Cas9 vektörü/hücreye nüfuz eden peptit kompleksi ile nicotiana benthamiana yapraklarinda GSH2 geninin knock out edilmesi ve γ-GC üretimi

Gamma glutamate cysteine (γ-GC) is a compound with the potential for therapeutic and nutritional supplements, but current production methods are costly and inefficient. This thesis aims to produce this compound more efficiently by gene editing techniques in Nicotiana benthamiana plant leaves, This innovative method is believed to be an important parameter in the field of plant and health biotechnology. A novel technique was used to increase γ-GC production on N. benthamiana plants. In the first step, a specially designed CRISPR/Cas9 vector (pKI1.1R) and KLA-10 cell penetrating peptides (CPP) were used. The designed gRNA was combined with KLA-10 peptide to form a CPP-pDNA complex and the CPP-pDNA complex was infiltrated into N. benthamiana leaves at different N/P ratios. At this stage, the Agrobacterium-based transformation method was also used, and the efficiency of the selected method was checked. In our research, it was found that ratios above N/P:1 caused a necrosis in plant leaves. In both the Agrobacterium-based plant transformation method and the method developed, the desired mutation in the glutathione synthetase 2 (GSH2) gene was detected. However, molecular analyses showed that all results were mixed profile and a significant decrease in GSH amount was observed.