جستجوی مقالات مرتبط با کلیدواژه « in planta » در نشریات گروه « بیوتکنولوژی و ژنتیک گیاهی »

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 mega-primer method and one tube approach. Glycine in position 96 was modified to Alanine which was sub-cloned 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.

Tomato leafminer (Tuta absoluta) is one of the important pests of tomato in Iran. It causes serve losses to tomato yield between 50 to 100% in the world. The cryIAb gene has been introduced into many plant species, including maize resulting in protection of the maize plants against corn borer larvae. In most studies, constitutive promoters such as CaMV35S were employed for genetic transformation; however the constitutive expression of genes led to changes in plant metabolic pathways due to permanent energy consumption in plants. Since, wound inducible promoter MPI (Maize Protease Inhibitor) posses more efficiency and strength than CaMV35S promoter. Therefore, in the current study, transgenic tomato (cv. Falat) plants harboring cryIAb gene under control of the MPI promoter were developed for the first time. The MPI promoter was isolated from maize and cloned into pPZP122 expression vector replacing the CaMV35S promoter. The cryIAb gene was isolated from pCIB4427 and cloned in pPZP122:MPI:cryIAb and the resulting construct was transformed into Agrobacterium AGL1 strain using In planta approach. Initial selection of the transgenic plants was carried out in media culture containing gentamicin. PCR analysis confirmed the presence of transgene in gentamycin-resistance plants in the first and second generations by rate of 62.5% and 75.58%, respectively. Protein dot blotting using anti-CryIAb polyclonal antibody confirmed the presence of protein in the second generation of transgenic lines. Based on the result of Tuta bioassay, transgenic plants demonstrated an enhanced resistance against Tuta. Thus, the wound inducible promoter MPI can be used in genetic transformation of crop plants if insecticidal protein-encoding genes (such as different types of cry) are used and therefore, it is important to be used when plants asked to express only when are being attacked by insect pests.

The monoterpenoids comprise a family of structurally and pharmaceutically diverse alkaloids. Strictosidine synthase is a key enzyme in monoterpenoid indole alkaloid biosynthesis pathway. In spite of the apparent lack of complex alkaloids in Arabidopsis, a gene family called Strictosidine synthase like (SSL) has been found in the genome. SSL6, a member of SSL family, has been induced significantly by various stresses and signaling molecules. An overexpressed mutant is a powerful tool for functional characterization of an unknown gene. In view of that, SSL6 overexpressed mutant have been generated in order to study the possible role of the gene in Arabidopsis defense against biotic and abiotic stresses. The open reading frame from SSL6 was amplified and cloned into intermediate pJET vector before subcloning into pPZPY122 plant vector. Plant transformation was made by floral dip method using Agrobacterium tumefaciens strain GV3101 (PMP90). The putative transgenic plants were isolated on selective MS medium containing gentamycin. Transgene integration was further analyzed by PCR using SSL6 and gentamycin resistance gene specific primers. The transcription level of SSL6 in the T2 plants was measured using q-PCR and indicated an overexpression in transgenic compared to wild type Col-0 plants. Segregation ratio of plants in T2 and T3 generation on selection medium proved that some of the genotypes contain single T-DNA insert. The SSL6 Expression level in response to salt stress was measured in Col-0 and indicated an up-regulation of the gene 3, 6 and 12 hrs after treatment.