(1. Institute of Biotechnology , Zhejiang University, Hangzhou 310029，China； 2. College of Life and Environmental Sciences，Zhejiang Normal University, Jinhua 321004, China)
Abstract：Abstract: The cryptogein (Crypt ) gene was obtained by PCR amplification of genomic DNA of Phytophthora cryptogea and verified by DNA sequencing. A promoter of a rice (Oryza sativa ) phenylalanine ammonia-lyase (PAL ) gene, which was low-level constitutive induced by pathogen, and specially expressed in epidermal tissues, was selected to control the expression of Crypt gene. This promoter may exhibit a potential to inhibit the infection of pathogen from epidermal tissues. Meanwhile, for functional interaction of cryptogein protein with its outside membrane binding sites, the Crypt gene was fused to signal sequence of the extracellular pathogenesis-related PR1b protein of tobacco(Nicotiana tabacum ), The final construction was subcloned into a binary vector and transformed into tobacco by useing Agrobacterium-mediated transformation method. Twenty-two kanamycin-resistant tobacco plants were obtained by screening in selective medium with 100 mg/L kanamycin. PCR amplification and Southern blot analysis demonstrated that Crypt gene was integrated into tobacco genome. The transgenic plants were challenged separately, with black shank fungi (Phytophthora parasitica var. nicotianae ) , brown spot fungi (Alternaria alternata ), and wild fire bacterium (Pseudomonas syringae pv. tabaci ). More than half of the plants（68.2%）showed that the resistance was strongly enhanced to these pathogens. Results suggested that a low-level constitutive expression of Crypt gene in tobacco should have a potential use to generate a broad-spectrum disease resistant plants.
