salt tolerant lines. The lines along with original wild cultivar were also evaluated at whole plant stage under field condition with 7 dS m-1 soil EC, which three lines viz. MT41, MT189 and MT196 based on stress tolerance index (STI) were identified as salt tolerant mutants. However, regarding yield loss under salinity, three mutants viz. MT184, MT189 and MT196 were identified as salt tolerant lines. ISSR markers were used to assess genetic variability induced by mutation in mutant lines, which 50 percent of them detected polymorphism between wild cultivar and mutant lines. Cluster analysis using UPGMA method could classify studied genotypes into two distinct groups which mutant lines group showed over 2 times yield and stress tolerance index more than wild cultivar group. Based on this research, two mutant lines (MT189 and MT196) are introduced as salt tolerant lines at both seedling and whole plant stages. Also, it can be concluded that EMS-induced mutagenesis had a desire effect on the development of salt tolerant lines and that ISSR markers are applicable for differentiating salt tolerant mutants in rice mutant populations.

Microbial life is present not only in our familiar world but also in extreme environments. Salt lakes with near or at saturating salinity are extreme environments that common all over the world. The study in detail of such environments would permit to determine not only the microbial diversity but also the gene pools and potential use of this information for biotechnological applications. Urmia Salt Lake in the northwestern of Iran is the second saltiest lake in the world and resembles the Great Salt Lake in the western USA.Water, soil, sediment and salt samples were taken from east and western sites in Urmia Salt Lake in July 2012. Direct plating, dilution plating and long incubation period were used to isolate organisms on MGM, MH, SWN medium. Isolates were taken from the samples by using the conventional culture-dependent methods. Of these, 36 isolates were selected for sequencing and phylogenetic analysis, based on their growth characteristics and colony morphology. Two hundred and twenty-eight of microorganisms were obtained from soil, salt, water and sediment samples collected from the east and western of the lake. Of these, 36 isolates were selected for sequencing and phylogenetic analysis.Results showed that 36 strains represented 8 species, belonging to 3 generaHalorubrum, Haloarcula, Haloterrigena. As total, bacterial isolates were belonged to Salicola, Pseudomonas. All strains showed 96.5 to 100 % similarity in 16S rDNA sequencing. Of these, 5 strains showed less than 98.7% sequence similarity to the closest known strains and were representatives as new taxa of Urmia Lake.The phylogenetic analysis of sequences of Urmia Lake indicated in overlaps with 16S rDNA sequences from other lakes with similar habitats.

biotic stress is a limiting factor for plant products. The most common reaction under drought and saline stresses is proline accumulation as a protective osmotic action. Drought stress causes to produce reactive oxygen species by reducing plant water. This reactive oxygen species injured the cell by demolition proteins, amino acids, chlorophyll and DNA. To reducing these injurious, plants possess antioxidant defense systems such as catalase (CAT) which its main obligation is to decompose hydrogen peroxide. The best way to confrontation with drought stress is using the plant growth regulators that not only cause to plant growth and cell division, but also is a contributing factor for gene expression, producing nucleic acids and proteins. In this assay we conducted an experiment on the effect of Cytokinin and Brassinosteroied hormone's on proline content, catalase activity and their gene expression under drought stress in two Cultivars called OKAPI and GKH. This experiment was conducted as factorial based on CRD design with three replicates in greenhouse at laboratory of Agricultural Biotechnology Research Institute of Iran (ABRII) in 2012. The results showed that both varieties of rapeseed, GKH and OKAPI have the highest CAT activity in term of drought stress on Brassinosteroied hormone levels, moreover it cleared up that there is a significant difference with the control (non-hormone spray). The results of Cat16 gene expression assay showed that the both cultivars in drought stress had the maximum gene expression in hormone levels Brassinosteroied and Cytokinin 100 mM+Brassinosteroied. Also the results showed that the highest mean values for cat54 gene expression was for Brassinosteroied hormone with expression level more than three times relation to non-hormone spray. The content of proline and p5cs gene expression in levels of hormone spray was rising in GKH and reduced in OKAPI, respectively.

