Study On Transferring Oxalate Oxidase Gene (OxO) Into Nicotiana Tabacum And Double-low Brassica Napus By Agrobacterium-mediated Transformation

Sclerotinia sclerotiorum is an important disease worldwide. In China, it is the first of the threemost serious diseases of rape and affects the rape production greatly. In recent years, thestrategy of genetic engineering has been applied in enhancing rape resistance along with thedevelopment of technique of genetic engineering and establishment of rape transformationsystem. Sclerotinia can infect more than 400 plant species. It is a fundamental way to seekcontrol method based on the determinant factor of pathogenesis. Aiming at oxalic acid, themajor pathogenicity factor of S. sclerotiorum, the introduction and expression of genesencoding enzymes that degrade oxalic acid is a potential powerful strategy to enhanceresistance to the fungi that secrete oxalic acid. In order to study the possibility of utilizingoxalate oxidase gene (OxO) to increase plant's resistance and obtain the resistantrape, Nicotiana tabacum and Double-low Brassica napus were transformed by usingAgrobacterium tumefaciens harbouring the plasmid containing OxO from wheat. The mainwork and results were as follows:1. Establishment ofAgrobacterium tumefaciens-mediated transformation systemsOrdinary Nicotiana tabacum and Double-low Brassica napus were used for transformationexperiments. The leaf explants from Nicotiana tabacum and petioles from Brassica napuswere transformed as acceptor. Optimum Agrobacterium tumefaciens-mediated transformationsystems were established.Optimum medium for Nicotiana tabacum transformation:Ⅰ. differentiation medium: MS+4 mg/L6-BA+0.2 mg/L NAA, pH 5.8Ⅱ. selective medium: differentiation medium+100 mg/L Km(kanamycin)+500 mg/L Cb ??(Carbenicillin), pH 5.8Ⅲ. root-inducing medium: 1/2 MS+2 mg/L IBA+100 mg/L Km+300 mg/L Cb(Carbenicillin), pH 5.8Optimum medium for Double-low Brassica napus transformation:Ⅰ. pre-culturemedium: MS+1 mg/L 2,4-D+0.2 mg/L 6-BAⅡ. differentiationmedium: MS+2.5 mg/L6-BA+0.1 mg/LNAA, pH 5.8Ⅲ. selective medium: differentiation medium+10-15 mg/LKm+500 mg/LCb+6 mg/LAgNO3, pH 5.8Ⅳ. root-inducingmedium: 1/2 MS+2 mg/LIBA+20 mg/LKm+300 mg/LCb, pH 5.82. Obtaining of kanamycin-resistant regenerated plantsBacterial culture (optimum density: OD600 0.3～0.4) of Agrobacterium strain was used tohandling the transformation experiments. After cocultured with Agrobacterium, selected bykanamycin, 11 kanamycin-resistant regenerated plants of Nicotiana tabacum and 12kanamycin- resistant regenerated plants of Double-low Brassica napus were obtained.3. PeR identification of kanarnycin-resistant regenerated plantsPCR identification of the kanarnycin resistant regenerated plants with gene-specificprimers showed 9 of 1 lkanamycin-resistant regenerated plants ofNicotiana tabacum and 7of 12 kanamycin-resistant regenerated plants of Brassica napus gave the expected fragment of470 bp as the positive control.4. Identification of resistanceInculcated by detached leaves with mycelia of Sclerotinia sclerotiorum and treated withdifferent concentrations of oxalic acid, transgenic plants showed a stronger tolerance againstSclerotinia sclerotiorum than wild type, so the introduction and expression of OxO couldenhance the resistance to fungi which secrete oxalic acid. It has been demonstrated thatrepresentation ofBrassica napus treated with oxalic acid is highly identical to that of infectedby Sclerotinia sclerotiorum, so the oxalate oxidase can surely improve the resistance of rapeplant to Sclerotinia sclerotiorum.