GENETIC ENGINEERING AND BIOSAFETY JOURNAL
Year:2019 | Volume:8 | Issue:1 |Papers Count:10

Tomato (Solanum lycopersicum L. ) is an important vegetable in the world, it exposes to a wide range of pathogens and plant pests attack. Fruit worm is one of the most important pest of tomato that mainly damage the fruit and causes the yield reduction. In this study, cry1a105 gene was cloned to pBI121 plasmid (under 35S promoter and Nos terminator), and was transferred to tomato by Agrobacterium tumefaciens LBA4404 strain. The cotyledons of different tomato cultivars were used for gene transformation. The candidate transgenic plants have been selected on MS medium complemented with vitamins of B5 and 0. 3 mg/L NAA, 1. 5 mg/L BAP, and 50 mg/L of kanamycin antibiotic. Molecular analysis using polymerase chain reaction (PCR) confirmed the presence of cry1a105 gene in transgenic plants (T0). The candidate plants were cultivated until T2 generation and then the expression of gene were studied by molecular analysis (RT-PCR). The gene expression in the plants were confirmed, then they were examined by bioassay analysis. Bioassay analysis showed that the transformed Karun cultivar had a high resistance (70%) to tomato fruit worm than the control treatment.

Rhizoctonia solani is an important soil-borne pathogen which has a broad host range and cause damping-off or rottenness in root and crown of cultivated plants. The combined use of different control methods in IPM programs is an effective strategy against pathogens and is useful especially in resistance management programs. In this study, EC50 and EC25 values of three conventional chemical fungicides Zineb, Captan and Carboxin thiram and two biocontrol antagonist Pseudomonas fluorescens 73 and Trichoderma harzianum T22 were tested against R. solani in vitro and greenhouse condition. The results revealed that Carboxin thiram was effective than other fungicides with EC50 values of 0. 05 ppm in vitro. The best control (75%) were obtained in combined use of Carboxin thiram fungicides with EC50 value of T. harzianum T22. and EC50 and EC25 values of P. fluorescens 73 in combination with Captan fungicide showed the best control (near 75%) in vitro. In green house tests that the control agents individually and in combination with each other were evaluated showed that, the best control of the disease were observed in positive control (without infection) and EC50 values of fungicides in combination with each of biocontrol agents. Overall results of this research showed that combined use of two antagonists with selected fungicides in both in-vitro and green house condition are effective than individual use of each control agent and may be it is possible to control the R. solani derived disease using this integrated method in cotton crop.

Fusarium Head blight (FHB), caused by Fusarium graminearum known as a destructive disease of wheat has a significant impact not only on global wheat production, but also on food safety because of grain contamination with mycotoxins during infection, a serious threat to the human and animals’ health. To achieve to non-chemical control of FHB, the study of molecular mechanism of resistance to FHB in wheat varieties is important. In this research, in order to investigate the effect of FHB on spike proteome pattern, six bread and durum wheat cultivars were inoculated in a greenhouse with F. graminearum. In addition to measuring the morphological traits, 14 days after inoculation, changes in their proteome pattern were investigated. Two-dimensional electrophoresis technique was used to study the response of wheat spike to Fusarium. Protein extraction was performed by TCAacetone method and two-dimensional electrophoresis was performed in the first dimension by strip gel method and in the second dimension was performed by using sodium dodecyl sulfate polyacrylamide gel. The results of gel analyses showed that out of 57 repeatedly found protein spots, 13 protein spots had a significant difference between treatments, among which 11 protein spots, showed increased expression and two protein spots showed reduced expression. Proteins with changes in expression based on biological function were categorized in different groups that proteins involved in the metabolic pathways which involved in infected plants (Hypothetical protein and PR proteins) had the highest contribution to proteins with significant changes. The results of this study can be used in wheat breeding programs.

Salinity stress is one of the most important abiotic stresses that can reduce the yield of crops such as maize. In order to evaluate the effect of salinity stress on maize hybrid SC704 an experiment was performed in completely randomized design with three replications under hydroponic cultural system. The applied salt stress (0 and 200 mM NaCl) decreed dry weight. leaf proteins were analyzed on two-dimensional gel electrophoresis. Comparison of leaf proteins showed that abundance of 16 protein spots was significantly altered under salinity stress. Identification of these spots showed that these proteins were divided into five functional groups: metabolism and energy production, translation and processing, defense, nitrogen cycle, and other protein-rich changes. V-type proton ATPase subunit F protein decreased significantly and EGG APPARATUS-1 protein had the most abundance, which can play an important role in maintaining plant fertility under stress at reproductive stage and crop production.

