Study On Construction Of Expression Vector PCAOxO-PMI And Transformation Of Rapeseed

Rapeseed(Brassica napus L.) is one of the most important economic crops in the world.However,the production and quality of this crop has been greatly affected by the destructive disease,Sclerotinia sclerotiorum,worldwide.Meanwhile,in China,it is the top one of the three most devastating diseases of rapeseed.After long time practice,no completely resistant or immunized cultivars have been identified in rapeseed,as well as in the kindred species.Resistance to Sclerotinia sclerotiorum in rapeseed is quantitative trait and easily affected by environmental condition.No widely accepted resistance identification method exists.These questions make breeders difficult to select qualified resistant plants.Fortunately the successe on plant genetic engineering has brought light to the difficulty.People began to resort to it and several successful cases 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's resistance and obtain the resistant rapeseed,plant expression vector pCAOxO-PMI containing OxO from barley and PMI,which is consided as a safe selective marker from E coli,constructed successfully and Brassica napus were transformed by using agrobacterium tumefaciens harbouring the plasmid pCAOxO-PMI.The main work and results were as follows:1.Isolation OxO and PMI genesWe made use of RT-PCR and PCR to clone OxO and PMI from barley and E coli. respectively.After sequenced and blasted,the homology between genes we isolated and the sequences published is up to 100%,and both of them have complete ORF.2.Vector construction and transform into agrobacteriaPlant expression vector pCAOxO-PMI containing OxO and PMI was constructed based on the plasmid pCAMBIA 1301,then it was transformed into agrobacteria. 3.Definition of mannose content in selective-mediaAter tested the sensitivity of rapeseed to mannose,we definite mannose content in selective-media should gradually raise from 0.5%,1%to 1.5%.4.Obtaining of mannose-resistant regenerated plantsBrassica napus ChuanYou21 were used for transformation experiment.The hypocotyls from Brassica napus were transformed as acceptor.Bacterial culture(optimum density:OD600≈0.3～0.4) of Agrobacterium strain was diluted to 3～5 times,explants immerged in it for 3～5 mins.After cocultured with Agrobacterium,selected by mannose, ChuanYou21 mannose-resistant regenerated plants of Brassica napus were obtained.PCR identification of the mannose-resistant regenerated plants with gene-specific- primers showed 11 of 14 mannose-resistant regenerated plants of Brassica napus gave the expected fragment as the positive control.OxO had been integrated into Brassica napus genomes definitely.