The Construction Of The Transformation System For Sclerotinia Sclerotiorum Mycelia And The RNA Interference Vector For ISO & OXA Gene

In China,brassica napus's quality and product suffered a severe negative influence coursed by Sclerotinia sclerotiorum,a fungus disease,chronically.It was hard to study disease-coursed mechanism of Sclerotinia sclerotiorum through spore transformation,because indoor condition restrained the product of spore.We were eager to instruct a convenient Sclerotinia sclerotiorum -genetic transformation and get further knowledge of disease-coursed mechanism.Therefore,we,using plasmid B7 as exogenous gene and Sclerotinia sclerotiorum mycelia as explant,completed the system of Sclerotinia sclerotiorum mycelia-genetic transformation,through the Agrobacterium tumefaciens Mediated Transformation(ATMT) that help us to gain the fluorescently-labeled-protein mycelia and sclerotia,which grew as normal ones.To demonstrate the relationship between oxalic acid and infected-plant,we cloned two the two fragments by use of 3'-end-linking-to-5'-end way to form a longer complex fragment involving oxaloacetate hydrolase and Isocitrate lyaserespectively. Their length were 303bp and 167bp.Then,we construct their both RNAi vector for silencing the expression of the two genes.It certified success of construct RNAi vector,through digest by enzyme analyse and PCR.