Evolutionary Analysis And Functional Study Of BnaSDG26 And BnaRING1a/B In Brassica Napus L

Brassica napus(A_nA_nC_nC_n),one of the most important oil crops in China,was formed by natural hybridization between Brassica rapa(A_rA_r)and Brassica oleracea(C_oC_o),a process known as allopolyploidy.The reproductive development of rapeseed directly affects the yield that could closely relate to the safety of edible oil.Previous studies have shown that histone methylation and recognition played an important role in reproductive development.Histone methyltransferase,AtSDG26,as a“writer”catalyzing H3K27me3 where was read by PRC1 as a“reader”(one of core component is AtRING1a/b)to regulate the expression of target genes,further effected on the flowering time and flower organs formation.In this study,the evolutionary characteristics of BnaSDG26 and BnaRING1a/b during polyploidization in Brassicas and their functions as well as the effects on the development of rapeseed were systematically demonstrated by bioinformatics analysis and molecular biology techniques.The result follows:Evolutionary analysis and functional research of BnaSDG26:(1)Three BnaSDG26 homologous genes were identified in B.napus,BnaSDG26.A1,BnaSDG26.A2 and BnaSDG26.C.During the formation and evolution process of rapeseed,it is concluded that BnaSDG26 gene in A_n subgenome had been replicated and this replication formed an incomplete product,BnaSDG26.A1.(2)The BraSDG26,BolSDG26,BnaSDG26.A2 and BnaSDG26.C genes were cloned,and the full-length CDS of each gene were highly consistent with the Brassica Database.Compared with AtSDG26 protein sequence,they were largely different.There were 8 and 2 amino acids changed in the core functional domain SET and post SET respectively,and partial deletions in the non-domain region.(3)The BnaSDG26 homologous genes were subjected to CRISPR/Cas9-mediated genome edit,and 16 gene-edited mutants of T1 generation were obtained.11 of them with three genes(BnaSDG26.A1/A2/C)were effectively edited at the same time,accounting for72.72%of the total number of mutants.In the T1 population,transformants harbored homozygous,heterozygous,or bi-allelic mutations.The genetic segregation ratio of heterozygous mutant in T2 conformed to the law of Mendelian segregation.(4)The four SDG26 homologous genes in(2)were heterogeneously expressed in Arabidopsis T-DNA mutant sdg26-1 and wild type Col-0,respectively.It was found that none of the four heterologous SDG26 could rescue the late flowering phenotype of sdg26-1,and it could not significantly change the number of rosette leaves before Col-0 bolting.The expression pattern of key flowering time control gene At FLC and At SOC1,showed that SDG26 homologous genes had no significant effect on the flowering time control in Arabidopsis.The function of BnaSDG26 may be different.T2 gene-edited mutants showed the same bolting time with the control without any obvious phenomenon of earlier or later flowering.The expression relationship of flowering inhibitor Bna FLC and flowering promoter Bna FT,Bna SOC1 was not found.It could explain and corroborate with the results of Arabidopsis functional complementarity experiment.This result indicated that BnaSDG26 also had no significant effect on flowering time control in B.napus.(5)At the same time,it was found that the inflorescence of gene-edited mutants was more developed than the wild-type,and the number of secondary branches was more than control(XY15/1.5,T2-H4/12,T2-H27-1/11,T2-H10/5.3 branches).It was suggesting that BnaSDG26 may be involved in regulating the development of inflorescence and secondary branches of rapeseed.Based on the transcriptome sequencing analysis,there were 24differentially-expressed genes enriched in hormone signal transduction pathway based on KEGG analysis,suggesting that the function of BnaSDG26 genes may be related with plant hormone.Based on the above results,we concluded that BnaSDG26 was functional divergent compared to AtSDG26 in flowering time control.Evolutionary Analysis and functional Research results of BnaRING1a/b:(6)RING1a gene replication was active in evolution.Two genes(Bra RING1a-1 and Bra RING1a-2)and 1 gene(Bol RING1a)were found in B.rapa and B.oleracea.There were five copies(BnaRING1a.A1/A2 and BnaRING1a.C1/C2/C3)found in B.napus.BnaRING1a.A1/Bra RING1a-1,BnaRING1a.A2/Bra RING1a-2,BnaRING1a.C1/Bol RING1a were orthologous,respectively.BnaRING1a.C2/C3 were paralogous,and the replication source was BnaRING1a.A2.RING1b was relatively conservative compared to RING1a,with two orthologous genes in B.napus which came from B.rapa and B.oleracea.(7)BnaRING1a.A1/A2 and BnaRING1a.C1/C3 genes were cloned respectively,and the similarity compared with Brassica napus database were higher than 98%.The BnaRING1b.A and BnaRING1b.C genes were cloned,and the similarity compared to the database were 99.2%and 97.6%,respectively.The analysis of homologous genes showed that both RING1a and RING1b proteins had a significant conservative RING domain which was the C3H1C4 finger structure.(8)The transgenic rapeseed of BnaRING1a and BnaRING1b genes was obtained by RNAi technique.The phenotype of RNAi transgenic rapeseed showed that it is extremely similar to the Arabidopsis double mutant ring1a;b,indicating that the function of BnaRING1a/b was relatively conservative and there was no functional divergence in the process of polyploidy.