Study On The Function Of Brassica Napus BnNPR1 In Resistance To Sclerotinia Sclerotiorum

Brassica napus is the important oil crop that has a high economic value and is widely applied in food,chemical,mechanical,energy and other industries in China.Sclertinia sclerotiorum(Lib.)De Bary causes one of the three major diseases of rape which restricts the production of rape severely.Till now,we still know little about the the mechanism of oilseed rape in resistance to S.sclerotiorum.NPR1(nonexpressor of pathogenesis–related genes 1),a key node in plant defense signaling networks,is an important regulatory factor for plant defense response and plays an important role in a variety of host defense resistance mechanisms.And studies on resistance to pathogens in various plant hosts such as Arabidopsis thaliana,rice and tobacco have been explored.However,in the interaction between B.napus and S.sclerotioru,the role of NPR1 are still not studied or have not been reported.In order to investigate the role of B.napus NPR1(BnNPR1)in resistance to S.sclerotiorum.In this study,we firstly cloned the cDNA sequence of BnNPR1 from B.napus,and sequence analysis showed that the cDNA encodes a 579 amino acid residues protein with an ankyrin-like repeat domain and a BTB/POZ domain.qRT-PCR experiments showed that BnNPR1 was up-regulated obviously when B.napus was infected by S.Sclerotiorum.Further,the pharmacological approach showed that the gene was significantly responsive to SA and JA treatment in oilseed rape.Subcellular localization experiment in Nicotiana tabacum cv.Xanthinc leaves showed that BnNPR1 is a protein that is predominantly located in the nucleus.These data indicated that BnNPR1 is a response gene for S.Sclerotiorum infection,and it may be involved in SA and JA signal pathway.In order to further study the function of BnNPR1 in Sclerotinia sclerotiorum,the BnNPR1-inhibited RNA interference vector was constructed and transformed into the rape by In plant Agrobacterium-mediated method.By Kanamycin,PCR detection,we got 17 transgenic lines in which four independent transgenic lines(R-19-4,R-20-8,R-25-34,R-17-21)showed much lower levels of expression of the BnNPR1 gene than the untransformed control by qRT-PCR analysis,and the R-19-4?R-20-8?R-17-21 were used for further analysis.S.sclerotiorum inoculation infection experiments showed that the lesion area of BnNPR1-RNAi was bigger significantly than that of untransformed,indicating that the inhibition of BnNPR1 expression significantly reduced the resistance to S.sclerotiorum.DAB staining showed that the accumulation of H2O2 is increased in response to S.sclerotiorum infection evidently in BnNPR1-RNAi.Further experiments showed that while BnNPR1 inhibited,the expression of BnPR1,the marker gene for the SA-mediated defence pathways,was significantly down-regulated,whereas PDF1.2,EIN3 and ACS2,ACS7,the marker genes of JA and ET pathways,respectively,all showed up-regulated expression.The results show that BnNPR1 plays an important role in B.napus to S.sclerotiorum and suggest that the inhibition of SA defense response may be one of the main reasons that the depression of BnNPR1 results to decreased resistance to S.Sclerotiorum in BnNPR1-RNAi,and that SA signaling pathway plays an important role in resistance to S.sclerotiorum in B.napus.