Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (i) - transformation vectors

Abstract

Coat protein gene of beet necrotic yellow vein virus (BNYVV) was isolated from inoculated sugar beet roots and leaves of Chenopodium quinoa and Tetragonia expansa, by RT-PCR and imuno capture RT-PCR. Specific primers were made to complement coat protein gene and untranslated leader sequence, so that two fragments were obtained: long (731 bp), which contained coat protein gene and leader sequence, and short (587 bp), with coat protein gene. Fragments were cloned in two plant transformation vectors: pCAMBIA 3301M and pCAMBIA 1304M, which were modified by removing multicloning site and NcoI restriction site at the 5' end of the reporter genes. Vector pC3301M had bar gene which confers resistance against the herbicide gluphosinate ammonium as selectable marker, and pC1304M had gene for resistance to antibiotic hygromycin. Three constructs were made from each vector: CPL, containing coat protein gene with leader sequence; CPS with gene for coat protein, and CPSas with coat protein gene in antisense orientation. All constructs were transfered to Agrobacterium tumefaciens strain LBA4404.