همسانه سازی و تولید dsRNA همولوگ با ژن P6 ویروس موزائیک کلم گل

The cauliflower mosaic virus (Caulimovirus tessellobrassicae) is one of the important plant viruses that infect hosts from the Brassicaceae family, such as turnip, canola, broccoli, and cauliflower. This virus belongs to the genus Caulimovirus in the Caulimoviridae family, with a DNA genome of approximately 8000 base pairs. The cauliflower mosaic virus causes symptoms, including mosaic, vein clearing, vein banding, and leaf deformation. The P6 protein of this virus disrupts the activity of proteins DCL4 and DRB4, preventing gene silencing at the post-transcriptional level. In the present study, dsRNA homologous to the P6 gene and a non-coding region of cauliflower mosaic virus, designated P6-NC, was employed to induce the pathogen-derived resistance. The topical application of dsRNA was employed as an alternative to genetic transformations. The P6-NC sequence was first amplified using polymerase chain reaction, and cloned it into the pL4440 plasmid using the XhoI and NcoI restriction sites then it was transformed into Escherichia coli strain Top 10, resulting in the construct named pL4440-P6-NC. The construct pL4440-CaMV-P6-NC was then transferred to E. coli strain HT115 cells to produce the corresponding dsRNA. To confirm the accuracy of the cloning, colony PCR was performed using specific primers for the vector, and an expected fragment of approximately 600 nucleotides was amplified from three colonies. Following the induction of the T7 promoter with IPTG, dsRNA was extracted using the phenol/chloroform method. The treatment of Nicotiana benthamiana plants with dsRNA, followed by inoculation with the virus and assessment of viral symptom severity and virus replication, is currently underway.