Preliminary Functional Analysis Of Two Resistance-related Genes Against Sclerotinia Sclerotiorum In Brassica Napus

Our research mainly focuses on the genes BnaC03g17120D and BnaA 04g05470D according to the former selection of the resistance-related genes against Sclerotinia sclerotiorum in Brassica napus.BnaC03g17120D belongs to the GSTF family and BnaA04g05470D belongs to the SYP1 family.We cloned BnaC03g17120D,constructed two overexpression vectors(one has N-terminal tag and the other one has C-terminal tag)and transformed into the model plant Arabidopsis thaliana by Agrobacterium-mediated floral-dip transformation protocol.Then the T1 generation transgenic lines were selected with the soil containing BASTA.Expression and localization of the proteins in the overexpressed(OE)lines were observed by fluorescence microscope.It could be found stable expression of the aim protein.For BnaA04g05470D,on the basis of the former selection,we identified the positive OE transgenic T1 generation lines by means of fluorescence microscope.Then we inoculated S.sclerotiorum on T2 generation OE lines and stained with Trypan blue and NBT.The inoculation results showed that the average lesion size of 35S::BnaC03g17120D-GFP OE lines was 6.6 mm2,the average lesion size of 35S::YFP-BnaC03g17120D OE lines was 5.4 mm2,the average lesion size of BnaA04g05470D OE lines was 6.7 mm2,while the average lesion size of wild type was 11.5 mm2,so the lesion sizes of OE lines were apparently smaller than that of wild type.The staining results showed more dead cells and O2-accumulation in the wild type leaves than that in the transgenic ones.Because we haven’t got OE lines of B.napus effectively until now,we transient expressed 35S::YFP-BnaA04g05470D in B.napus and used western blot to detect the protein.The result showed the size of aim protein was right.These results indicates that the two genes are related to the plant disease resistance,and deserved to be further studied.In addition,we used SA,MeJA,ABA and OA to treat B.napus respectively.The relative expression level of SYP1 subfamily genes were quantitated through real-time quantitative PCR.The results showed that some genes were up regulated with specific chemical induction.It’s most likely that they are the signaling pathway related genes.It provides the basis and material for our further exploration of the S.sclerotiorum-B.napus interaction.