مجله بیوتکنولوژی کشاورزی
سال هشتم شماره 1 (پیاپی 21، بهار 1395)

Human tissue-type plasminogen activator (tPA) is one of the most important therapeutically proteins involved in the breakdown of blood clots of brain and heart blood vessels following stroke. Therefore, several modern approaches in plant biotechnology such as nuclear and chloroplast transformation have been used to produce the valuable protein. Plant transient expression is a rapid, flexible and reproducible approach to obtain high level of expression of valuable recombinant pharmaceutical proteins. It is shown that posttranscriptional gene silencing (PTGS) process influences the expression level. Therefore, the effect of co-expression of gene silencing suppressor gene P19, derived from tomato bushy stunt virus (TBSV), has been studied on transient expression of tPA in Nicotiana tabacum cv. Xanthi. To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pTRAc-tPA-ERH with Agrobacterium containing pCambia-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pTRAc-tPA-ERH. ELISA results from co-agroinjection experiments have shown that the expression level of tPA using p19 was 10μg/g of leaf fresh weight, which this expression level was about two times more than that from leaves injected with Agrobacterium without the vector harboring p19. Therefore, comparing to the stable transformation which needs more time to produce the protein, in transient expression, depends on the number of the injectable leaves per plant and production of 10μg of tPA per gram leaf fresh weight, a higher level of expression can be achieved less than one week. In conclusion, it seems transient expression could be utilized to express tPA usefully and the expression level could ameliorate using virus coded gene silencing suppressor to achieve the expression proportion of 10μg/g of leaf fresh weight.

The study was designed to characterize the genetic diversity of 95 rice genotypes from various countries by 67 microsatellite markers which covered all rice chromosomes. Number of 303 alleles was detected with an average of 4.52 alleles per locus. The average polymorphism information content value across the marker dataset was 0.51, and it was varied from 0.81 (RM 10346) to 0.11 (RM 4862). The cluster analysis based on UPGMA and Nei genetic distance grouped the studied population in four main subgroups with a significant tendency to cluster by geographical origins. Phylogenetic relationships among the genotypes were in agreement with cluster analysis results. Principal co-ordinates analysis (PCoA) with considering two first co-ordinates that explained about 71.15% of the total variation also confirmed cluster analysis results. Accuracy of cluster analysis was assessed by analysis of molecular variance and results revealed significant difference between clusters. The present study by evaluation genetic distances in part of Iranian germplasm against foreign inbreed lines and varieties obtain appropriate results for applying Iranian germplasm in rice breeding projects.

In order to identify QTLs associated with grain yield and yield components in Bread Wheat under limited irrigation condition using ICIM method, 305 pure lines (FR8R) generation of bread wheat derived from cross between Roshan and Falat along with four standard cultivar namely Roshan, Falat, Mahdavi and Shahpasand, were evaluated based on Augmented design. Stress treatment was cut off irrigation at heading stage. Grain yield and other agronomic traits were measured as the phenotypic traits. The result revealed that there are transgressive segregations for all under studied traits. A genetic map was constructed with 808 DArTs markers which these markers covered 5395.69 CM of the genome with the average of 7CM. In total 24 QTLs were identified on 13 chromosomes with additive effect for 12 traits. Four QTLs had RP2P>10. The qAL6A was identified for awn length that explained 26.24 percent of the total phenotypic variance. qPL4B was found for peduncle length and contributed 17.15 percent of phenotypic variance and is locating at the same interval with flag leaf sheath length QTL. This phenomenon could be due to polytrophic or due to linkage effect of those genes which control these two traits. Major QTLs associated with grain yield and yield components under limited irrigation condition after validation can be used in plant breeding programs and marker-assisted selection in order to produce tolerant and high performance wheat varieties.

