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

Brassica is the most important genus among the more than 300 Brassicaceae genera.It contains mainy vegetable,oilseed and ornamental crops of world importance,such as B.rapa,B.oleracea and B.napus.B.napus is an amphidipoid species of B.rapa of B.oleracea generagted by natural inter-species hybridization and successive tetraploidization.Also from Brassicaceae,Arabidopsis thaliana is a model plant with deepist exploitation.Chromosomal molecular marker colinearity studies have shown that both genomic and genic conservations exist among Brassica species as well as between Brassica and Arabidopsis thaliana.So,achievements on functional genomic studies in A. thaliana are important references to promote studies on molecular mechanisms of important traits and on comparative genomic studies of Brassica species.Anthocyanins are nature water-soluble flavonoids widely existing in plant kindom.They are important pigmentation substances of flowers,fruits and vegetables.Anthocyanins are safe, non-toxic,resource-rich natural pigments,and it is a trend to use natural pigments to substitute toxic artificial pigments.Anthocyanins/nthocyanidins also have health-promotive and medicinal effects, e.g.oxidatant resistance,mutation prevention,prevention of cardio-cerebral-vascular disease,liver protection and turnout inhibition.So anthocyanin/nthocyanidin extracts have huge utilization potential in foods,cosmetics,medicines and so on.Anthocyanins are biosynthesized via the phenylpropanoid-flavonoid-anthocyanin compound pathway,while the phenylpropanoid-flavonoid-proanthocyanidin compound pathway biosynthesizes another group of flavonoids,proanthocyanidins which are main pigments in the seed coat of rapeseed and other plants.In A.thaliana,the highly homologous genes PAP1(AtPAP1),PAP2 (AtPAP2),MYB113(AtMYB113)and MYB114(AtMYB114)encode transcription factors and positive regulators of the flavonoid-anthocyanin pathway,acting on the promoters of key enzyme genes F3'H, DFR,ANS,UGT75C1 and GST12PAP.In view of functional similarity,here these 4 genes are denoted as the AtPAP gene family.In natural plants,their in planta functions do not regulate proanthocyanidin pathway-specific genes such as ANR and LAC15,i.e.they guide the flux into biosynthesis of anthocyanins other than proanthocyanidins.However,when they are fused with the EAR repression domain to form negative regulators by CRES-T technology,ectopic expression of these fusion genes in the transformed A.thaliana plants not only modifies the anthocyanin profiles but also suppresses proanthocyanidin synthsis which leads to transparent testa(yellow seed) phenotype.Anthocyanin traits are important biological and economical trats in Brassica plants,and yellow seed is a focus high quality trait in rapeseed.Cloning of PAP genes from Brassica species has significance in dissecting the molecular mechanism of anthocyanin trait and in molecular breeding of anthocanin/proanthocyandin traits of these species.In this research,full-length cDNAs and genomic sequences of a total of 12 gene members of the PAP gene families from B.napus and its parental species were isolated and bioinformatically and comparative-genomically analysed.1)Cloning and structural features of full-length sequences of PAP gene families from B.napus and its parental species.Using RACE technology,full-length cDNAs and genomic sequences of a total 12 gene members of the PAP gene families from B.napus and its parental species were isolated.The 6-member B.napus PAP(BnPAP)gene family:BnPAP1,BnPAP2,BnPAP3,BnPAP4, BnPAP5 and BnPAP6 are 1793,1804,1979,1541,1522 and 1956 bp respectively,with sequenced longest mRNAs 872,1011,1001,873,1005(putative)and 1003 bp respectively(not including the poly(A)tail,and the same for the following).The 3-member B.rapa PAP(BrPAP)gene family:BrPAP1,BrPAP2 and BrPAP3 are 1854, 1515,and 1973 bp respectively,with sequenced longest mRNAs 932,998(putative)and 1001 bp respectively.The 3-member B.oleracea PAP(BoPAP)gene family:BoPAP1,BoPAP2 and BoPAP3 are 1548, 1792,and 1957 bp respectively,with sequenced longest mRNAs 873,1001 and 1000 bp respectively.All the 12 PAP genes from the 3 Brassica species are composed of 2 introns and 3 exons,and all the introns conform to standar GT...AG splicing boundaries.Except BnPAP5 and BrPAP2,their mRNAs all have an ORF(open reading frame,including the stop codon)of 744～753 bp,a 5' UTR of 32～86 bp,and a 3' UTR of 93～181 bp,Upstream the latest poly(A)tailing site,there are 1～2 sites of canonical polyadenylation signial AAATAAA.BnPAP5 and BrPAP2 both have an ORF of only 18 bp because of prestop mutation at the 6th codon.Their original(ori)ORFs before the taking place of this mutation are both 744 bp,with 5' UTRs of 86 bp in both and 3' UTRs of 175 and 168 bp respectively.This is the first time of isolating full-length PAP genes from Brassica.It will promote the functional,evolutionary and regulatory studies of Braasica PAP genes,and lay a base for modifying the anthocyanin and seed coat pigment traits through transgenic manipulation such as antisense, RNAi and CRES-T means in these species.2)Protein structural features of PAP families from B.napus and its parental speciesFor the convenience of phylogenetic