The button mushroom commercial strains are supposed to be genetically very similar because they have originated from a limited heritage lines. Identification and strain-typing of this mushroom could solve issues of breeding and recording legal ownership. Molecular markers and sequencing of specific regions in the genome have provided appropriate tools for evaluating genetic diversity and relationship. ISSR marker has proved to be a promising marker for identification of close genotypes. On the other hand, sequences of ITS and IGS regions of ribosomal DNA in fungi are unique areas that offer useful evolution information. The objective of this work was to evaluate the potential of molecular tools for genotype identification in the button mushroom. For this purpose, 23 genotypes of three species of button mushroom were assessed for their similarity using 20 ISSR primers. ITS and IGS regions of ribosomal DNA also were amplified by specific primers and sequencing were done. Out of 20 ISSR primers, 13 proved to be discriminative in button mushroom, producing 245 scorable and 242 polymorphic bands. According to the similarity matrix, the lowest polymorphic coefficient (0.92 and 0.95) belonged to the primers 841 and 809, respectively and the highest (1.0) belonged to the other primers. Genotypes P4 and Dezful with similarity coefficient of 0.100 and genotypes yousefi and 512 with similarity coefficient of 0.961, were accordingly the least and the most similar genotypes. Data aggregation from primers No.808, 809, 841 and 842 showed that these four primers are able to isolate all the genotypes with close relationship. On the other hand, sequencing results of ITS and IGS regions indicated that these areas are identical between genotypes of one species but are different between species. Therefore they are able to discriminate the button mushroom species. Our result demonstrated that ISSR markers compared with ITS and IGS regions are powerful enough for detection of polymorphism among closely related genotypes of button mushroom and can be used for fingerprinting and legal ownership.

In this study, the induced responses of acclimated and non-acclimated genotypes of bread wheat (Triticum aestivum L.) (Norstar) and durum wheat (Triticum turgidum L.) (Gerdish and SRN) to cold stress have been assayed by detecting electrolyte leakage index (ELI), malondialdehyde content (MDA) and the activityof antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and guiacol peroxidase). Wheat genotypes showed different responses to thermal treatments (23 and -5°C). After exposing plants into cold stress, along with decreasing of damage indices the antioxidative activities increased particularly in acclimated plants so that the maximum and minimum activity of enzymes belonged to Norstar and SRN plants. Acclimated plants showed more readiness in facing up cold stress in Norstar plants compared to durum wheat so that the minimum damage indices and the maximum enzyme activities were observed. The results have shown a potent variety in the level and speed of cell responses in these genotypes. Simultaneous changes of damage indices (ELI and MDA results) and antioxidative activities showed that these enzymes accompanied with other defense mechanisms increased cold tolerance and/ or enhance plant survive in wheat and can cause plant recovery even after sever cold stress. The short-term cold acclimation increased genetic capacity in wheat genotypes so that the degree of tolerance in Norstar was higher than that of durum genotypes. Such defense and damage indices may be used as cold tolerance marker in evaluation of genotypes in a short-term cold stress profitably in a short time and low cost.

Different investigations have shown that salicylic acid (SA) plays a key role in plant resistance induction against environmental stresses specially diseases. Pathogen attack induced systemic acquired resistance (SAR) in plants accompanied with increasing of endogenous SA, which activates wide signal transduction pathways such as transcription of the pathogenesis related proteins (PRP), enzymes related to phytoalexins biosynthesis and ABC transporter pump. Therefore, the effect of salicylic acid on the expression of defense-related genes includingPDR8, PDR5, PDR4, PDR3, NPR1, Thionin, PDF1.2and PR1 was investigated using Real Time PCR technique in two cultivars Khazar (as resistant to blast) and Hashemi (as susceptible to blast) in 0, 6, 12, 24 and 48 hours after SA treatment.Results showed that the expression of the all studied genes was increased by SA treatment. The expression of thePR1, PDF1.2 and PDR3 was increased in cultivar Khazar while no significant differences was observed among different times of SA treatment in Hashemi. Also the expression ofPDR5 and PDR8 was increased significantly in cultivar Hashemi 48 h after SA treatment. Totally, it could be concluded that SA exogenous treatment of rice might increase the expression of the defense genes and probably induce plant resistance against pathogen attack. In addition, the expression pattern of the defense related genes largely depends on plant genetic background.

