جستجوی مقالات مرتبط با کلیدواژه "pvx" در نشریات گروه "بیوتکنولوژی و ژنتیک گیاهی"

The use of plant viruses as carriers of anti-cancer drugs in medicine is increasing day after day. One of the most important potato viruses is Potato X Potexvirus (PVX) which is globally expanded and is one of the first plant viruses used as a carrier of anticancer drugs. In this research, in order to produce PVX particles, its Coat protein (CP) gene was expressed. First, the RT-PCR reaction was performed for CP amplification using specific primers containing the endonuclease digestion sites. Electrophoresis of the PCR product on agarose gel confirmed the amplified fragment with a length of 714 bp. Then the CP gene and pGBKT7 plasmid were digested by BamHI and EcoRI enzymes and after purification, ligation was done between them. Recombinant plasmid pGBKT7-CPPVX was transferred to E. coli bacteria by electroporation. After selecting the bacteria containing the recombinant plasmid by PCR and extracting the plasmids from them, sequencing confirmed the correctness of the cloned gene sequence. Then, the CP gene was cloned into pET-21a expression plasmid by digestion and ligation method. The recombinant plasmid was transferred to BL21 strain of E. coli and cultured. By adding IPTG in the bacterial culture medium, CP protein was expressed from the plasmid and bacterial proteins were extracted. Protein electrophoresis showed the expression of CP. After purification, this protein can be used as a carrier of anti-cancer drugs.

Viruses are the main causes of potato yield loss. Potato X Potexvirus (PVX) is one of the most important potato viruses and the use of resistant varieties is the principal way to control it. In this study, we tried to produce potato plants resistant to this virus using an RNA silencing technique. To this end, a hairpin construct of P25 of PVX was made in pFGC5941 under control of the CaMV 35S promoter with two consecutive cloning steps. The P25 protein is a suppressor of RNA Silencing in viral host plants. The recombinant vector was transferred into Agrobacterium tumefaciens LBA4404 strain. Leaf and internode pieces of potato (Agria variety) were transformed by Agrobacterium. Following induction of callus, shoot and root, regenerated transgenic plants were selected and micro-propagated. Molecular analysis by PCR on DNA of plants and by RT-PCR on their RNA confirmed the presence and the expression of the transgene. Transgenic plants were propagated and then inoculated mechanically by PVX in the greenhouse. ELISA results using PVX-coat protein antibody showed resistance of two transgenic lines to PVX. Molecular confirmation of these potato lines was performed using PVX coat protein primers by RTPCR on plant RNAs. The results of this research have led to the production of two independent lines of potato that are resistant to PVX and future work will involve analysis of resistance in field conditions and the production of their mini-tuber.