Expression Of Glucose Oxidase Gene And Oxalate Oxidase Gene In Oilseed Rape (Brassica Napus L.)

The fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary distributes widely and infects more than 400 plant species worldwide. In China, this disease causes significant yield losses of various important crops including oilseed rape, soybean, sunflower and Brassica vegetables. Oxalic acid (OA), a non-host-specific toxin, secreted by S.sclerotiorum, has been demonstrated to be a major phytotoxic and pathogenic factor. It is regretful that no germplasm resources in host plant species are found to be immune or effective to S. sclerotiorum.Glucose oxidase (GO), catalyses the oxidation of -D-glucose resulting in the production of gluconic acid and H2O2- Oxalate oxidase (OXO), a class of detoxification enzyme, catabolize OA and O2 into CO2 and H2O2. CO2 is a substrate of plant photosynthesis and can be refixed in plant. Gluconic acid can be reused through glucuronic acid cycles or phosphopentose pathway. H2O2, one of reactive oxygen species (ROS), are very important to plant both as antimicrobial substance to inhibit pathogen growth, and as signal molecules to trigger a series of plant defense events including hypersensitive response, expression of defense reaction genes and induction of systemic acquired resistance.Therefore, the introduction of GO gene into plants may become a new genetic engineering avenue to produce transgenic plants with broad -spectrum resistance to a wide range of plant pathogens. Similarly, there is significance in practice of making use of OXO gene to construct engineered plants resistant to S.slerotiorun.The plant expression vectors pBOXO and pBGO-OXO were constructed harboring the germin gene and germin+GO genes, respectively, driven by CaMV 35S promoter. pBOXO, pBGO-OXO, and a third plant expression vector pBGO, were used for Agrobacterium-medizted transformation of Brassica napus cv. 84039. A total of 27 independent transgenic plants have been obtained, including 12 oxo-transgenic plants, 9 go-transgenic plants, 6 oxo+go-transgeic plants.PCR and Southern assay demonstrated that the target genes were integrated into the plant genome.Enzymatic activity or/and ELISA analysis showed that the germin gene and the GO gene were expressed in transgenic oilseed plants, respectively. The amount of H2O2 in the GO-transgenic plant can be as high as 656 M per gram of leaves, which is more than 4.3 times higher than that in control plant. The value of OXO relative enzyme activity, expressed as OD550, is as great as 0.4512, which is more than 4.2 times greater than that in the non-transgenic plants.Detached leaf inoculation test was undertaken in order to assess the resistance to S.sclerotiorum of the transgenic oilseed rape lines. The average lesion area of GO-transgenic plants 3 days after inoculation was 12.9 cm2 while the control lesion was14.5 cm2. Variance analysis indicated that the disease resistance difference was significant at level of 5%between the go-transgenic line and the control. The evaluation of S. sclerotiorum-resislant performance of the oxo-transgenic lines as well as the oxo+go-transgenic lines is still in progress.