JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2013 | Volume:5 | Issue:2|Papers Count:11

Phytase (myo -inositol hexakisphosphate) is the hydrolysis enzyme of phytic acid that produces inorganic phosphate. The genephyC encoding phytase was isolated from Bacillus subtilisATCC12711 genomic library and sequenced. The nucleotide sequence of the phytase gene contained an open reading frame of 1089 bp, which codes for 90 bp signal peptide and a mature protein with a deduced molecular mass of 42 kDa. Target gene was inserted in pET32a (+) as expression vector and transformed to Escherichia coli BL21 (DE3) as host expression. The fusion phytase phyC-Trx gene was successfully overexpressed in E. coli as an active and cytoplasmic phytase. Recombinant protein production was induced with 1mM IPTG in shaking flasks at 300C in presence of 10 mM calcium. Samples were taken at 0-5 h after induction by 1 hour time intervals, and then protein electrophoresis was done.phyC -Trx protein was expressed in the cytoplasm ofE. coli successfully. Molecular weight of recombinant phytas was estimated about 64 kDa. Phytase activity was 7.44 U/ml with using standard phosphates method. Optimum pH for the degradation of phytate was 7. The results of current study showed that thermal stability and cost effective production make this enzyme attractive for using in feed supplements.

The results of multiple QTL mapping design in recent years, quantitative trait loci for milk fat percentage and milk yield was described in BTA14. With the coding of diacylglycerolacyltransferase enzyme, DGAT1 gene has the main role in the triglyceride synthesis and finally fat milk synthesis. In this research, 398 blood samples were collected from Tehran and Esfahan province. In PCR reaction, 411 bp fragment of exon 8 DGAT1 gene was amplified.Finally, genotyping population was performed using RFLP-PCR technique. Three genotypes such as KK, AA and KA were detected. Frequencies of DGAT1 alleles (K and A) were estimated 0.37 and 0.63 respectively. Using of fitted fixed model and animals ‘records significant traits were recognized. Finally using of parameters coding and regression model prediction models were presented. Correlation coefficient was utilized for validation of prediction models. The results of this study indicated presented model can be prediction of Milk305 (milk production adjusted for milking in 305 days), EBVFP (estimated breeding value for milk fat percentage, %) and FATP2X (milk fat percentage adjusted for two milking per day, %) traits in population.

Considering the economic importance of tobacco and its role as a model plant in molecular researches, in the present study, 50 tobacco genotypes from Barley and Virginia group were evaluated. In order to determine of genotype, 21 primer combinations of AFLP were used.According to the results, out of 480 total numbers of bands with average of 17.76 bands for each combination, 373 bands showed polymorphic pattern. Due to high polymorphic percentage of used marker (77.45%), it can be expected to consider, this marker as a powerful tool for assessment of genetic diversity in tobacco breeding programs. Investigation of Jaccard’s genetic coefficients revealed Badisher Burley E, Pennbel69, R9 and Coker 176 had the highest genetic distances than the others. Therefore, it seems the genotypes to be useful for breeding programs including hybridization and developing of segregating mapping population. The results of AMOVA showed 4% of the total genetic variation was estimated between two groups of Barley and Virginia flu-cured and 96% was related to within groups. Also diversity statistic revealed primer combinations of E060-M160, E070-M140, E070-M150, E070-M160, E080-M150, E080-M160, E100-M140 and E100-M150 were the most powerful markers in the evaluation and identifying of relationships among tobacco genotypes that can be considered in the other related studies.

Due to necessity of salt tolerance in rice (Oryza sativa L.) and availability of different approaches of molecular breeding in identification of major QTLs controlling salinity tolerance, it is very important to evaluate the presence of these QTLs in Iranian cultivars and the roles in breeding strategies. In this study, 20 different rice cultivars and two tolerance (Pokkali) and sensitive (IR29) controls were evaluated under salinity stress at, seedling stage in greenhouse conditions (EC=0, 6 and 10 dSm-1) using 6 polymorphic SSR markers. Analysis of variance showed the high significant difference among genotypes of different traits, rice genotypes showed different salinity tolerance at seedling stage. Obtained dendrogram by UPGMA method categorized genotypes in to 3 different groups.20 rice cultivars based on major QTLs of Saltol region on chromosome 1 were arranged in separate haplotype groups. RM8094 marker was distinguished as the best marker for identification of salt tolerance genotypes in seedling stage.

