Application Of Host Induced Gene Silencing In Improving Resistance Of Rapeseed To Sclerotinia Sclerotiorum

Oilseed rape?Brassica napus L.?is one of the most important oil crops in China,which has high edible value and economic value.Sclerotinia sclerotiorum is one of the most important diseases in rapeseed,which causes great losses to rapeseed production.At present,no natural germplasm resources with complete resistance to Sclerotinia have been found,so breeding resistant varieties is the necessary way to control this disease.Host induced gene silencing?HIGS?is a technique based on RNA interference.In the process of interaction between the host and the pathogen,the small interfering RNA?siRNA?or double stranded RNA?dsRNA?produced by the host is ingested by the pathogen,and the corresponding gene expression of the pathogen is silenced,so as to reduce the pathogenicity of the pathogen and make the host obtain resistance to the pathogen.In order to explore whether the HIGS technology can be used to improve the resistance of rapeseed to S.sclerotiorum,the cell wall degrading enzyme and oxalate synthesis related genes,which are important pathogenic factors of S.sclerotiorum,were selected as the target genes in this study.The main results of this study are as follows:1.Through transcriptome analysis,we found that when S.sclerotiorum infects the stems of susceptible line?S-line?and resistant line?R-line?,the genes related to the synthesis of two major pathogenic factors?cell wall degrading enzyme and oxalic acid?of S.sclerotiorum:polygalacturonate endonuclease gene?SS1G₁0167,PG?,cellobiose hydrolase gene?SS1G₀9020,CBH?and oxaloacetate hydrolase gene?SS1G₀8218,OAH?was highly expressed.The expression of these three genes has been increasing with the time of infection,indicating that they play a very important role in the pathogenicity of S.sclerotiorum;2.We designed the interference fragments based on these three genes,and successfully synthesized the corresponding dsRNA It was found that all the three dsRNA could inhibit the expression of the corresponding genes,but not affect the growth of S.sclerotiorum;The dsRNA of the above three genes was dripped on the tobacco,and then inoculated with S.sclerotiorum.At the same time,the dsRNA of GFP was used as the control.It was found that after the application of the dsRNA of PG,CBH and OAH,the lesion was significantly smaller than control.;3.We constructed the corresponding RNAi expression vector and transformed it into rape to obtain transgenic rapeseed with stable expression of interference fragments.Through small RNA sequencing,a large number of corresponding siRNA were found in transgenic rapeseed,indicating that transgenic materials could successfully express corresponding siRNA.The RNAi interference materials of the three genes and the transgenic receptor material‘J9712'were inoculated respectively.By comparing the lesion size,we found that the lesion size of the transgenic material,whether cotyledon or leaf,was significantly smaller than that of the receptor material.In the experiment of cotyledons inoculated with bacteria,we took cotyledons of different periods after inoculated with bacteria for microscopic observation.Microscopically,it was found that the colonization efficiency of S.sclerotiorum was low,the formation of infection mat was inhibited,and the mycelial expansion was also inhibited.Therefore,the resistance of transgenic rapeseed to S.sclerotiorum was enhanced.4.OAH is involved in oxalic acid synthesis of S.sclerotiorum.We add OAH-dsRNA to the liquid medium to culture S.sclerotiorum and detect the accumulation of oxalic acid.Compared with the control?GFP-dsRNA?,the oxalic acid synthesis of S.sclerotiorum was significantly inhibited by adding OAH-dsRNA,and the oxalic acid content per unit volume of culture medium was significantly lower than that of the control.At the same time,the accumulation of oxalic acid in the infected parts of transgenic materials was detected.It was found that the oxalic acid content in the diseased leaves per unit mass was significantly lower than that of the control materials.In conclusion,using HIGS strategy to interfere with the important pathogenic genes of S.sclerotiorum can significantly inhibit its infection ability and improve the resistance of rapeseed to this disease.HIGS technology provides new ideas and new germplasm for the improvement of rapeseed's disease resistance.