and comparative genomic studies,here the ori ORF-encoded proteins of BnPAP5 and BrPAP2(BnPAP5ori and BrPAP2ori)are also included.Except BnPAP5 and BrPAP2,all the PAP family members from B.napus and its parental species encode polypeptides of 247～250 amino acids(aa),with molecular weights(Mw)of 27.73～28.53 kDa and isoelectric points(pIs)of 8.73～9.01.Leucine is the most abundant residue, and basic residues are more than acidic ones.BnPAP5 and BrPAP2 are both only 5 aa in length.All the 12 Brassica PAPs(including BnPAP5ori and BrPAP2ori,and the same for the following) are predited with 9～17 potential phosphorylation sites,so phosphorylation might participate in their activity regulation.They all have no signal peptide and transmembrane,but they are predicted to be localized in the nuclear.Their secondary structures are much similar to each other,random coil being the most abundant(47.97%～53.41%)followed by alpha helix(31.71%～41.30%).From N-terminal to the near central region,they all have 2 SANT/MYB-DNA-binding conserved domains,and they each have 3 alpha helices in both R2- and R3-MYB domains.The tertiary structures of the R2R3-MYB domains prediected by EsyPred3D conform to typical features of R2R3-MYB.NCBI BLASTs on both nucleotide and amino acid levels,sequence pariwise alignments and phylogenetic analysis all indicate that the 12 PAP genes from B.napus and its parental species share the highest homologies to AtPAP gene family members,implying that they also encode AtPAP-like nuclear-located R2R3-MYB regulators of anthocyanin biosynthesis.3)Clues from PAP loci indicate that B.rapa and B.oleracea are gene donors of B.napusSystamic clues from nucleotide and amino acid sequence alignments,phylogenetic clustering, intron identities and featured base/amino acid mutations all indicate that BnPAP1,BnPAP4,BnPAP2, BnPAP5,BnPAP3 and BnPAP6 are phylogenetically from BrPAP1,BoPAP1,BoPAP2,BrPAP2, BrPAP3 and BoPAP3 respectively.Not only each BnPAP gene can find its corresponding donor gene from a parental species,but also the number of BnPAP gene family members is just a sum of those of BrPAP BoPAP gene families.This implies that,at least at the PAP loci,B.rapa and B.oleracea are gene donors of B.napus.This research provides direct and concrete evidences from full-length sequence cloning and comparative study of PAP gene families for revealing the evolutionary relationships between B. napus and its parental species.4)The PAP locus was duplicated for more times in A.thaliana than in basic Brassica speciesThe homologies among the AtPAP members(AtPAP1,AtPAP2,AtMYB113 and AtMYB114)as well as among the 12 Brassica PAP genes are all obviously higher than those between the AtP AP members and the Brassica PAP genes.It is speculated that the multiple PAP genes in both Brassica and Arabidosis are results of duplications of one Brassicaceae ancestor PAP gene in respective genera after their split,in Brassica it triplicated to 3 PAP genes,while in Arabidopsis it quadruplicated to 4 PAP genes.In the past,studies on C-value,molecular marker colinearity and loci comparative cloning showed that during the evolution the Arabidopsis genome tends to shrink while the Brassica genomes tend to expand.This research shows that the PAP locus in Arabidopsis uncexpectedly duplicated to times more than those occurred in Brassica.The reason of this abnormal gene dosage relationship might be that:1)anthocyanin trait in Arabidopsis is as important as in Brassica,and it was the pressure from trait evolution that caused PAP gene duplication,or 2)the chromosomal position of the PAP gene(s)in Arabidopsis was somewhat special,which was prone to causing duplication event.5)Regional fast evolution versus relative conservation of PAP genesThe 12 PAP genes from B.napus and its parental species share whole-gene identies of 59.8%～99.3%,mRNA identities of 85.2%～100%,whole-protein identities and positives of 77.7%～100%and 82.3%～100%,and R2R3-MYB identities and positives of 91.8%～100%and 94.8%～100%,respectively.Between the 12 Brassica PAP genes and the Arabidopsis PAP genes,the whole-gene identies are 59.5%～67.1%,the ORF identies are 77.8%～83.6%,the whole-protein identities and positives are 65.9%～77.6%and 71.9%～83.7%,the R2R3-MYB identities and positives are 88.7%～96.9%and 91.8%～97.9%,and the C-terminus identities and positives are 47.9%～65.0%and 55.7%～74.1%, respectively.On both orthologons and paralogous levels,PAP proteins are highly conservative at R2R3-MYB region,while the C-terminus is of much less conserved and diverges faster.6)Transcriptional/posttranscriptional alternative actions of PAP genesBnPAP2 and BrPAP2 both have a kind of mature mRNA resulting from alternative splicing of the second intron.With a deletion of the first 21 bp of the third exon,the alternatively spliced mRNA encodes a protein lacking 7 aa at the second alpha helix region of the R3-MYB.Theoretically this deletion will influence the DNA binding ability,but the extent needs functional identification.Many Brassica PAP genes have alternative transcription start sites and alternative poly(A)tailing sites,thus diverse mRNAs with different lengths are generated from one gene.It needs to be cleared whether they represent cis regulations or allowable action arrors.