In the present study to investigate the effect of dietary methionine restriction on IGF-1 gene expression in breast muscle6T of 6 TJapanese quail, 200 pieces of 6Tthe birds were 6Trandomly 6Tdivided into two different dietary groups containing recommended methionine supplement and without the methionine supplement. IGF-1 gene expression levels were measured using the Real time PCR techniques in breast muscle of the Japanese quail at 24 days of age. Also in this age, the body weight and bone characteristics such as 6TFemur, Humorous and tibia were recorded. The results indicate that 6TIGF-1 gene expression significantly increased when the methionine supplement was removed from the diet (P

HA1 antigen is the major host protective immunogen of the highly pathogenic H5N1 avian influenza A virus (AIV), which leads to great pandemics and epidemics throughout the world. Plants are now gaining widespread acceptance as a general platform for the large scale production of recombinant proteins like influenza vaccines. In present study HA1 antigen of H5N1 was expressed in Alfalfa (Medicago sativa), soybean (Glycine max) and Lettuce (Lactuca sativa) leaves using agro-infiltration. Also the effect of three signal peptide including KDEL, Extensin and ZERA which cause protein accumulation respectively in endoplasmic reticulum, apoplastic space and protein bodies, were analysed. 72 hours after the agro-infiltration, qRT-PCR results showed that lettuce and soybean respectively had the maximum and minimum of HA1 transcripts. The expression of HA1 was detected in leaves extracts by ELISA method. The highest expression was detected in alfalfa leaves with apoplastic signal peptide while the lowest expression was found in lettuce despite the highest transcription rate. The results showed that using agro-infiltration system in alfalfa plant with apoplastic signal peptide was the appropriate strategy for HA1 antigen production in the assayed plants.

Myrosinase is a β-thioglucoside glucohydrolase enzyme which catalyzses the separation of glucose from glucosinolates and produces an unstable intermediate, aglycone. This intermediate will be rearranged and then converted to different compound such as thiocyanate, isothiocynate and nitrile, depending on the environmental conditions. Among them, isothiocyanates are more important compounds due to the pharmaceutical applications. Sulforaphane is the most isothioyanates which produces from glucosinolate glucoraphanin through myrosinase hydrolysis. In this study, in order to investigate the effects of Cu and Zn ions on myrosinase activity and rearrangement of aglycone fragment in Lepidium draba, glucose and sulforaphane content were measured respectively. The results showed that myrosinase activity significantly promoted at treatment with 8 µM ZnP2 in compared to the control and significantly reduced by the increase ZnP2 concentration in media. Elevated of the enzyme activity was seen at all Cu concentration which was more significant at 4 µM. On the other hand, sulforaphane content was reduced at presence of all concentrations of the both ions especially Zn ions. Overall, it deduced that the mentioned ions, especially Cu at low concentrations have stimulatory effects on myrosinase activity. These ions also affected rearrangement of the aglycone fragment particularly Zn which turned the rearranged pathway into other compound except isothiocyanates, therefore the sulforaphane content reduced at presence of these metals.

Oxylipins the products of fatty acid oxidation, to acclimate biotic and abiotic stresses. In this study, we evaluate the effects of cold stress by measuring manlodialdehyde (as membrane damage index), the activity of lipoxygenase (LOX) and allene oxide synthase (AOS) enzymes, the levels of transcript LOX, AOS and allene oxide cyclase (AOC) genes using quantitative reverse-transcriptase polymerase chain reaction (qPCR) under cold stress (4 C) in tolerant (Sel96Th11439) and susceptible (ILC533) chickpea (Cicer arietinum L.) genotypes. The data analysis revealed that under cold stress, MDA content significantly increased in both genotypes so that MDA content in susceptible plants was maximum on day 6 of cold stress ( by 57%) whereas in tolerant genotype, after an increase on day 1 of cold stress its content did not significantly change. Such increase in MDA content showed signaling role and induction of defense mechanisms in tolerant genotype. Under cold stress, the activity of LOX and AOS significantly increased in susceptible genotype compared to tolerant one. Therefore, the activity of AOS pathway, as main pathway of oxylipin was considered in chickpea under cold stress, indicating probably its role in creating jasmonates and induction of defense genes. The analysis of transcription level of LOX, AOS and AOC genes with REST software confirmed changes in oxylipin components. Thus degree of cold tolerance in tolerant plants was related with cell damages content and the activity of oxylipin pathway under cold stress.

