Differential Gene Expression Analysis Of Brassica Napus To Leptosphaeria Biglobosa Infection And Functional Study Of BnNAC61 Gene

Oilseed rape blackleg is a fungal disease,has caused serious yield loss in world.In China,only Leptosphaeria biglobosa was found,no L.maculas right now,L.biglobosa belongs to necrotrophic fungus,the incidence rate is about 10%,and the trend of gradual spread,but there is no corresponding high resistance variety in China.It is necessary to reveal the molecular mechanism of the interaction between oilseed rape and the pathogen L.biglobosa and to find the key resistance genes.In this study,The RNA-Seq technology was used to obtain differentially expressed gene in oilseed rape response of blackleg,we analyzed resistance genes and KEGG pathway,and combined the gene co-expression network,the function of resistance related gene BnNAC61 was selected and researched.The research results are as follows:(1)There were 3384,2270,3802,5811,6155,7153 host genes differentially expressed at 4,12,24,36,48,96 h respectively by RNA-Seq,using the soft of Short Time-series Expression Miner(STEM)and KEGG enrichment of differentially expressed genes,the results showed that dynamic change trend of genes in plant-pathogen interactions,protein kinases,glucosinolate synthesis and jasmonic acid synthesis pathway were firstly rising then decline.Further analysis of differentially expressed genes revealed the signaling cascade response pattern,and the transcription factors WRKY,NAC,bHLH and MYB played a key role in the disease resistance response of oilseed rape.The plant hormone Jasmonic acid/Ethylene signal plays a leading role in resistance,and the secondary metabolite Glucosinolates is an important substance involved in disease resistance.(2)The weighted gene co-expression network was constructed through the system biology method,and a total of 20 characteristic gene expression modules were obtained.Among them,6 modules were characteristic modules at different inoculation time points,reflecting the sequence of resistance reaction dominated by cell wall strengthening,hypersensitivity reaction,protease inhibitor expression and resistance gene expression,and the hub genes of three feature modules(12,48,96h)were excavated.(3)Based on the above results,it was found that BnNAC61 was up-regulated and had a high degree of connection with disease-resistant genes.BnNA C61 gene sequence was cloned from Brassica napus.Through bioinformatics analysis,gene CDS was 846bp long,encoding 281 aa,containing two copies of the same protein.The promoter of BnNAC61 was analyzed,and it was found that there were cis-acting elements in response to methyl jasmonate,abscisic acid,auxin and resistance.The fusion expression vector was constructed,and the subcellular localization of BnNAC61 protein was performed,and its expression was found in the nucleus and cytomembrane.The transcriptional self-activation activity of BnNAC61 was analyzed by yeast two-hybrid system,and it was found that the transcriptional activation region was located at 136-208aa.(4)The expression of BnNA C61 was analyzed and found to be expressed in roots,stems,leaves,flowers,silix and pistil in different tissues,with the highest expression in leaves.Under the stress of PEG6000,NaCl and 4℃,the expression of BnNA C61 increased first and then decreased.Under the treatment of ABA,salicylic acid(SA),methyl jasmonate(MeJA)and ethylene(ACC),except the ABA,expression level of BnNAC61 was basically up-regulated,but the expression level was the highest under the induction of methyl jasmonate and ethylene.(5)The over-expression vector and RNAi vector of BnNAC61 were constructed respectively to obtain transgenic plants through genetic transformation.Compared with the wild type,the over-expression transgenic plants had enhanced resistance to L.biglobosa,while the resistance of RNAi transgenic plants was weakened,indicating that BnNAC61 had a positive regulatory effect on the resistance of Brassica napus to L.biglobosa.To sum up,based on the number of differentially expressed genes,this research revealed oilseed rape and pathogen L.biglobosa interactions molecular mechanism,clarify disease-resistant mechanism of rapeseed in response to necrotrophic fungus L.biglobosa infection,the results provided theory basis to fully uncover the molecular mechanism of oilseed rape and L.biglobosa interactions,and accumulate oilseed rape genetic resources information for disease-resistant breeding.