Cloning And Comparative Genomic Study Of MYB4 Gene Families From Brassica Napus And Its Parental Species

Phenylpropanoid pathway produces a large number of biologically important secondary metabolites though several important branch pathways.One of them synthesizes lignins,which play fundamental roles in mechanical support,solute conductance and disease resistance in higher plants.Another important branch pathway synthesizes various flavonoid compounds.In addition to attracting pollinators and protecting plants from UV irradiation and attacks by fungi and animals,flavonoids also possess anti-inflammatory,antiallergenic,antioxidant or cancer preventive functions in human.Phenylpropanoid pathway also synthesizes sinapate esters,coumarins,chlorogenic acids,stilbenes and salicylic acid to act as antagonistic ingredients or signaling molecules.Manipulation of phenylpropanoid pathway metabolites has long been a hotspot.Arabidopsis thaliana MYB4(AtMYB4)is a R2R3-MYB family transcription repressor,at least it negatively regulates C4H gene expression in phenylpropanoid pathway.AtMYB4 negatively modulates sinapatc ester formation in the absence of UV-B light.So manipulation of MYB4 gene may provide some encouragenent for the use of overexpression of regulatory geness to elucidate the regulatory mechanism of plant secondary metabolites formation.Brassica napus,B.rapa and B.oleracea are important oil or horticaultural crops.In them,many agronomically imporant traits related to phenylpropanoid pathway are focuses of genetic improvenment pursued by researchers for many years.For example,the commonly occurred lodging problem calls for stronger stems and branches. Improvement of resistance to diseases needs quicker and enhanced cell wall lignification in response to invasion.Genetic engineering of lignin pathway flux,monolignol ratio and lignin composition provides a promising strategy to cope with these problems.In recent years yellow seed trait of oilseed rape has attracted many researchers due to its good quality.However,lacking of yellow-seeded genotypes together with instability of yellow seed phenotype has largely retarded breeding and application of yellow-seeded rapeseed.The mechanism of yellow seed trait formation of oilseed rape especially B.napus is still not clear,The most typical feature of yellow seed trait is the reduction of lignin and pigment contents in the seed coat.As has been revealed,plant seed coat pigments are polymers of proanthocyanidin,a metabolite of flavonoid pathway.Cloning of Brassica MYB4 genes and expression of them specifically in seed coat may came modification of seed coat pigments and seed coat lignins of rapeseed.In family Brassicaceae,except the characterized MYB4 gene from Arabidopsis thaliana(AtMYB4),no other full-length MYB4 gene has been cloned,though many important oilseed and vegetable crops are included in this family.This dissertation reports the cloning,molecular characterization,and comparative genomic analysis of MYB4 gene families from B.napus and its parental species B.rapa and B.oleracea.In this research,full-length cDNAs and/or genomic sequences of 3 members of B.napus MYB4(BnMYB4) gene family,4 members of B.rapa MYB4(BrMYB4)gene family and 2 members of B.oeracea MYB4(BoMYB4) gene family were isolated and systemically analysized.The main results are as follows:1)Cloning of MYB4 genes from B.napus and its parental speciesUsing rapid amplification of cDNA ends(RACE)technology,full-length cDNAs and/or genomic sequences of MYB4 genes from B.napus and its parental species were isolated.BnMYB4-1,BnMYB4-2 and Bn MYB4-3 are 1393, 1364 and 1405 bp,with the corresponding mRNAs of 1271,1219 and 1253 bp(not including poly A tail), respectively.BrMYB4-1 and BrMYB4-2 mRNAs are 1280 and 1223 bp respectively.Gneomic sequences of BrMYB4-3 and BrMYB4-4 are 1407 and 1370 bp,with the corresponding mRNAs of 1278 and 1224 bp, respectively.BoMYB4-1 mRNA is 1005 bp.BoMYB4-2 gene is 1421 bp,and its mRNA is 1275 bp.These results will lay the base for study of function,evolution,regulatory mode of MYB4 genes of Brassica.2)BnMYB4,BrMYB4 and BoMYB4 genes share typical gene and protein features of AtMYB4They all have one intron with positions the same as that of AtMYB4.All the introns conform to canonical intron splicing boundary "GT...AG".At 250～1133,165～1076,231～1079,249～1136,232～1083,249～1133,232～1083, 