Atropa belladonnais one of the most important medicinal plants in Solanaceae family.It contains tropani alkaloids that often are synthesised in root. Present study was conducted in order to induction of hairy root in A.belladonna using gene transfer via Agrobacterium rhizogenesmethod. The stem explants with Agrobacterium suspensions harbouring rolB gene were inoculated. Then, in order to direct regeneration, five compounds (MS, MS+3 mg/l Kin , B5, MS+12 mg/l Kin, B5+5 mg/l Kin)) in a completely randomized design with three replications was used. The statistical analysis indicated that among treated hormone, 12 mg /l KIN level in MS medium showed the most shoots direct regeneration. Two methods for the analysis of transgenic plants, phenotypic (observed in hair roots of transgenic seedlings), gene amplification of the rolB (using PCR) was used.

Introduction: Black cumin (Bunium persicum Boiss.) belongs to Apiaceae family.Despite the importance of this medicinal plant in pharmacognesis, due to problems of germination and growth over long periods, yet in the country has not been domesticated and grown in nature wildly. Techniques such as tissue culture can reduce the period of growth in this plant effectively. The aim of the research: optimization of somatic embryogenesis toward production and commercialization of this plant for the pharmaceutical usage and artificial seed production were studied. Hypocotyl and cotyledon leaves of Esfahan ecotype were used as explants. MS medium supplemented with 14 different hormonal treatments for the production of callus and somatic embryogenesis were applied. To optimize the growth culture for conversion of somatic embryos to seedlings MS medium supplemented with 17 different hormonal treatments were used. Experiment was conducted in completely randomized design factorial layout based on multi-observations and 4 replications. Hypocotyl identified as the best explant for somatic embryogenesis. Hormonal treatment of 0.5 mg/l Kin+1 mg/l NAA and 1 mg/l NAA revealed the highest efficiency for callus formation while medium containing 0.2 mg/l 2ip+0.01 mg/l IBA+1/2 MS showed the highest regeneration amount of somatic embryos. Produced embryos in MS medium containing auxin IBA and Cytokinin 2ip showed the normal growth. Due to problems in the caraway seed germination, production of artificial seeds is very important. Considering the successful production of somatic embryos in this research, synthetic seed production of black cumin can be followed in future.

Rice (Oryza sativa L. cv. IR64) was grown in split-root systems in order to analyze long-distance drought signaling within root systems. This in turn underpins how root systems in heterogeneous soils adapt to drought. The approach was to compare four root tissues: Well watered (WW), water deficit (WD), split-root systems where one half was well watered (spWW) and the other half water deficit (spWD). This was specifically aimed at identifying how drought root tissues altered the proteome of adjacent wet roots by hormone signals and secondly, how wet roots reciprocally affected dry roots hydraulically. Using a 2-DE based proteomics approach, 738 protein spots were reproducibly detected, Thirty-two proteins showed reproducible and statistically significant changes in response to drought. Of them, 13 protein spots up-regulated in WD and spWD treatments. Of them 9 protein spots up-regulated in WD, spWD and spWW treatments, 8 protein spots not detected in WW and 2 protein spots just detected in WD. Specific functional groups changed consistently in drought.Pathogenesis-related proteins were generally up-regulated in response to drought and heatshock proteins and thioredoxin were totally absent in roots of fully watered plants.

