Functional Analysis And Exploration For Candidate Genes Of Self-incompatibility Response In Brassica Napus

Self-incompatibility has been evolved to avoid self-fertilizing and promote outcrossing in higher plants,which helps to prevent self-degradation.Self-incompatibility is an important system which could be applied in breeding and genetics.Great improvements have been made in the molecular mechanism of self-incompatibility in Brassica napus recently.However,the downstream components involved in self-incompatibility signaling pathway remain largely unknown.Here,to study on the pollen-pistil interactions,we employed sequence analysis,genetic transformation,expression analysis,yeast two-hybrid,RNA-seq and other techniques.Bn CIMS1(Cobalamin-Independent Methionine Synthase 1)was identified and proved to be the positive regulator of self-incompatibility in B.napus,which provides a new insight into the study of self-incompatibility.Besides,we also demonstrated that At CIMS1 had a conversed function in regulation of self-incompatibility in Arabidopsis thaliana,which supports the possibility that Brassica and Arabidopsis species share a common self-incompatibility system.The main results are shown in this list:1.The role Bn CIMS1 plays in self-incompatibility of B.napusBoth the genetic region and the full-length amino acid sequences of CIMS1 and CIMS2 are highly conserved in Brassicaceae species analyzed by synteny and phylogenitec tree.Bn CIMS1 is expressed at the highest levels in stigma and stem tissues in B.napus Westar plant,while it is expressed at relative lower levels in other tissues.The expression of Bn CIMS2 could be hardly detected in all the tissues of Westar.Self-incompatibility was partially broken down by down-regulation of Bn CIMS1 through RNAi in B.napus,indicating that Bn CIMS1 is a positive regulator in self-incompatible response.Subcellular localization analysis showed that Bn CIMS1 is expressed in cytosol.To identify whether Bn CIMS1 interacts with self-incompatibility related genes,Bn SRK-1 and Bn ARC1,yeast two-hybrid was conducted.It turned out that Bn CIMS1 did not interact with either Bn SRK-1 or Bn ARC1,indicating that Bn CIMS1 might regulate self-incompatibility in an unknown pathway.2.Establishment of transgenic self-incompatible A.thaliana lines and the functional analysis of At CIMS1To creat self-incompatible A.thaliana lines,Ah ARC1(involved in self-incompatible response),together with Ah SRK13 and Ah SCR13 which belong to the most dominant S13 haplotype in self-incompatible A.halleri,was transformed into A.thaliana Col-0 plant.Several transgenic self-incompatible Col-0 lines were obtained.However,the transgenic lines were all self-compatible when they were transformed with just Ah SRK13 and Ah SCR13.These results indicated that ARC1 was required for self-incompatible response.To identify the functions of these two genes,hybrid F2 populations were established between the transgenic self-incompatible A.thaliana lines and cims1 or cims2 mutants,respectively.The individual lines of both the two F2 populations showed self-compatibility phenotype,implying that At CIMS1 and At CIMS2 played roles in maintaining the self-incompatibility in A.thaliana,and the role of CIMS1 was conserved in regulating self-incompatible response in both B.napus and A.thaliana.3.Functional analysis of BC and other candidate genes? Down-regulation of BC caused abnormal development of leaf in B.napus and A.thalianaLeaves in both of the transgenic Bn BC RNAi Westar lines and At BC RNAi Col-0 lines developed abnormally,with a severe shrunk phenotype.Besides,stigmas of the transgenic At BC RNAi lines showed defect in pollen adhesion.? The specificity analysis of SLR1 promoterThe promoter of SLR1 was cloned in B.napus Westar line.To test if SLR1 promoter functions specially in stigma in A.thaliana,it was fused to GUS reporter protein and transformed into A.thaliana.It showed that the expression of GUS could be visualized in stigma near anthesis,GUS was also expressed in tissues of whole seedlings except the cotyledons and in mature leaves,which proved that SLR1 promoter did not function specially in A.thaliana stigma.? Functional analysis of ANN1,FBA2 and MUR5The expression levels of ANN1(Annexin),FBA2(Fructose-Bisphosphate Aldolase 2)and MUR5(Reversibly Glycosylated polypeptide 2)were down-regulated specially in stigma of B.napus Westar line.However,no significant changes in self-compatibility were observed in all the transgenic RNAi lines.Besides,no other obvious phenotypes were observed,either.4.Time-course transcriptome analysis of compatible and incompatible responsesA time-course transcriptome analysis was employed to explore the gene expression characteristics following compatible and incompatible responses.The pollination stages were defined as early stage events and late stage events according to the distribution of differentially expressed genes(DEGs).It was also found that the signaling pathway of incompatible response might be more complicated than that of compatible response.We proposed hypothesis on the regulation mechanisms in pollen-pistil interactions based on the function annotation of DEGs.Among the most representative GO terms in metabolic pathways,all the genes,including Bn CIMS1 identified in this study,involved in SAM cycle were expressed highly in stigma.