47～938 and 279～1190 bp,BnMYB4-1,BnMYB4-2,BnMYB4-3,BrMYB4-1,BrMYB4-2,BrMYB4-3,BrMYB4-4, BoMYB4-1 and BoMYB4-2 mRNAs have an open reading frame(ORF)of 885,915,852,888,852,885,852,885 and 912 bp(including stop codon),respectively.Their 5' UTRs are 248,164,230,248,231,248,231,46 and 278 bp, and their 3'UTRs are 135,140,171,144,140,139,141,67 and 85 bp,respectively.The deduced BnMYB4-1,BnMYB4-2,BnMYB4-3,BrMYB4-1,BrMYB4-2,BrMYB4-3,BrMYB4-4, BoMYB4land BoMYB4-2 proteins are 294,304,283,294,283,294,283,294 and 303 aa in length,with a Mw of 33.10,34.37,31.65,33.10,31.65,33.07,31.66,33.12 and 34.27 kDa,and a pI of 8.78,9.13,8.61,8.78,8.61,8.79, 8.73,8.88 and 9.13,respectively.They all are typical basic proteins.They all have many predicted potential phosphorylation sites:19 for BnMYB4-1 and BrMYB4-1,23 for BnMYB4 and BoMYB4-2,18 for BnMYB4-3, BrMYB4-2 and BrMYB4-3,and 17 for BrMYB4-4 and BoMYB4-1,respectively.They were predicted with no signal peptide and transmembrane domain,but they are possibly targeted to the nuclear.LsrGIDPxT/SHPI/L and pdLNLD/ELXIG/S are two consered motifs in the C-terminal domain of BnMYB4, BrMYB4 and BoMYB4 proteins,which are the same as in AtMYB4.All these Bmssica MYB4 proteins belong to subgroup 4 of the Arabidopsis MYB superfamily.And they may share similar function with AtMYB4 in negative regulation of phenylpropanoid pathway.They share very similar secondary structures.Random coil is the most abundant proportion(47.85～56.80%,by frequency),followed byαhelices(25.17～32.67%),and supplemented with a little extended strand andβ-turn.They have 5～7 largeαhelices at the middle region and one bigαhelix near-C-terminus.The also share very similar tertiary structures of the R2R3-MYB domain,but no crystal model can predict their C-terminal regioin.Clues from gene structure,protein structure and sequence identities all suggest that BnMYB4-1,BnMYB4-2, Bn MYB4-3,BrMYB4-1,BrMYB4-2,BrMYB4-3,BrMYB4-4,BoMYB4-2-1 and BoMYB4-2-2 are orthologous genes of AtMYB4.3)Gene donor-receptor relationship between B.rapa/B,oeracea and B.napusBnMYB4-1 and BrMYB4-1 share 100%of identities on ORF scale,and 100%between BnMYB4-3 and BnMYB4-2.These homologies are much higher than those among intra-species paralogous BnMYB4 genes.On phylogenetic trees of both nucleotide and amino acid sequences,Bn MYB4-1 groups with BrMYB4-1 first, BnMYB4-3 groups with BrMYB4-2 first,and BnMYB4-2 groups with BoMYB4-2 first.Furhermore,clues from intron similarities and featured mutation sites on both nucleotide and amino acid sequences,all point to the corresponding relationships of BnMYB4-1 to BrMYB4-1,BnMYB4-2 to BrMYB4-2 and BnMYB4-3 to BoMYB4-2,suggesting that B.rapa and B.oeracea have provided genetic materials for B.napus.This research provided straight and concrete evidence for revealing the evolutionary relationships among B.napus,B.rapa and B. oeracea,based on a profile of comparative cloning of full-length MYB4 gene family.4)Numbers of BnMYB4,BrMYB4 and BoMYB4 gene family members,and "triplication" as compared with A.thalianaThe genomic DNA of typical black-seeded lines of B.oleracea var.acephala L.ftricolor Hort.and B.rapa ssp. oleifera was subjected to DraI,EcoRI and EcoRV digestions respectively to carry out Southern hybridiation.The results indicated that there may be 4 members in BrMYB4 family and 3 members in BoMYB4 family.Clues from Southern hybridization and gene cloning indicated that the MYB4 locus was really triplicated in Brassica ancestor as compared with A.thaliana.Southern blot of the BnMYB4 family has not been done,and only 3 and 2 members have been isolated from BnMYB4 family and BoMYB4 family respectively,so further study is neeeesaary.5)Vector constrution for sease expression of BrMYB4 genes and antisense suppression of Brassica MYB4 genesBrMYB4 genes(drived by CaMV 35S promoter)were integrated into intermediate vector pCambia2301G to replace the GUS gene.The constructed plant expression vectors were named them as pCBrMYB4-1,pCBrMYB4-2, pCBrMYB4-3 and pCBrMYB4-4.A 465-bp antisense fragment(drived by CaMV 35S promoter)conserved in all Brassica MYB4 genes was integrated into intermediate vector pCambia2301G to replace the GUS gene,and the antisense plant expression vector was named as p2301G-MYBYA.These vectors will facilitate the functional identification of Brassica MYB4 genes and related trait modification.