The Apple chlorotic leafspot virus (ACLSV) is among of the most common virus infecting pome and stone fruit trees around the world. The virus has spread throughout the world and has been reported in many hosts such as; apples, pears, peaches and cherries. The purpose of this study is to express the coat protein (CP) of ACLSV in E. coli. CP expression in E. coli has been developed to prepare antigen for antibody production without needs to purify the virus from infected plant. In this study, complete CP gene (582 bp) from an isolate of ACLSV (SSa) was amplified by specific primers, MF and MR. The amplified CP gene was ligated to TA cloning vector (pTG19-ACLSV CP) and transformed into E. coli strain DH5α . Transformed cells were selected on LB containing ampicillin, X-Gal and IPTG, and recombinant plasmids were confirmed by restriction analysis. Then, the pTGACLSV CP was sub-cloned into pET28a (+) as expression vector that digested by BamHl and XhoI restriction enzymes. Finally, the pET28a-ACLSV CP was transformed into E. coli strain BL21 by heat shock method. For ACLSV CP expression, the cells containing pET28a-ACLSV CP were induced by 1 mM of IPTG and the protein was extracted two and four hours after induction and analyzed in SDS-PAGE. The SDS-PAGE result showed that ACLSV CP have been expressed that is expected based on additional tags from plasmid.

Hairy root cultures are an effective method for production of secondary metabolites, because the hairy roots are genetically and biologically stable and they are able to produce metabolite within a short time without needs to hormone. Chicory (Cichorium intybus L. ) is one of the important medicinal plants that contains a number of important medicinal compounds. In this research, hairy root induction was established through the mediation of the ATCC11325 strain of Agrobacterium rhizogenes. In first experiment, the effects of type and age of explant and co-culture times on the efficiency of hairy root induction were investigated. In the second experiment, we studied the effect of different hairy root lines on growth rate. In third experiment, the effects of various concentration of NAA (0, 0. 5, 1 and 1. 5 mg/l) and sucrose (3, 4, 5 and 6%) on biomass accumulation were investigated. Results showed that maximum hairy root induction (75. 55 percent) and number of roots (7. 26 roots per explant) obtained from 5-day-old cotyledons. The results revealed that 1. 5 mg l-1 NAA in combination with 3 and 4% sucrose were superior for highest fresh (1. 4 and 1. 3 g) and dry weight (0. 107 and 0. 100 g) productivity and growth index (21. 42 and 19. 96). The highest total phenolic content (6. 76 mg g-1 DW) and flavonoid content in 270 nm wavelengths was observed in hairy roots that were grown in medium supplemented with 1. 5 mg l-1 NAA and 4% sucrose while the maximum flavonoid content in 300 and 330 wavelengths was achieved in 1. 5 mg l-1 NAA and 5% sucrose.

Root architecture modification can increase grain yield and seed quality. These can be achieved by better anchorage of the plant in soil, more effective use of water and nutrients, and through biosynthesis of amino acids and hormones. These will result in more effective use of nutrients and drought tolerance. Considering drought stress in Iran, production of drought tolerant plants is important. In this study three genes involved in modification of root length, number, thickness and growth angle were cloned into a construct for Agrobacterium-mediated transformation of rice plant. The genes, OsEXPA8, OsNAC5, and DROI, from native cultivars of rice were cloned under the control of constitutive and root specific promoters; afterwards, they were placed in T-DNA region of Agrobacteriummediated transformation binary vector. The resulted construct, pUhrN5ExDro, was transferred into Agrobacterium tumefactions strain EHA105 and used for transformation of immature embryo of rice cultivar Hashemi. Selective medium containing 50 mgl-1 hygromycin B was used for selection of transgenic plants. Construct specific and gene specific PCR confirmed three putative transgenic lines and one copy of transgene were detected in one of them using Real time-PCR. Inverse PCR revealed that T-DNA was integrated in chromosome 10 of this rice event. Comparison of the phenotype and root structure of the transgenic plants with the control in the Root Box and in the same growth conditions showed that transgenic plants displayed stronger root phenotype compared with non-transgenic ones. The resulting multi-genic construct can be used for transformation of other crops in order to root structure modification and drought tolerance. It is hoped that the production of rice with modified root structure can increase drought tolerance in this important crop and reduce water consumption in rice cultivation.

