GENETIC ENGINEERING AND BIOSAFETY JOURNAL
Year:2018 | Volume:7 | Issue:1 |Papers Count:10

Tomato yellow leaf curl virus (TYLCV) is a supreme pathogen in tropical and subtropical areas. During 2014-2015, a total of 393 tomato leaf tissue samples showing tomato yellow leaf curl disease (TYLCD) symptoms were collected from six different provinces of Iraq. In double antibody sandwich enzymelinked imunoserbent assay (DAS-ELSA) 55 out of 393 samples (14%) reacted positively with TYLCVspecific antibodies. Following total DNA extraction, the presence of TYLCV was verified in 21 out of 55 ELISA positive samples by PCR and coat protein gene (V1) of 16 TYLCV isolates was amplified using specific primers and cloned. Nucleotide sequences of Iraqi TYLCV isolates of V1 gene showed the highest nucleotide sequences identity of 94. 1-99. 6% in CP gene with Kuwait (KJ830841) and Iraq (JQ354991) isolates. In phylogenetic analysis based on V1 nucleotide sequences, Iraqi isolates were separated into two main groups and four subgroups, without correlation with geographical origin. Genetic diversity parameters based on V1 nucleotide sequences indicated a high genetic variability in Iraqi populations of TYLCV. The proportion of non-synonymous to synonymous nucleotide diversity was <1. 0, indicating that this gene is under negative selection. A low gene flow was observed between southern and centric Iraq subpopulations, demonstrating genetic differentiation among Iraqi subpopulations. This is the first study dealing with the distribution and genetic variation of TYLCV in Iraq. Fulllength viral genome sequencing of TYLCV isolates of Iraq is necessary to differentiate virus strain(s) in Iraq.

The diamondback moth, Plutella xylostella L. has been developed resistance to many groups of pesticides including of pyrethroids. The present study was conducted to evaluate the toxicity of deltamethrin on the third instar larvae of six populations of the pest using leaf dipping method. The results showed that different populations had different susceptibilities to deltamethrin. At the LC50 level, the resistance ratios of the Urmia, Flaverjan, Karaj, Tehran and Naqadeh populations to deltamethrin were 20. 75, 21. 84, 2. 00, 3. 08 and 26. 26-fold. Resistant populations were selected for 15 generations and their susceptibility to deltamethrin was evaluated. Resistance levels were noticeably increased in these strains and equaled with 91. 87, 82. 84 and 70. 42-fold in Naqadeh, Urmia and Flaverjan populations, respectively. Although, DEM and TPP had no synergistic effect on deltamethrin, treatment with PBO significantly decreased the toxicity of deltamethrin in the tested resistant strains. DEF also exhibited a moderate synergism with deltamethrin. Enzyme analysis proved that the activity of monooxygenase and esterase enzymes in the resistant strains were much stronger than that of glutathione S-transferase. The results showed that the high resistant strain (Naqadeh) of P. xylostella selected by deltamethrin exhibited high cross resistance to hexaflumuron and indoxacarb. This strain also had moderate positive cross resistance to flubendiamide and thiodicarb.

Strictosidine synthase-like (SSL) is a group of gene family in the Arabidopsis thaliana genome, which their orthologous in other plants, such as Catharanthus roseus, are key enzymes in the monoterpenoid indole alkaloid biosynthesis pathway. Arabidopsis SSL gene, a member of SSL family, has been induced significantly by various stresses and signaling molecules. In this study, the reverse genetics approaches (T-DNA insertion) are used to determine the function of the SSL gene in response to the salt stress. The experiment was conducted using a factorial experiment based on the completely randomized design with two factors and three replications. These factors were included two genotypes (Col-and ssl ) and three NaCl treatments ( mM, mM and mM). Compared with the wild-type, the ssl mutant shows significantly increased proline accumulation, anthocyanin content and reduced malondialdehyde (MDA) content in response to the salt stress. Real-Time PCR analysis revealed that the expression levels of SOS genes were upregulated significantly in ssl compared with the expression in Col-in response to mM NaCl at h and h after the treatment. Totally, these results suggest that SSL might have a negative regulatory role in response to salt stress in Arabidopsis.

