Genetic engineering of nitrite reductase gene improves uptake and assimilation of nitrogen dioxide by Rhaphiolepis umbellata (Thunb.) Makino

Abstract

Hypocotyl sections of Rhaphiolepis umbellata (Thunb.) Makino were transformed by Agrobacterium tumefaciens bearing a binary vector pIG121-AtNiR, which contains cDNA of the nitrite reductase gene from Arabidopsis thaliana (Atni) under the control of a modified cauliflower mosaic virus 35S promoter and chimeric hygromycin phosphotransferase gene (hph). A 4% of the hypocotyl explants transfected with Agrobacterium formed hygromycin resistant adventitious shoots, and most of them rooted upon root induction treatment. The presence and expression of the introduced transgene in putative transgenic plants (12 months after transfection) were respectively confirmed by polymerase chain reaction (PCR) analysis using primers specific to Atni and by western blot analysis using anti NiR antibody. A total of 37 transgenic plant lines were obtained. Plants 33 months after the transfection were fumigated with 200� 50 ppb 15 NO2 under the natural light for one week in the fumigation chamber in a confined glasshouse. The amount of total nitrogen derived from NO2 (reflecting uptake of NO2) and that of Kjeldahl nitrogen derived from NO2 (reflecting assimilation of NO2) were determined using a mass spectrometer. One of the 9 transgenic plants tested was 1.6-2.0 times higher both in the uptake and assimilation of NO2 than non-transformed wild-type ones.