Construction Of Double-stranded RNA Interference Vector Against Potato Leafroll Virus

In this research,we extracted the potato leafroll virus(PLRV) RNA which was used for cDNA synthesis from virus-infected potato leaves,some specific PCR fragments which we had expected were obtained by reverse transcription and polymerase chain reaction(RT-PCR) amplification. With the insert these fragments in RNA interference zone on binary vector plasmid use two-step cloning method,we successfully construct double-strinded RNA interference vector for PLRV. The main results were showed as following:(1) The PLRV RNA were extracted form virus-infected potato leaves use the method of Trizol.(2) Six pair of DNA primer were disigned by analyzed the nucleotide sequence of potato leafroll virus. Five specific fragments about 240bp,270bp,360bp,380bp and 400bp were obtained by reverse transcription and polymerase chain reaction(RT-PCR) amplification.(3) Recombined the plasmid vector pFGC5941and pCAMBIA1300.Two specific fragments about 3.5kb and 8.9kb were obtained by cuting up the new vector with restriction endonucleases EcoRI and PstI, showed the proper construction of RNA mediate plasmid vector for potato transformation.(4) Insert amplified fragments to the pCADS1341 use two-step cloning method.After cut up these recombined plasmid with enzyme in order to ensure its correctness,we abtained three RNA interference plasmid vector ,named pCADS1341-PLRV1,pCADS1341-PLRV2,pCADS1341-PLRV3...