Physalis alkekengi L. is a medicinal plant belonging to the Solanaceae family. This plant is rich in phytochemicals such as: physalins, withanolides, sterols, polysaccharides and flavones. P. alkekengi has many uses in traditional medicine and pharmaceutical industry. Hairy root cultures of P. alkekengi can be used to produce secondary metabolites. In this research, the effects of strains of Agrobacterium rhizogenes (GM, C, A, MSU and ) and leaf and stem explants were studied on induction of hairy root in P. alkekengi. The highest transformation rate was related to explants which were inoculated with A, MSU and strains. Leaf explants produced hairy roots per explant and were better than stem explants (produced hairy roots per explant). To confirm the transformation of hairy roots, PCR was performed with specific primers of rolA gene. Electrophoresis of PCR products confirmed the integration of rolA gene ( bp) to the plant genome. This article is a first published study about induction of hairy root in P. alkekengi.

Rapeseed (Brassica napus) is one of the most important oil seed crops in the world. The weeds are the most important threats to cultivating this plant. Glyphosate is a general herbicide that inhibit the EPSPS enzyme. One of the most effective methods to make glyphosate herbicide resistance is the transformation of the EPSPS enzyme-encoding gene. In the present study, a 3-point mutation in the E. coli aroA gene was created and then this gene with a wild-type gene was cloned in the pUC18 and pBI121 plasmids and transformed to the RGS003 spring straw cultivar of rapeseed by the Agrobacterium tumefaciense strain LBA4404 strain. The gene was expressed with the CaMV35S promoter and NOS terminator. Gene cloning in cloning and expression vectors, plant transformation confirmation through PCR and other molecular tests were carried out. In this research, 142 independent T1 transgenic lines were screened for glyphosate treatment and then 10 lines were selected for later tests in the T2 generation. Seeds of transgenic T2 generation under in vivo and in vitro conditions were studied in a factorial experiment in different concentrations of glyphosate herbicide. The percentage of burn and some morphological traits such as plant height, number of branches, number of pods per sub-branch, number of pods per main branch and number of pods per plant were measured. The results showed that transgenic plants can tolerate glyphosate herbicide up to 76. 8 mM, while the control plant has a lifetime of 0, 1. 2, 2. 4 mM.

Bean root rot caused by soil pathogenic fungus, Fusarium solani f. sp. phaseoli, is considered as one of the most important diseases of bean in the world and in Iran. The use of resistant cultivars is the most effective method of its control. For this reason, the response of five cultivars and 14 lines of white beans were evaluated to Fusarium root rot. Firstly, a morphological evaluation of disease symptoms and then real time PCR quantification were performed. Based on the results of morphological evaluation the KBC43106 line and Dorsa cultivar, were found to be sensitive and tolerant, respectively. For quantitative assessment and growth trend of the pathogen in sensitive and tolerant genotypes, the bean seedlings were inoculated by 107 suspension of relevant pathogen with root dip method, then DNA was extracted from root of inoculated and control seedlings at regular sampling intervals. The gradual increase in the amount of fungal DNA of roots was quantified by Real time PCR technique and utilizing of SYBR Green system with specific primers for elongation factor 1-α gene. The results showed that there is a significant difference between the amount of pathogen's DNA in the roots of susceptible and tolerant plants, therefore, the Real time PCR technique is suitable for resistance screening of bean genotypes against Fusarium root rot.

