Expressing A Gene Encoding Oxalate Oxidase Shows An Involvement Of Antioxidant Enzymes And Higher Accumulation Of Nonenzymatic Antioxidant In Arabidopsis Thaliana

For some cereal plants, gene oxo that codes for the enzyme oxalate oxidase(OxO)(EC1.2.3.4) which catalyzes the degradation of oxalic acid (OA) to carbondioxide (CO2)and hydrogen peroxide (H2O2), plays an significant role not only inthe process of growth but also in that of defense response reaction. And theproduction of H2O2acts asa signal molecule involved in acclimatory signalingtriggering tolerance to various biotic and abiotic stresses,but it leads to programmedcell death (PCD) at high concentrations. More specifically, H2O2has been supposedto be involved in plant defense to stress by different means such as inducing defencegenes, reinforcing cell-wall, changing the membrane integrity and so on. Therefore,OxO was widely applied for improving plant disease resistance to Sclerotiniasclerotiorumthrough genetic engineering techonology.Sclerotinia sclerotiorum is akind of necrotrophic ascomycete which attacks more than400plant speciesthroughout the world especially crucifer. However, plants expressing oxo can notonly contribute to the resistance to Sclerotinia sclerotiorum but also help to defenseother types of pathogens which can secret OA. And also there were some reportsindicated that these plants could improve plants tolerance to abiotic stress such asdrought, heavy metal and so forth. Whereas, the important questions that remain tobe answered are how OxO works during the process of plant defense againstenvironment stress. So far, some cDNA microarrays and other molecular biologicalanalyses between transgenic plants carrying oxo gene and their susceptible parentline support that, the pathogen elicts lignin synthesis and not-yet studied G proteinsignaling pathways. And the induced denfense genes are mostly related to ophotosynthesis and oxidation, highlighting the importance of redox. Others alsoemphasized the H2O2play multiple functions in plant defence against pathogens, oroxidation genes/Salicylic acid (SA) was also involved.Herein, we designed an experiment to find out the relationship between plants’ own defense system including two parts: enzymic defense system and non-enzymicantioxidants in the basis of theory that ROS resulting in oxidative stress, is promotedin plants exposed to biotic and abiotic stressful conditions, and to protect themselves,plant cells are equipped with oxygen radical detoxifying enzymes, which includesuperoxide dismutase (SOD), catalase (CAT), peroxidase (POD)and non-enzymicantioxidants which including soluable sugar, proline and others. Thus, in order toclarify wheather the exogenous oxo gene had an effect on the plant originaldefenseresponses, we constructed transgenic Arabidopsis thaiiana.Firstly, through Agrobacterium-mediated transformation method and followedby selecting in PPT selective medium, staining in GUS buffer solution, verifying byPCR and RT-PCR, we finnaly got the transgenic Arabidopsis thaiiana expressingOXO stably. Then, we have detected their resistant ability to Sclerotiniasclerotiorum comparing with the wild type by two methods:Sclerotinia sclerotioruminoculation and OA resistance evaluation. After those tests, we subsequentlyinvestigated changes in antioxidant enzyme activities, proline and soluable sugarcontents using nitroblue tetrazolium (NBT), guaiacol, H2O2, acid ninhydrin andanthrone-sulfuric acid.Our main results are exhibited as follows:1. The Agrobacterium-mediated transformation method was optimized.We separately considered how did Agrobacterium growth phase and theintensity of Agrobacterium in the inoculaton medium affect the transformation rates.And the results were that when Agrobacterium grew to the phase that OD600was1.6, and the intensity (OD600) of Agrobacterium in the inoculaton medium was1.0,the Arabidopsis Floral-Dip transformation rate was the highest.2. The transgenic Arabidopsis plants steadily expressing OxO weresuccessfully constructed. And their resistance to Sclerotinia sclerotiorum was alsoconfirmed.The combination of Flor-dip and Drop-by drop transformation methodwere used in our experiments. And after the treatment of Agrobacteriuminoculation medium, seeds were selected in the PPT selective medium, and theresistant seedling was determined by GUS histochemistry stain method, PCR and RT-PCR techonique. And after acquiring transgenic plants, their enhanced resistanceto Sclerotinia sclerotiorumwas also proved through Sclerotiniasclerotioruminoculation and OA resistance evaluation.3. The induced changes of antioxidant enzyme activities, proline and soluablesugar contents were find12h after inoculated with Sclerotinia sclerotiorum intransgenic plants comparing with the wild type.Upon inoculation with pathogen, transgenic plants displayed considerably increasedSOD and POD activities (with up to173and209.9%comparing withnon-inoculated wild plants), SOD activity of the wild plants after inoculationwere also increased to106.3%, which was66.7%lower than the former,Whereas POD activity of those reduced slightly to90.5%comparing with that ofnon-inoculated wild plants. However, CAT activity in both of transgenic andwild plants were inhibited, which were only69.5%and61%comparing withthat of non-inoculated wild plants respectively. Relatively, proline and soluablesugar contents of the two groups were consistent, the former were up to113.6%and137.7%, and the latter were raised to204.4%and324.4%, respectively.But the contents of them in transgenic plants were significantly higher thanthe wild type.