Motherwort (Leonurus cardiaca) is the only species of the genus Leonurus in Iran. It has many different compounds such as flavonoids, iridoids, triterpene, tannins, sterols, carotenoids useful to treat heart and therapy stomach diseases, any neurological disorders. In this study, RAPD molecular markers were used to evaluate genetic diversity among 30 accessions from six wild populations of motherwort in Iran. Totally 60 random primers were tested initially where 28 produced polymorphic and high resolution bands. In total, 364 DNA fragment were obtained, from which 325 were polymorphic. Cluster analysis of genotype based on the Dice similarity coefficient and UPGMA method was performed. The min and max population’s values of genetic similarity were recorded between Dargaz 1 and Khansar 5 (0.12) and Sari 2 and Sari 3 (0.89) respectively. In the Cluster analysis genotypes were divided into two main groups at 20 distances, on included Dargaz and the other contained Sarab, Khansar, Kerman, Sari and Taleghan. Population diversity using Nei's genetic diversity (h) and Shannon index (I) showed that genetic variation within populations Sarab and Taleghan (h=0.10, I=0.15) was the highest and within Kerman population was the least (h=0.06, I=0.10). crosses genotypes of Dargaz population with other populations according to genetic distance determined in this study can be good option for Production Hybrid with desired functions.

Study of the morphological characteristics and molecular markers for genetic diversity determination is one of the basic steps in the most breeding programs. For this purpose, current investigation was carried out with ten cultivars based on randomized complete blocks design (RCBD) with three replications, during the period of October 2010 to June 2011 cropping season.Statistical analysis of agronomy data showed that there were significant differences between genotypes for almost studied traits. Cluster analysis by UPGMA method for agronomic traits divided genotypes into three groups. High genetic gains were observed for flag leaf length.Correlation between morphological and molecular traits was assessed using Mantel test and significant positive correlation was observed between them. Thus according to genetic distances between the genotypes and heterosis value based on morphological markers according to similarity coefficient, the cross between 212 with 217, 201, 202, and 204 is recommended. In conclusion, replication of such experiment with more varieties for more accurate results is recommended.

Cucumber mosaic virus(CMV) is one of the most important viruses of cucurbits worldwide.To study the phylogenetic relationships of several CMV isolates, a number of samples were collected from Kerman and Yazd provinces. The CMV infection of the samples was tested using DAS-ELISA method. Based on the host and geographical regions, six ELISA positive samples were chosen for molecular characterization. A 946 bp fragment comprising fulllength of coat protein gene with its flankes was amplified by RT-PCR using specific primer pairs, cloned and sequenced. Sequence comparisons were done using DNAMANsoftware on CP gene of Iranian isolates and those of available in GenBank. Phylogenetic analysis showed that CMV isolates were divided into two subgroups I and II in which the subgroup I was further divided into two subgroups of IA and IB. Two Iranian isolates sequenced in the present study were placed in the subgroup IA and the rest of isolates in the subgroup IB. The higest nucleotide sequence identity of 99.1% was obtained for the isolates Ker.Ker.Pep and Ker.Ker.Mel.2 (subgroup IB) while the lowest for the isolates Ker.Jir.Cu isolate (subgroup IA) and the Yaz.Yaz.Tom of 92.1 %. Isolates of subgroup IB belong almost exclusively to East Asia. It is the first report of occurrence of subgroup IB of CMV isolates in Iran and Middle East.

Today, genetic engineering is an indispensable component of modern biological research and DNA cloning, as one of the most important applications of this technology, has a wide-range application. Plasmids are the most commonly used vectors that provide replication of a desired gene usually in a bacterial host. T/A cloning vectors are one type of plasmids which facilitate cloning of a DNA fragment provided that the DNA is amplified by Taq DNA polymerase through polymerase chain reaction (PCR). In cases where the resources are limited, purchasing commercial T/A cloning kits may be hardly possible. So, availability of a home-made T/A vector with a good performance in the laboratory would be important. In this study, after preparation of competent Echerchia coli cells, the circular plasmid pTZ57R (without insert) was transformed into the cells. After plasmid extraction by alkaline lysis method, the plasmid was cut with Eco RV to make it linear. Then, the enzyme was inactivated and thymine nucleotide was added to the free 5’ ends of the linear plasmid. The efficiency of the vector was demonstrated during ligation reactions and subsequent transformations. In addition, a segment of potyvirus genome that was amplified by a pair of universal primers was inserted into the T/A vector and subjected to sequencing. Comparison of the sequencing data with that of the counterpart regions available in GenBank has ended in identification of the virus asSoybean mosaic virus.