068 Development of the Bacterial Disease Resistant Potatoes by Introduction of Shiva Gene

Abstract

Antimicrobial peptide gene (shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) was transformed into potato (Solanum tuberosum L.) plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified its nucleotide seqnence to increase antimicrobial activity. Phenylalanine ammonia lyase 5 (PAL5) gene was known to express highly by wounding, irradiation, and infection by pathogens. It also expresses specifically on vessel tissues of young roots, stems, and leaves. The vector with shiva and CaMV35S promoter was also introduced into potatoes. The efficiency of regeneration was maximized at the medium containing Zeatin 2 mg/L, NAA 0.01 mg/L, GA3 0.1 mg/L. Putative transgenic potato plants were cultured on the media containing kanamycin 50 mg/L. From the tissue extracts of putative transgenic plants, GUS activity was assayed using 4-methylumbellyferyl glucuronide (MUG) as a substrate for GUS enzyme. In several transformant, GUS activity was 20- to 40-fold higher than non-transformants. Especially, one clone with CaMV35S promoter expressed ≈400-fold higher GUS activity than nontransformants. For histochemical in situ localization of GUS activity, chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide(X-gluc) was used for staining. GUS was highly expressed in the whole tissue of the transformants under CaMV35S promoter, but the other side GUS was expressed especially in the vascular tissues of stems and leaves of transformants with tPAL5 promoter. PCR was carried out at 94 °C for 20 s, 52 °C for 20 s, and 72 °C for 60 s with 45 cycles, using NPTII gene-specific primer set. PCR amplification by NPTII-specific primers confirmed 0.5-kb band in most transformants.