Enhanced Resistance To Disease In Tobacco Via Agrobacterium-mediated Transformation Of GbNPR1 Gene

It is generally known that tobacco disease can result in the losses of yield and quality, and one of practical channels is to improve the resistance of tobacco by using disease resistance gene engineering, including the application of key factors closely related to disease resistance in plants such as non-expressor of pathogenesis-related genes1(NPR1). In the present study, GbNPR1 gene was isolated from sea island cotton and two vectors, 35S-GbNPR1 and CUT1-GbNPR1 were constructed. The target gene was separately drove by constitutive promoter 35 S and epidermal-specific promoter CUT1. The two vectors were transferred into tobacco via Agrobacterium-mediated transformation. The result of resistance assays demonstrated that transgenic tobacco have been significantly improved in resistance to brown spot, anthracnose and black death diseases. Transgenic tobacco plants with CUT1-GbNPR1 vector showed slightly lower resistance to those three diseases than the transgenic plants with 35S-GbNPR1 vector, but both showed better resistance than the wild-type plants. The results are listed as following:1.CUT1 promoter was successfully isolated from rice, and used to construct two vectors of 35S-GbNPR1 and CUT1-GbNPR1. The two vectors were transferred into tobacco via Agrobacterium-mediated transformation in obtaining two different type of transgenic tobacco plants.2. PCR analysis of T0 andT1 transgenic plants showed that GbNPR1 gene and CUT1 promoter have been integrated into nuclear genome of tobacco. RT-PCR analysis of T1 plants displayed GbNPR1 gene already expressed at transcriptional level. The bar test strip result of PCR-positive plants also confirmed that bar gene closely linked with GbNPR1 gene can normally transcript and translate into protein.3. The T1 plants with positive result of RT-PCR were subjected to conduct in vitro innoculation experiment of three species of tobacco diseases. The leaf of wild-type plants infected with Alteraria alternate turned yellow and gradually wilted, but transgenic plants with two vectors had no apparently changes. The wild-type plants infected with Colletotrichum micotianae appeared white spots in leaves and the center of infected areas sank obviously. Transgenic plants with 35S-GbNPR1 vector, however, didn’t show any changes. The leaves of transgenic plants with CUT1-GbNPR1 vector turned slightly yellow around the infected areas where also sank in the middle, but didn’t appear lethal spots. The wild-type plants inoculated with Colletotrichum capsici wilted obviously in the infected areas, and tend to spread around. The transgenic plants performed similarly like the plants infected with Colletotrichum micotianae. All above results demonstrated that transgenic tobacco plants exhibited better resistance to the three fungal diseases compared to the wild-type palnts despite the difference in resistant level. In the present study, the two kinds of transgenic tobacco plans with GbNPR1 displayed better resistance to diseases than the wild-type tobacco plants. However, the constitutive promoter showed better performance than epidermal-specific promoter when comparison of disease resistance between the two kinds of transgenic plants.