| Brassica napus L.is an important source of edible vegetable oil in china,Sclerotinia sclerotiorum（Lib.）de Bary is the most restricting factor for the production.At present,molecular breeding is an important strategy to solve S.sclerotiorum.However,for a long time,people have not clear the molecular mechanism of B.napus-S.sclerotiorum disease resistance,which greatly restricts the molecular resistance breeding work of B.napus-S.sclerotiorum disease.The research on various plant-pathogen interactions shows that plant WRKY transcription factors play an important role in plant defense.In Arabidopsis,researchers have found that WRKY75 transcription factor can enhance the stress resistance to S.sclerotiorum and oxalic acid.However,in the research on the molecular mechanism of B.napus-S.sclerotiorum disease interaction,the biological role of WRKY transcription factor in plant resistance is still rarely researched.In this study,we cloned BnaWRKY75from B.napus,and carried out research on its resistance to S.sclerotiorum disease.The main results are as follows:1.First,cloned BnaWRKY75 from B.napus.BnaWRKY75 consist of 147 amino acids with an isoelectric point of 9.30 and a molecular weight of 16.98 KDa.Sequence analysis showed that this protein contains a highly conserved WRKY domain and Cx4Cx23Hx H zinc finger structure,which belongs to the Group II WRKY transcription factor;BnaWRKY75 is a relatively unstable hydrophilic protein,without transmembrane domains and signal peptides,and contains 12 Potential O-glycosylation sites,1 N-glycosylation site,36 phosphorylation sites.2.The expression BnaWRKY75 was rapidly and significantly increased at 6 hpi（hour（s）post inoculation）after S.sclerotiorum treatment.BnaWRKY75 had the highest expression in leaves,followed by flowers.These results indicate that BnaWRKY75 is a rapid response gene for S.sclerotiorum infection and may play a role in organs such as leaves and flowers.3.According to the analysis and prediction of Plant-PLoc Server,BnaWRKY75 is a nuclear localization protein.For further experimental verification,we fused BnaWRKY75 with e GFP to transform rapeseed,and conducted subcellular localization analysis in the transformed plant.The results showed that BnaWRKY75 was located in the nucleus,which was consistent with the predicted results.4.In order to further study the function of BnaWRKY75 in nuclear disease resistance,an overexpression vector was constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation.The evaluation and analysis of disease resistance showed that the disease spot area was significantly smaller than wild type.The trypan blue staining experiment showed that the hyphae growing on the leaves of the transformed plants were short and curly,while the hyphae on the untransformed plants were long and stretched.These results indicated that BnaWRKY75overexpression significantly improved the resistance to S.sclerotiorum.5.In order to test whether BnaWRKY75 has the function in S.sclerotiorum resistance,an overexpression vector was transformed into rapeseed by hypocotyl tissue culture,and 9 positive transformed lines were obtained.The q RT-PCR analysis showed that the overexpression of BnaWRKY75 significantly upregulated the defense response marker genes Bn PAL,Bn PR1,Bn MYC2,Bn PDF1.2 and Bn EIN3 mediated by salicylic acid（SA）,jasmonic acid（JA）and ethylene（ET）.Therefore,our results suggest that overexpression of BnaWRKY75 may enhance SA,JA and ET mediated defense responses.6.Furthermore,the disease resistance evaluation analysis of transformed rapeseed lines showed that the disease spot area was significantly smaller than WT.DAB and NBT staining experiments showed that overexpression of BnaWRKY75 inhibited the accumulation of H2O2,but promoted the production of superoxide anion(O2·-).Our experiments showed that BnaWRKY75 overexpression not only increased the defense response mediated by SA,JA and ET after S.sclerotiorum infection,but also significantly improved the resistance,which indicated that BnaWRKY75 played an important role in the resistance to S.sclerotiorum7.To obtain the inhibited expression strain,we constructed RNAi and CRISPR/Cas9 vector,obtained 3 BnaWRKY75-RNAi positive transformants and 4CRISPR-Cas9-BnaWRKY75 gene editing plants,which laid a material foundation for further experimental analysis. |
