Overexpression of Alfalfa Glutamate Semialdehyde Aminotransferase Gene in Tobacco and Analysis of its Physiological Results

The chlorophyll biosynthesis pathway is a main target for genetic modification to change plant photosynthesis and growth rate to support a greater demand for food in the growing world population. In this study, the effect of overexpression of GSA gene one of gene involved in biosynthesis pathway of chlorophyll on physiological condition of tobacco plant was investigated. 5-Aminolevulinate (ALA) is product of GSA gene. ALA is a precursor for all tetrapyrrole, these components have impotent roles in living cell, as pigments, light receptor (Phytochrome), prosthetic group of many different proteins (like cytochromes, hemoglobin, myoglobin, and leghemoglobin) and enzymes (for example Catalase, Ascorbate, Peroxidase and etc.). Nowadays, ALA has received wide attention for its widespread usage in agriculture, forestry and medication. ALA at low concentrations increases photosynthesis, growth, development, yield and productivity, also promoted fruit color appearance and quality and taste of products in treated plants under both normal and stressful conditions. ALA also improves antioxidant features, absorption of nutrient, water use efficiency and osmotic balance in plants.

 In this research, according to bioinformatics studies, MsGSA gene cDNA of Alfalfa (Medicago sativa L. cv. Isfahani) was selected to transfer to Xanthi tobacco (Nicotiana tabacum) plant, the binary expression vector pBI121 which has Kanamycin antibiotic resistance gene for selection in bacteria and plants, cutting sites for SacI and BamHI enzyme, CaMV35S promoter (cauliflower mosaic virus promoter), nos transcription termination sequences and ß-glucuronidase (GUS) reporter gene was used. After constructing the gene construct pBI121-GSA and confirming the transfer of the construct using PCR cloning methods, enzymatic digestion and sequencing were performed, then the corresponding construct was transferred to Agrobacterium tumefaciens strain LB4404 using Agrobacterium with the gene construct. The corresponding gene was transferred to the tobacco plant genome and the transgenic plants were selected on the medium containing kanamycin and the presence of the gene was confirmed by performing PCR in the regenerated plants. The rooted transgenic sprouts were transferred to the soil. The level of GSA gene expression in the resulting transgenic plants was evaluated by real-time PCR, and their growth rate and biochemical content were also evaluated.

 It was observed that the growth of transgenic plants increased significantly depending on the level of GSA gene expression, and the content of ALA (aminolevulinic acid) and chlorophyll a, b and total chlorophyll, which are the products of the corresponding gene expression. The results also showed the content of anthocyanin, flavonoids and phenol of plants have significantly increased in proportion to the increase in GSA gene expression compared to wild type tobacco plants.

 The growth rate as well as the content of chlorophyll a, b, total chlorophyll, ALA, anthocyanin, flavonoids and phenol of transgenic GSA plants is proportional to the increase in the expression of the GSA gene. The results from this study indicate that an increase in transgenic growth rate as well as an increase in the secondary metabolites content in transgenic plants were influenced by GSA transferred gene and an increase in the content of ALA. These results imply that transgenic tobacco plants expressing MsGSA gene had higher resistance potential to stresses than the wild type tobacco plants.