This study was carried out to identify new salt induced genes from wheat involved in salt stress tolerance using EST analysis and study the role of gene encoding BAX Inhibitor 1-like protein (BI-85) in response to salt stress. Using EST analysis, it is also cleared that BI-85 is one of the salt induced genes whose expression increased under salt stress condition. Hence, this gene was selected to determine its expression pattern in response to salinity stress in tolerant (Arg) and sensitive (Alamout) genotypes of wheat and its wild relative, Aegilops crassa (DD). Gene expression analysis at 0, 12 hours and 3 weeks after salt treatment using Real-time PCR revealed that the expression ofBI_85 gene under salt stress in the root was markedly greater in Aegilops crassa compared with salt-tolerant and saltsensitive cultivated genotypes although its expression was not significantly different between the three genotypes under unstressed conditions. Twelve hours after salt treatment, inAegilops crassa the expression ofBI-85 significantly increased in comparison with before treatment and its expression level was dramatically decreased after 3 weeks in these genotypes while the expression level of this gene was not different before and after salt treatment in cultivated genotypes. It looks there are some signal transduction pathways only in tolerant genotypes that increased salt tolerance in these genotypes and the product ofBI-85 gene plays a critical role in these pathways and the protein encoded by this gene may contribute to a negative feedback regulation ofBI-85 expression.

This study was conducted to determine the effects of Betaine (Bet) substitution with a part of Methionine (Met) on malic enzyme gene expression in laying hens under heat stress. Ninety six hens (strains of commercial laying 65-wk-old Hy–Line Leghorn in second cycle of production) were performed as a completely randomized design (CRD) with a 2 ×2 factorial arrangement of treatments with three replicate cages and eight hens per cage. There were 4 dietary treatments: two levels of Bet (0 and 13%) substitute to Met combined with two environmental conditions (heat stress (HS) and thermoneutral (TN) conditions). To apply stress, the hens of a salon were exposed under 35 °C, for six hours, daily. Two experimental levels including 100% Met diet as control group and 87% Met+13% Bet diet as trial group were surveyed. After two month feeding, four hens euthanasia by cervical dislocation and the livers were removed, frozen in liquid N2 and stored at−80 °C. After extracting the total RNA, quality was measured and was used to cDNA synthesis. Finally, mentioned genes expression evaluated by specific promoter and Real- time PCR set. The results indicated that supplementation of the basal diets with Betaine lead to decrease significantly (2.47 unit) in enzyme gene expression in liver tissue (P<0.01). Also, heat stress decreases significantly (1.96 unit) in the malic enzyme gene expression (P<0.01). Therefore, Betaine and heat stress could repress malic enzyme gene expression in mRNA level and play important role in decreasing liver fat.

Six highly variable microsatellite loci were used to investigate the genetic diversity and population structure of theOxynoemacheilus kiabii in the Gamasiab River in between the Hamedan and Kermanshah provinces. A total of 60 samples were collected from these areas. All of the 6 microsatellite loci screened in this study showed polymorphism. A total of 70 alleles were observed for six loci. The number of alleles per locus ranged from 6 to 16. The averages of observed (Ho) and expected heterozygosity (He) recorded as much as 0.515 and 0.849, respectively. Most cases deviated significantly from Hardy-Weinberg equilibrium (p ≤0.01). It seems that there is a low differentiation between two populations of this species through the studied areas due to high level of gene flow (Nm=14.554) between two regions and low Fst (0.021).