Study On Transformation Of Nicotiana Tabacum And Brassica Napus With Oxalate Oxidase Gene

Sclerotinia sclerotiorum is an important disease worldwide. In China, it is the first of thethree most 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 resistant rape,Nicotiana tabacum and Brassica napus were transformed by using Agrobacteriumtumefaciens harbouring the plasmid containing OxO from wheat. The main work and resultswere as follows:1. Establishmentof Agrobacterium tumefaciens-mediated transformation systems Nicotiana tabacum 97131 and Brassica napus 01701 were used for transformationexperiment. The leaf explants from Nicotiana tabacum and hypocotyls from Brassica napuswere transformed as acceptor. Optimum Agrobacterium tumefaciens-mediated transformationsystems were established. Optimum medium for Nicotiana tabacum transformation:I．differentiationmedium: MS + 4 mg/L 6-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 Cef (Cefotaxime), pH 5.8 Optimum medium for Brassica napus transformation:I．pre-culturemedium: MS + 1 mg/L 2,4-D + 0.2 mg/L 6-BAII．differentiationmedium: MS + 4 mg/L 6-BA + 0.2 mg/L NAA, pH 5.8 iiiIII．selective medium: differentiation medium + 25 mg/L Km + 500 mg/L Cb + 6 mg/L AgNO3, pH 5.8IV．root-inducingmedium: 1/2 MS + 2 mg/L IBA + 20 mg/LKm + 300 mg/L Cef, pH 5.82. Obtaining of kanamycin-resistant regenerated plants Bacterial culture (optimum density: OD600 0.3～0.4) of Agrobacterium strain was dilutedto 3～5 times, explants immerged in it for 3～5 min. After cocultured with Agrobacterium,selected by kanamycin, 15 kanamycin-resistant regenerated plants of Nicotiana tabacum and7 kanamycin- resistant regenerated plants ofBrassica napus were obtained.3. PCR identification of kanamycin-resistant regenerated plants PCR identification of the kanamycin resistant regenerated plants with gene-specificprimers showed 14 of 15 kanamycin-resistant regenerated plants of Nicotiana tabacum and 3of 7 kanamycin-resistant regenerated plants of Brassica napus gave the expected fragment of470 bp as the positive control.4. Sequencingof PCR products The PCR products cloned into PGEM?-T Easy Vector were sequenced. The sequencecomparison results showed sequence identities were 100%,OxO had been integrated intoNicotiana tabacum and Brassica napus genomes.5. Identification of resistance Treated with different concentrations of oxalic acid, transgenic plants showed a strongertolerance against oxalic acid than wild type, so the introduction and expression of OxO couldenhance the resistance to fungi that secrete oxalic acid. It has been demonstrated thatrepresentation of Brassica 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.