Function And Regulation Mechanism Analysis Of BnGLIP1.CO7 Gene On Sclerotinia Sclerotiorum Resistance

Rapeseed is one of the most important oil crops in China.Sclerotinia stem rot(SSR)is a host-nonspecific soil-borne fungal disease,caused by Sclerotinia sclerotiorum(Lib.)De Bary,which occurs in rapeseed-growing countries and regions all over the world.It is also a major disease restricting the high,stable,high quality and safe production of rapeseed in China.At present,breeding new rape varieties resistant to S.sclerotiorum is still the fundamental way to overcome SSR,but the lack of resistance sources and the complexity of resistance genetic mechanism have greatly restricted the breeding of disease resistance.Therefore,it is particularly important to excavate new genes for resistance breeding in rapeseed.In our previous study,the resistance-related gene BnGLIP1.C07 was screened by candidate gene association analysis,and its function was preliminarily verified in Arabidopsis thaliana.In this study,BnGLIP1.C07 overexpression,RNAi suppression and CRISPR/Cas9 knockout rape were used as materials to further verify the effects of the gene and key SNPs loci on S.sclerotiorum resistance,screen and identify upstream interaction transcription factors and their regulatory networks involved in disease resistance,and provide theoretical basis and potential candidate genes for molecular breeding of rape disease resistance.results are as follows.1.In order to verify the role of BnGLIP1.C07 gene in S.sclerotiorum resistance in Brassica napus,we first constructed BnGLIP1.C07 overexpression and RNAi suppression lines using hypocotyl genetic transformation system of B.napus and analyzed their resistance phenotype in T2 generation.The results showed that compared with the wild type,the overexpression lines of BnGLIP1.C07 inhibited the growth and cell death of S.sclerotiorum,and significantly enhanced the resistance of plants to S.sclerotiorum.The interference lines enhanced its susceptibility.In order to further verify the role of BnGLIP1.C07 in enhancing the resistance to S.sclerotiorum,we continue to use the hypocotyl genetic transformation system of B.napus to create CRISPR / Cas9 knockout lines of BnGLIP1.C07,obtain CRISPR / Cas9 knockout mutant plants,and conduct S.sclerotiorum inoculation on the T2 generation.It is found that glip1 mutant is more susceptible than the wild type.In order to further verify the role of BnGLIP1.C07 in enhancing the resistance of B.napus to SSR,we continued to use the hypocotyl genetic transformation system of B.napus to create BnGLIP1.C07 CRISPR/Cas9 knockout lines,obtained CRISPR/Cas9 knockout mutant plants and inoculated S.sclerotiorum in T2 generation,and found that knockout mutant was more susceptible than wild type.2.In order to further study the regulation mechanism of BnGLIP1.C07 in SSR resistance,we analyzed the promoter of BnGLIP1.C07 and screened and identified the upstream interaction transcription factors.Firstly,by cloning the promoter ProBnGLIP1.C07 and bioinformatics analysis of core transcription elements,many motif elements related to transcription and disease resistance were screened.The promoterdriven GUS reporter gene expression vector ProBnGLIP1:GUS was constructed and transformed into A.thaliana.GUS staining showed that the gene was expressed in roots,stems and leaves of A.thaliana.Transgenic Arabidopsis lines with ProBnGLIP1 : GUS fusion were treated with S.sclerotiorum and hormones,and it was found that GUS could be induced by S.sclerotiorum,ABA,Me JA and ACC.Three potential candidate transcription factors bZIP44,WRKY15 and RAP2-3 were obtained by screening the transcription factors combined with the core transcription motif of the promoter ProBnGLIP1 through yeast single hybridization and Next-generation sequencing.Furthermore,the point-to-point experiment of yeast single hybridization verified that these three transcription factors interacted with ProBnGLIP1.C07.3.In order to explore the changes in lipid composition and lipid molecules mediated by BnGLIP1.C07 after S.sclerotiorum infection,we used the non-targeted lipidomics analysis platform to compare and screen the differential metabolites between the wild type and the over-expression transformation strain of BnGLIP1.C07 gene and the CRISPR/Cas9 knockout strain after S.sclerotiorum infection.It was found that these differential metabolites were mostly glycerol phospholipids,glycerol lipids,and sphingolipids.Through KEGG enrichment analysis,it was found that the main pathways affecting lipid metabolism of B.napus after infection by BnGLIP1.C07 were autophagyother,glycosylphosphatidylinositol(GPI)-anchored biosynthesis,glycerophospholipid metabolism and glycerolipid metabolism.4.Based on the analysis of functional SNPs in BnGLIP1.C07,we speculated that the three SNPs A/G,C/G and C/T,which located in the exon of BnGLIP1.C07 and caused non-synonymous substitution,may be important mutation sites affecting the resistance of SSR.Therefore,we used two single-base editing systems CBE and pTF101-BE to transform B.napus through hypocotyl genetic transformation technology and obtained some positive transformants.