The Evolution And Function Analyses Of BrMAM-3 And BrAOP2 Genes Involved In Glucosinolate Biosynthesis In Brassica Rapa

Brassica rapa,comprising a variety of vegetables and oil crops,has played important roles in people's daily life and has a long cultivation history in China.With the improvement of living standard,more and more attention has been focused on the nutritional qualities of vegetables.Glucosinolates are a class of nitrogen-and sulfur-rich secondary metabolites common in the family Brassicaceae(or Cruciferae).Some glucosinolates and their breakdown products(e.g.sulforaphane)act as anticarcinogenic compounds.However,progoitrin would cause goiter and has other harmful effects on animal nutrition.Thus,it will be advantageous to manipulate glucosinolate structures to alter the nutritional and economic,value of Brassica vegetables.The elongation and modification of the side chain during the process of glucosinolate biosynthesis generates diverse glucosinolate compounds.In Arabidopsis,Methylthioalkylmalate synthases(MAMs)are involved in amino acid chain elongation,and give rise to glucosinolates with diverse chain-lengths during the biosynthesis of methionine-derived glucosinolates.The AOP2 gene encodes an enzyme that plays a crucial role in catalyzing the conversion of beneficial glucosinolates into detrimental ones.B.rapa contains five syntenic MAM genes and three AOP2 genes via comparative genomics analysis.In the current study,we focused on the evolution and function analyses of the BrMAM-3 and BrAOP2 genes.The main results of this study are as follows:1.We analyzed the phylogenetic and synteny relationships of MAM genes from 13 sequenced Brassicaceae species.Based on these analyses,we propose that the syntenic loci of MAM genes,which underwent frequent tandem duplications,divided into two independent lineage-specific evolution routes and were driven by positive selection after the divergence from Aethionema arabicum.In the lineage ?species Capsella rubella,Camelina sativa,Arabidopsis lyrata,and A.thaliana,the MAM loci evolved three tandem genes encoding enzymes responsible for the biosynthesis of aliphatic glucosinolates with different carbon chain-lengths.In lineage ? species,the MAM loci encode enzymes responsible for the biosynthesis of short-chain aliphatic glucosinolates.2.Mutant complementation assay showed that BrMAM-3 increased the amount of total aliphatic glucosinolates.Detailed expression analysis revealed that BrMAM-3 was abundantly expressed in the accession of L143,but showed trace or none accumulation of this transcript both in seedling and reproductive stages of Z16 accession.The further study identified a natural occurring insertion(-1.2Kb)in the exonl of BrMAM-3 resulting in a non-functional allele.The insertion was then used to develop a PCR-based marker to screen a collection of B.rapa accessions finding that the species derived from Caixin,Turnip and Yellow Sarson,with high total aliphatic glucosinolates,has a functional allele of BrMAM-3.While the species derived from Chinese cabbage with lower total aliphatic glucosinolates has a non-functional allele.Furthermore,the similar results were found in the BILs population,which was used to position the QTL of controlling of aliphatic glucosinolate accumulation in B.rapa.Finally,the other three candidate genes(BrMYB28.1,BrMYB34.1 and BrMAM-5),involved in the main QTL controlling the aliphatic glucosinolates accumulation,were cloned and sequenced,finding that several SNP were just existed between L143 and Z16 accessions.These SNPs were either synonymous mutations or could not affect the characteristic of amino acids.According to the expression profile data,we found that BrMAM-4 expressed abundantly in Z16 accession,while it had trace expression in L143 accession,which was negative with their corresponding glucosinolates content.Thus,we got the conclusion that BrMAM-3 was the key candidate gene controlling the accumulation of aliphatic glucosinolates in B.rapa,and the natural occurring insertion(?1.2Kb)of BrMAM-3 could lead to the distinct glucosinolates accumulation among different accessions.3.The amino acid sequence alignments confirmed the presence of two conserved domains(DIOX_N and 2OG_Fe?_Oxy)at the N-terminal and C-terminal regions of the BrAOP2s respectively.In contrast to the two highly conserved domains,the middle part of the BrAOP2 proteins showed patches of homology.The phylogenetic analyses showed that all AOP genes in the sequenced species formed two major relatively distinct groups,AOP1 group and AOP2/AOP3 group.The gene structure analysis showed that the AOP1 genes shared high sequence similarity with DIOX_N and 2OG_Fe?_Oxy two conserved domains retained.The AOP2 and AOP3 genes had further evolved some specific motifs in the middle part of the protein.We confirmed that the three BrAOP2 proteins,located in the cytoplasm,were encoded by paralogs derived from the Arabidopsis AOP2 ortholog.Heterologous expression and enzyme assays as well as Arabidopsis mutant complementation studies showed that all three BrAOP2 genes encoded functional BrAOP2 proteins that converted the precursor methylsulfinyl alkyl glucosinolate to the alkenyl form.Our detailed expression analysis revealed that the BrAOP2 genes displayed overlapping but distinct tissue-and cell-specific expression profiles in B.rapa.