EFFICIENCY OF CO-SUPPRESSION AND RNA-SILENCING TECHNOLOGIES FOR IMPROVING PLUM POX VIRUS RESISTANCE OF TRANSGENIC PLUM (PRUNUS DOMESTICA L.)

Abstract

Two technologies based on co-suppression gene and RNA-silencing have been used for improving plants resistance to Plum pox virus (PPV). Binary vector pCamPPVcp which contained the selective hpt gene and ppv-cp gene in sense-orientation (driven by double 35S promoter) was used for realization of post-transcriptional gene silencing. Vector pCamPPVRNAi contained self-complementary fragments of ppv-cp gene (698 bp) driven by double 35S promoter and the hpt and gus genes. The fragments of the ppv-cp gene were separated by pdk-intron to produce a “hairpin” RNA structure in antisense-sense orientation. Seven independent transgenic lines with the ppv-cp gene and five transgenic lines with two inverted repeats of a ppv-cp gene fragment were produced. Stable integration of genes into plant genomes was confirmed by PCR analyses. We carried out infection of resulted plum plants by grafting of infected buds to them. The absence of PPV appeared for one line of St-pCamPPVcp and all lines of St-pCamPPVRNAi by RT-PCR and Western blot analyses. Detection by RT-PCR was based on primers targeting the 3’ untranslated region and the HC-Pro gene of PPV. Western-blot analysis was performed using rabbit polyclonal antibodies to PPV coat protein (Loewe, Germany). The field tests of transgenic plants are carried out in the testing areas of the All-Russian Research Institute of Fruit Cultivar Selection RAAS near Orel city now.