Beta 1, 3-glucanase gene derived from barley encodes an antifungal protein and hydrolyzes glucans which are major components of cell walls of many pathogenic fungi. This gene increases plants resistance against fungus infection. In order to transform beta 1, 3-glucanase gene into the cotton, at first a suitable gene cassette was designed by addition of the CaMV35S promoter and the Nos terminator to open reading frame of the glucanase gene in pCaMV vector. Subsequently the complete cassette of glucanase gene was subcloned into T-DNA region of binary vector pBI121 between nptII as a selective marker and Gus as a reporter gene. The pBI121-Glu recombinant plasmid was used for gene transformation via Agrobacterium into the cotton shoot apices. After isolation the cotton shoot apices and inoculation them by Agrobacterium solution, explants were transferred to selective medium containing kanamycin. The surviving and regenerated shoot apices in selective medium that were positive in histochemical assay for Gus gene expression were considered as putative transgenic plants. They should be cared to grow enough for carrying out further molecular analysis to confirm the integration and expression of the interest gene and transformation event.

Objective Oilseeds are the second-largest source of calories for human societies after cereals. Sesamum indicum has been considered recently due to its oil quality and content of 43-46% unsaturated fatty acids. To investigate the possibility of transferring the recombinant aroA-CYP81Q1 gene through Agrobacterium tumefaciens to the Sesamum indicum plant (reported for the first time in the country), after optimizing tissue culture and selecting the best hormonal combination, a factorial experiment was performed. Materials and methods In this experiment, the recombinant aroA-CYP81Q1 gene was synthesized and transformed into an Agrobacterium strain (LB4404). Gene cloning was confirmed by PCR and then confirmed by sequencing. The efficacy and frequency of transgenic Sesamum indicum were evaluated in the selected medium containing 50 mg/l kanamycin. Finally, to confirm the transgenicity of the regenerated plants, PCR analyzes were performed with specific primers on selected plants. Also, the expression of the sesamin-producing gene (CYP81Q1) in control groups and transgenic groups was measured at a significant level of P <0. 01. Results Statistical analysis showed that the percentage of regeneration of transgenic seedlings using Agrobacterium (LB4404) in the selected medium containing kanamycin was 33%. Also, PCR analysis of transgenic plants showed a prevalence of transgenics of 33%. In addition, amplification of the bp fragment indicated the accuracy of cloning in transgenic plants. The results indicate the successful transfer of this gene to the sesame plant to increase its industrial properties. The results of the analysis of variance showed that there was a significant difference in the expression of sesamin-producing genes between transgenic cultivar and control groups (P <0. 01). Conclusions Since pBI121-based expression vectors are more efficient than other expression vectors and are widely used in the transfer of recombinant genes to plants, the recombinant vector constructed in this study indicates successful gene transfer. Sesame to sesame plant to increase its industrial properties.

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.

Conventional pBI121-based binary vectors are widely used in transformation of higher plants mediated by Agrobacterium but they are useless in transformation of some monocots because of inefficiency of kanamycin in selection, while, hygromycin resistance gene is an important selectable marker that usually used in transformation of several plants, especially monocots. The aim of this study was to improve the pBI121 vector for transformation of monocot plants. For this purpose, the hygromycin resistance gene with the 35S terminator were isolated from p6-ubi-rnai plasmid and cloned into pBlueScript intermediate vector via SmaІ and NotI restriction enzyme digestion. The CaMV 35 promoter was isolated from pBI121 vector by using SmaI and HindIII enzymes and cloned upstream of the gene. By using HindIII and Eco53KI enzymes, the complete hygromycin resistance gene cassette was replaced the kanamycin resistance gene cassette (which digested by HindIII and MssI) of pBI121 vector. Construction of this vector was confirmed by PCR method, digestion pattern analysis, and sequencing. Due to the popularity of pBI121-based vectors than other binary vectors and the researchers' familiarity with their manipulation, the vector which is introduced in this study could be used in gene transfer studies of monocot plants.

Today, sucrose intake caused problems for people’s health. So, the demand for low-caloric natural sweeteners with good taste properties such as sweet proteins increased. Among the sweeteners, Brazzein is an attractive alternative to sucrose. Because of its intense sweetness, natural origin and good stability. Brazzein was isolated from the fruit of the African plant Pentadiplandra hrazzeana Baillon, that is impractical to produce economically on a large scale. Therefore, widespread commercial production of this protein will probably require the transfer of protein expression to a heterologous system by means of recombinant DNA technology. In this study, due to the unavailability of genome of P. brazzeana Baillon plant for Brazzein gene isolation, artificial synthesis of Brazzein gene was done by the assembly of PCR and SOEing PCR. Amino acid sequence of Brazzein protein was translated to 162 nucleotides based on the preferred codon usage of maize using the Emboss Backtranseq program. Then, by using six overlapping primers in five consecutive PCR reactions Brazzein gene sequence was synthesized. The results of the PCR reaction showed accuracy. Then the synthetic fragment was cloned into pBI121 plant expression vectors that contained a constitutive promoter CaMV35S and seed-specific promoter Napin, which was confirmed by nucleotide sequencing.

Exendin-4 (EX4) is a protein similar to glucan-like peptide (GLP-1) and has a longer half-life and greater effect in inducing insulin secretion. The fusion of EX4 to the non-toxic cholera subunit (CTB) increases its efficiency through absorption in the digestive system. In this study, CTB-EX4 gene was introduced into lettuce by Agrobacterium tumefaciens using vector pBI121. The results showed that 20.25% of the inoculated explants were successfully transformed and regenerated (T0 generation). To create T2 plants, seeds of T1 were cultivated, and 42% of the cultivated seeds germinated. T1 plants were analyzed using PCR test. The mRNA expression of the CTB-EX4 gene was examined through RT-PCR and Real-time PCR, while the protein expression was assessed using ELISA and Western Blot tests. The number of transferred copies to T1 plants was determined using Real-time PCR. This estimation was performed using both the absolute method and the standard curve, as well as the relative method, with Acitn gene as a reference gene. The results showed that 60% of the T1 plants were transgenic and the CTB-EX4 gene was expressed at the mRNA and protein levels. The results showed that the process of transferring the CTB-EX4 gene to the lettuce plant was successful and the lettuce plant was able to express the desired gene at the RNA and protein levels. Considering the subsequent processes, including the purification and evaluation of the biological activity of the produced protein, plants can be used as a suitable bioreactor for the production of recombinant proteins.

Morphinan group alkaloids in poppy are synthesized from two L-Tyrosine amino acids by 17 anzymatic stages through several continuous oxidative reactions. The first key enzyme on this path is L-Tyrosine decarboxylase (TYDC). Tyrosine decarboxylase is a common cyclic amino acid in plants which in different plant species, biosynthesis of a large number of primary and secondary metabolites depends on it. For over expression of this gene in opium poppy, a suitable gene construct named pBI121-TK was designed, so that Kozak Enhancer as a gene expression enhancer was added at upstream of TYDC2 gene’s start codon and was coloned in pTZ57R/T plasmid. Then, in GUS location under control of CaMV35S promoter and Nos terminator, it entered T-DNA area of pBI121 binary plasmid. Gene transfer optimization by GUS histochemical assay in agroinfiltrated plants was done by pBI121 plasmid containing GUS gene and eventually the agrobacterium containing pBI121-TK recombinant construct was transferred to six-week long plants according to the optimized condition. Amount of morphinan alkaloids was measured by HPLC device. The obtained results indicate 75.2% increase of morphine, codein and thebaine in recombinant sample relative to the control sample.