The most effective way to produce a plant resistant to glyphosate is manipulation of EPSPS enzyme in order to reduce its affinity to this herbicide. It is one of the vital enzymes in the shikimate pathway, which is responsible for producing aromatic amino acids and various secondary metabolites in plants and bacteria. Without an active enzyme, the organism will not be able to survive. In the present study, one of the important domains in the EPSPS enzyme was modified by site-directed mutagenesis using megaprimer method and one tube approach. Glycine in position 96 was modified to Alanine which was subcloned along with the wild-type gene into pPZPY122 plant binary vector. Hashemi cultivar of rice (Oryza sativa) was used for transformation using EHA105 strain of Agrobacterium tumefaciens by in planta method. T2 transgenic plants separately were evaluated to resistance to the gentamicin and glyphosate. Also, positive gene expression was confirmed by Quantitative PCR. Therefore, based on the results, these plants showed significantly a high degree of resistance to the herbicide.

Grapevine fanleaf virus (GFLV) has been reported from vineyards worldwide. The virus causes different symptoms categorized as three distinct syndromes including fan leaf degeneration, yellow mosaic, and vein banding. These variations in the symptoms can be addressed by analysis of genetic diversity of the virus. The aim of the present study was to estimate genetic diversity of the corresponding 2AHP gene in GFLV isolates especially the ones that are associated with the yellow mosaic syndrome. Accordingly, the Grapevine samples were collected from vineyards in Bonab, Shir-Amin, Hossein-Abad, and Tabriz in East Azarbaijan Province of Iran. After amplification of GFLV 2AHP gene from the infected samples via RTPCR, the products were cloned and sequenced. The sequenced GFLV 2AHP gene from 11 different isolates showed 0. 8-27% and 0. 8-35 % diversity at nucleotide and amino acid levels, respectively, denoting a higher diversity of 2AHP amino acid sequence than its nucleotide sequence. Likewise, presence of a hot spot for recombination events on 2AHP region was explored by the use of recombination analysis. Moreover, it was found that the selection pressure on 2AHP region is not uniformly distributed so that the ratio (dn/ds) for the N-terminus proximity, between the nucleotides 408-564, was more than one. Therefore, synonymous point mutations as well as recombination events might be reason for this evidence.

Because of limitations in antibody production against plant viruses, coat protein expression has been developed to prepare antigen for antibody production. In the recent research, Nucleocapsid gene of Tomato spotted wilt virus was amplified from a local strain in Iran. Cloned segment in TA vector (pTG19TSWV-N) was sub-cloned into pET32a as expression vector that digested by XhoI and BamHI. Then, pET32TSWV-N was transformed in two different strains of E. coli BL21(DE3) and BL21(DE3) pLysS by heat shock method. For protein expression, the transformed cells were induced by 1mM of IPTG in both strains. The protein was extracted four hours after induction and analyzed in SDS-PAGE. The SDS-PAGE result showed that a 48 kDa protein have been expressed that is expected based on additional tags from plasmid. The result revealed that nucleocapsid had been expressed in both strains whereas protein expression was little more in BL21(DE3) pLysS.

Although agriculture supplies the ever-growing need for food and other products of human, but at the same time it’ s one of the main causes of greenhouse gas emission, genetic diversity loss, chemical pollution as well as soil degradation. Concerns about the sustainability of traditional agriculture has drawn the attention to the alternative farming systems such as organic planting and transgenic products that are compatible with the environment. A review of the resources and various statistical analyzes have shown that although organic farming has less efficiency than traditional agriculture but it provides nutritionally valuable or even higher nutrients without pesticide residues. On the other hand, in the case of pest or disease damage occurrence in the organic product, a large amount of saprophytic fungi may develop which cause toxic and carcinogenic mycotoxins emergence. Therefore, in order to produce enough food at an affordable price for a growing population in the world that is expected to reach 7 billion by 2030, and also to provide livelihoods for farmers as well as to reduce the environmental impact of agricultural practices, the combination of old and new methods in agriculture, and the use of modern science and technology especially transgenic products can solve many of the food and environmental problems in the world. Transgenic plants resistant to pests and diseases can be used in integrative and organic farming to reduce the consumption of pesticides. The cultivation of transgenic plants is in line with organic farming goals including the reduction of pesticide usage. Hence, the term "organogenetic" is defined as the transplant of organic products and transgenic products.