Major flowering time genes including vernalization and photoperiod response play a crucial role in the geographical and agronomical adaptations, and potential yield in cereals. Assessment and understanding of the distribution of allelic variations for vernalization and photoperiod genes is of special importance in wheat breeding programs and parental selection. Therefore, the objective of this study was to evaluate 38 bread wheat varieties for allelic variations at the VRN1 and Ppd1 loci, using functional molecular markers. In this study, the frequency of vrn-A1, Vrn-A1a and Vrn-A1b alleles at the Vrn-A1 locus were 57.9, 39.5 and 2.6 percent, respectively. At the Vrn-D1locus, vrn-D1 and Vrn-D1alleles had the frequency of 73.7 and 26.3 percent, respectively. At the Vrn-B1locus, the most frequent allele was Vrn-B1a with 44.7 percent, while the frequencies of Vrn-B1b, Vrn-B1c and vrn-B1 alleles were 21, 18.4 and 13.2 percent, respectively. Allelic variations at the Ppd-1 gene were also detected in the studied population. As allelic frequencies of Ppd-D1a and ppd-D1b at the Ppd-D1 locus were 71 and 29 percent, respectively. At the Ppd-B1 locus, 18 genotypes had Ppd-B1a allele, while 20 others showed ppd-B1b allele. In general, Ppd-D1 was the most frequent allele, followed by Vrn-A1a, Vrn-D1, Ppd-B1b and Vrn-B1a. These results indicate that the frequency of dominant allele at the VRN1 and Ppd1 genes was the highest. Therefore, according to these results, most genotypes can be considered as spring and insensitive to photoperiod.

It was the first time that hairy roots were initiated from Salvia reuterana by infection with Agrobacterium rhizogenes strain ATTC 15834. Confirmatory studies were carried out by direct detection of inserted T-DNA by the polymerase chain reaction. These hairy root cultures had the ability to produce rosmarinic acid. The effect of six different basic media including B5 ¡½ B5 ¡MS ¡½ MS ¡White’s and WPM on the root growth and rosmarinic acid production was studied. Results showed that maximum fresh weight (41.4 g/LP-1P) and dry weight (3.7 g/LP-1P) were found in MS medium. Despite the maximum rosmarinic acid production (52.2 mg/g DW) was achieved in B5 medium, the highest amount of rosmarinic acid yield (0.13 g/LP-1P) was observed in MS medium. Therefore MS medium was suggested as the best culture medium for growth and rosmarinic acid production in hairy root culture of Salvia reuterana.

Gummy spike blight is a disease of wheat (Triticum aestivum) in several cereal growing areas of Iran. Rathayibacter iranicus, as the causative agent of the disease of wheat and barley ,appears to be confined to Iran. To evaluate possible phenotypic and genotypic diversity of strains infecting wheat and barley, infected spikes were collected from East Azarbaijan , Ilam, Isfahan, Fars and Khuzestan provinces and the associated bacteria were isolated from the spikelets and characterized. Fifteen strains were selected as representatives of groups displaying one or a few differences in phenotypic features or total protein electrophoretic patterns and were used in genotypic comprisons. DNA was extracted from these isolates and from the type strains of Rathayibacter spp. To evaluate the genetic diversity of the studied strains, minisatellite marker, with primers M13 and (GTG)R5R, was used. 7TFragments of the genes for 7T16S rDNA, ITS, gyrase (gyrB) and recombinase (recA) were amplified and sequenced. Combined PCR fingerprints obtained with the minisatellite primers indicated identity of the strains isolated from wheat with R. iranicus. Strains isolated from barley formed a distinct branch closer to that of R. iranicus and furthest from those of the other Rathayibacter species including R. tritici and R. rathayi. R. toxicus and other Rathayibacter species formed a distinctly different branch. Comparison of the sequences of the ITS, 16S rDNA, gyrB and recA genes with those deposited in GenBank, verified, further, the identity of the strains isolated from wheat and barley as R. iranicus.