"Witches' broom" Disease caused by the "Candidatus phytoplasma aurantifolia ” is one of the important citrus disease that causes significant economic losses in Iran, western Asia and North Africa. In this study the effect of disease stress on leaf proteome was investigated. Leaf samples were collected from plants and were analyzed using 2-DE coupled with MS. The power of 2DE for proteins separation makes it possible to study various qualitative and quantitative changes in protein expression under normal and stress condition. out of 801 leaf proteins from eight biological replicates of healthy and infected plants that were analyzed by melani6 software, 63 proteins showed a significant response to the disease. MS results indicated that 19 proteins were involved in oxidative stress defense photosynthesis, metabolism and the stress response. Identifying these proteins can reveal newer response pathways and the genes that control them. The results of this study can be used in mexican lime breeding programs.

Improving new high performance cultivars, with better quality and resistance to biotic and abiotic stresses are important objectives of rice (oryza sativa L. ) breeding through genetic engineering. This study, have been done to optimization of callus induction and, transformation protocol for some cultivar of Iranian Rice. In first experiment, effect of plant growth regulators (NAA, 2, 4-D, Kin), proline (200, 400 and 600 mg/L) and Rice genotypes (including Moosa tarom, Nemat, Dom sorkh, Garib, Ali kazemi and Derafk) in callus induction, bacterial concentrations (OD600 = 0. 2, 0. 6 and 1) and strain types (LBA4404, EHA105, AGL1) on selected cultivar transformation were evaluated. The results were analyzed in a factorial experiment based on a completely randomized design. Analyses of variance were showed significant difference between plant growth regulator and genotype in response to callus induction. Almost all of the cultivars induced callus up to 80% in majority of PGRs combinations. HasanSaraei, had the higher callus growth rate (over 0. 49 g) in the medium supplemented with 2 mg/l 2, 4-D, and DomSorkh and Garib had the highest callus fresh weight (above 0. 4 g) in media containing 2 mg/l 2, 4-D plus 2 mg/l NAA. Using prolin in callus induction medium decreased callus growth. Lowest callus biomass (0. 138 g) produced in medium containing 600 mg/L prolin. Among the three evaluated strain of Agrobacterium tumefaciens, only the EHA105 produced transgenic calli. Analysis of variance showed there was no significant difference between bacterial OD600 on transformation efficiency, but OD600=1 produced more transgenic calli. According to the results, the DomSorkh cultivar is more capable of callus formation in 2, 4-D medium and can be used for gene transfer for different purposes.

Β-lactoglobulin (BLG) is one of the main allergens in cow’ s milk that causes it to be digested poorly. BLG structure has two intramolecular disulfide bonds and one free cysteine in cys121 that disulfide bonds are responsible for its low digestibility. It seems that the digestibility of BLG will be increased via the reduction of disulfide bonds. In the present work, the aim was to study the effect of plant glutathione/thioredoxin systems on the digestibility of milk BLG. Thioredoxins (Trxs) are small and abundant disulfide reductase in all organisms. they are reduced with Trx reductase or glutathione reductase or glutathione. Previously the genes encoding plant Trx, OsTrx20 was cloned and heterologously expressed in E. coli. In this work this protein was produced and purified in considerable amounts. The interaction of Trx with glutathione (GSH) was confirmed by insulin assay. The result of this assay showed that GSH was able to reduce OsTrx20. Therefore, in this work the effect of GSH/OsTrx20 on the digestibility of BLG was studied. Finally, BLG was pre-treated by GSH/OsTrx20 at 4, 25 and 37° C and then digested with trypsin. The digestibility was studied by using SDS-PAGE and HPLC and allergenicity was studied by ELISA. The results revealed that the GSH/Trx system significantly affects on the BLG digestibility and allergenicity.