Study On Construction Of Expression Vector PCAMOXO And Transformation Of Brassica Napus

Rapeseed(Brassica napus L.) is one of the most important industrial crops in the world.However,this crop has been suffering from the destructive disease of Sclerotinia sclerotiorum for a long time.In China,Sclerotinia sclerotiorum is the first one of the three most serious diseases of rape and affects the rape production greatly.After long time practice,no completely resistant or immunizing cultivars have been identified in rapeseed,as well as in the closely related species.Resistance to Sclerotinia sclerotiorum in rapeseed is quantitative trait and easily affected by environment condition.There is no accepted widely identification resistance method.These questions make breeders difficult to select really resistant plants Fortunately the successe on plant genetic engineering has brought light to the difficulty.People began to resort to it and several successful results have been reported recently.It is a fundamental way to seek control method based on the determinant factor of pathogenesis.Aiming at oxalic acid,the major pathogenicity factor of S.sclerotiorum,the introduction and expression of genes encoding enzymes that degrade oxalic acid is a potential powerful strategy to enhance resistance to the fungi that secrete oxalic acid.In order to study the possibility of utilizing oxalate oxidase gene(OxO) to increase plant resistance and obtain the resistant rape,plant expression vector pCAMOXO containing OxO from wheat is constructed successfully and Brassica napus were transformed by using Agrobacterium tumefaciens harbouring the plasmid pCAMOXO.The main work and results were as follows:1.Plant expression vector pCAMOXO containing OxO from wheat is constructed by the plasmid pCAMBIA1301.2.Establishment of Agrobacterium tumefaciens-mediated transformation systems Brassica napus ChuanYou21 were used for transformation experiment.The hypocotyls from Brassica napus were transformed as acceptor.Optimum Agrobacterium tumefaciens-mediated transformation systems were established.Optimum medium for Brassica napus transformation:â… .pre-culture medium.MS+1mg/L 2,4-D+0.2mg/L 6-BA,pH 5.8â…¡.differentiation medium F₁:MS+2.5mg/L 6-BA+0.2mg/L NAA+400mg/L Cb+ 6mg/L AgNO₃,pH 5.8â…¢.selective medium S₁/S₂:F₁+10,15mg/L Hyg,pH 5.8â…£.root-inducingmedium:1/2MS+0.1mg/LIBA+15mg/LHyg+400mg/L Cef,pH5.83.Obtaining of hygromycin-resistant regenerated plantsBacterial culture(optimum density:OD₆₀₀≈0.3～0.4) of Agrobacterium strain was diluted to 3～5 times,explants immerged in it for 3～5 min.After cocultured with Agrobacterium,selected by hygromycin,ChuanYou21 hygromycin-resistant regenerated plants of Brassica napus were obtained.4.Molecular biology characteristic of hygromycin-resistant regenerated plants PCR identification of the hygromycin resistant regenerated plants with gene-specific-primers showed 8 of 3 hygromycin-resistant regenerated plants of Brassica napus gave the expected fragment of 470 bp as the positive control.OxO had been integrated into Brassica napus genomes initially.