Molecular Cloning And Comparative Genomic Analysis Of BANYULS (BAN) Gene Family From Brassica Napus And Its Parental Species

Oilseed rape (Brassica napus) is one of the most important oil crops. Its yellow-seeded type shows many quality advantages, and yellow seed trait is a hotspot of rapeseed breeding goal worldwide. Both parental speices, B. oeracea and B. rapa, have stable-phenotyped natural yellow seed genotypes, but B. napus has not. Yellow seed trait introgression through distant hybridization is time-consuming and low efficient, and the expression of the yellow seed phenotype shows drastic sensitivity to environmental factors. At present, it is highly necessary to simultaneously clone important functional genes involved in seed color determination from B. napus and its parental species, and then perform comparative studies. This will help to clarify the molecular genetic mechanism of yellow seed trait in these 3 species, and lay the base for improvement of seed color trait of B. napus through genetic engineering.The molecular mechanism of yellow seed trait formation is not clear still, but achievements on the TRANSPARENT TESTA (TT) trait in Arabidopsis thaliana can be referred The anthocyanins and the condensed tannin use common substrate. The BANYULS (BAN) gene coding anthocyanidin reductases (ANR) determines whether the substrate enters the condensed tannin synthesis pathway. BAN reducecs the anthocyanidin to 2,3-cis-flavan-3-ols which are then polymerized later to form condensed tannin. Study on BAN genes from B. napus and its parental species will help to reveal the molecular mechanism of yellow seed trait formation and lay the base for transgenetic creation of stably inherited yellow seed stocks.In this research, the full-length cDNAs and corresponding genomic sequences of 3 members of B. napus BAN gene family (BnBAN), 2 members of B. rapa BAN gene family (BrBAN) and 2 members of B. oeracea BAN gene family (BoBAN) were isolated and systemically analysized. The main results are as follows:1) Cloning of BAN genes from B. napus and its parental speciesUsing rapid amplification of cDNA ends (RACE) technology, full-length cDNAs and corresponding genomic sequences of BAN genes from B. napus and its parental species were isolated BnBAN-1, BnBAN-2 and BnBAN-3 are 1571,1612 and 1574 bp, with the corresponding mRNAs of 1178,1124 and 1180 bp (not including poly A tail), respectively. BrBAN-1 and BrBAN-2 are 1607 and 1623 bp, with the corresponding mRNAs of 1212 and 1174 bp, respectively. BoBAN-1 and BoBAN-2 are 1543 and 1642 bp, with the corresponding mRNAs of 1172 and 1150 bp, respectively. This is first report of full-length BAN gene cloning from Brassica species, and will lay the base for study of function, evolution, regulatory mode of BAN genes of Brassica.2) BnBAN,BrBAN and BoBAN conform to typical features of BANThey all have 5 introns with the same positions as those of AtBAN. All the introns conform to canonical intron splicing boundary fearture sequence "GT...AG". At 41-1069, 50-1066, 41-1078, 74-1111,46-1062,47-1063 and 41-1069 bp of BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 mRNAs have an open reading frame (ORF) of 1029,1017,1038,1038,1017,1017 and 1017bp (including stop codon). Their 5' UTRs are 40,49,40,73,45,46 and 40 bp, and 109,58,102,101,112,87 and 103 bp for 3' UTRs, respectively. Their possible non-canonical poly A tailing signals TATAAA and AAATAAT are located 22-42,2343,22-42,20-39,20-42,15-34 and 22-45 bp respectively upstream the poly A tailing sites.The deduced BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 proteins are 342,338,345,345,338,342 and 338 aa in length, with Mw of 37.850,37.680,38.277,38.266,37.614,37.927 and 37.554 kDa, and pI of 6.38,4.93,6.38,6.06,5.00,5.81 and 4.87, respectively. They all are typical acidic proteins. Serine is the richest one in their amino acid compositions. BnBAN-1, BnBAN-3 and BrBAN-1 have 24 potential phosphorylation sites; BnBAN-2, BrBAN-2 and BoBAN-2 have 18; and BoBAN-1 has 23. They were predicted with no signal peptide and transmembrane domain, with the higheset possibility to be located in cytoplasm.Like AtBAN and MtBAN, the 7 Brassica BAN proteins have classical Rossmann fold characterized by a conserved glycine-rich motif GXXGXXA. Within this consensus, they have redidues corresponding to N₂₁ and L₂₂ of AtBAN which may determine substrate specificities. Besides, they also have a perfect leucine zipper like AtBAN.They share very similar secondary structures.αhelix is the most abundant proportion (39.7742.60% by frequency), followed by random coil (30.04-32.84%), supplemented with a little extended strand andβ-tum. They have 2 bigαhelices at the middle of protein and 1 bigαhelix near-C-terminus. The also share very similar tertiary structure, resembling Dihydroflavonol reductase from Vtiis.Vinifera in general, and partilly similar to NADPH-Dependent carbonyl reductase from Sporobolomyces.Salmonicolor at local regions.Clues from gene structure, protein structure and sequence identities all suggest that BnBAN-1, BnBAN-2, BnBAN-3, BrBAN-1, BrBAN-2, BoBAN-1 and BoBAN-2 are orthologous genes of AtBAN.3) B. rapa and B. oeracea are donors of genetic materials for B. napusBnBAN-1 and BoBAN-1 share 97.5% and 99.1% of identities on whole genomic sequence and ORF scales respectively, and 96.5% and 99.5% between BnBAN-2 and BoBAN-2. These homologies are much higher than that between intra-species paralogs BnBAN-1/BnBAN—3 and BnBAN-2. On phylogenetic trees of both nucleotide and amino acid sequences, BnBAN-1 groups with BoBAN-1 first, and BnBAN-2 groups with BoBAN-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 BnBAN-1 to BoBAN-1 and BnBAN-2 to BoBAN-2, suggesting that B. rapa and B. oeracea have provided the genetic material for B. napus. This research provided straight and concrete evidence for revealing me evolutionary relationships among B. napus, B. rapa and B. oeracea, based on a profile of comparative cloning of full-length functional gene family.4) Number of members of BnBAN gene family, BrBAN gene family and BoBAN gene familyThe genomic DNA of line 5B was subjected to EcoRI, EcoRV, SphI, SacI and XbaI digestion respectively, and Southern blot result indicated that there may be at least 4 members of BnBAN family in B. napus. Because we haven't accomplished the cloning of the whole BAN gene sets from these 3 species, and Southern hybridization of the 2 prental species is still lack, so further exhaustive cloning is neccessary to identify whether the BAN locus has been triplicated in basic "diplioid" Brassica species as compared with A. thaliana.