Cloning And Comparative Genomic Study Of TTG1 And TT18 Gene Families From Brassica Napus And Its Parental Species

Oilseed rape(Brassica napus L.) is the most widely grown oilseed crop in the world,and takes important status in agricultural production of China.Traditional black-seeded oilseed rape has many advantages,such as pest-resistance,lodging resistance and high yielding,while its disadvantages are also obvious,i.e.low oil content and high erucic acid,polyphenol and pigment.The oil milled from black seed is not only dark and easily brings lamp fume,but also contains excessive erucic acid and glucosinolates.Both the two kinds of matters are useless even harmful for both human and animals. So,improving the oil content and quality,reducing the erucic acid,glucosinolates,polyphenols, pigment content in the seed of B.napus have become the new breeding targets.Compared with the black-seeded rapeseed at the same genetic background,yellow-seeded oilseed rape has advantages, such as thinner seed coat,lower pigment and fiber content,and higher protein and oil content. Though the yellow-seeded rapeseed has good quality and favorable market prospect,but the yellow seed trait is unstable,which is an urgent problem to be solved.However,creating yellow seed stocks through distant hybridization is time-consuming and low efficient,and the yellow seed phenotype also shows drastic sensitivity to environmental factors.At present,it is urgent to simultaneously clone important functional genes involved in seed color determination from B.napus and its parental species,and then perform comparative genomic studies.It is helpful to clarify the molecular genetic mechanisms of yellow seed trait in the 3 species,and lay the base for improvement of seed color trait of B.napus through genetic engineering.The amphidiploid species B.napus is assumed to have a genome fused from diploid basic species B.rapa and B.oleracea.It is important for investigating the evolution of Brassica gene structure, regulatory network,genetic traits and development by the comparative genomics study focused on Arabidopsis and other Brassica species.However,functional comparative genomics study based on comparative cloning and characterization of functional genes is rarely reported. Flavonoids are ubiquitous plant secondary products,which are well known as the typically red, blue,and purple pigments in plant tissues.The main seed coat pigments of plants like Arabidopsis are polymers of proanthocyanin(PA),which is synthesized via the phenylpropanoid- flavonoid-proanthocyanidin pathway.Among the genes in this pathway,TTG1 is one of the most important transcription factor,which encodes a WD40 repeat protein.The pleiotropic mutant ttg1 is glabrous, yellow-seeded,and affected in root hair differentiation.In Arabidopsis and other plants,TTG1 gene has been proved to play an important role in anthocyanin biosynthetic pathway.TT18 gene commonly named as anthocyanidin synthase or leucoanthocyanidin dioxygenase gene,a 2-oxoglutarate(2OG) iron-dependent oxygenase,catalyzes the penultimate step in the biosynthesis of anthocyanidin.This reaction is responsible for the formation of the colored anthocyanidin from the colorless leucoanthocyanidins,tt18 mutant is yellow-seeded too.Previous studies in Arabidopsis and other plants also show that TT18 gene is an important gene in anthocyanin biosynthetic pathway. In this study the full-length sequences of TTG1 and TT18 gene families from B.napus,B.oleracea and B.rapa were cloned and comparatively characterized.The overall expression of TTG1 and TT18 between black- and yellow-seeded stocks of B.napus,B.oleracea and B.rapa was also detected. The main results of this study are as follows:1) Simultaneous cloning of the full-length TTG1 gene families from B.napus,B.oleracea and B. rapaFull-length cDNAs and corresponding genomic sequences of TTG1 gene famlies from B.napus, B.oleracea and B.rapa were isolated based on Rapid Amplification of cDNA Ends(RACE) technology.The full-length genomic sequences of BnTTG1-1 and BnTTG1-2 are 1511 bp and 1555 bp,with 1355 bp and 1303 bp of full-length typical-type mRNAs respectively(not including the poly(A) tail, and the same for the following).The full-length genomic sequences of BoTTG1-2 is 1594 bp.The full-length typical-type mRNAs of BoTTG1-1 and BoTTG1-2 are 1355 bp and 1404 bp,respectively.The full-length genomic sequences of BrTTG1-1 and BrTTG1-2 are 1656 bp and 1541 bp,with 1478 bp and 1364 bp of full-length typical-type mRNAs,respectively.The Brassica TTG1 genes share an ORF(open reading frame,including the stop codon) of 1014 bp,with 53～169 bp of 5'UTRs and 162～617 bp of 3'UTRs.This is the first time to isolate full-length TTG1 genes from Brassica species.It will lay a base for further studying the functional,evolutionary and regulatory pattern of TTG1 genes and for artificial modification of seed color,thichome,root hair,seed coat mucilage of Brassica speices by manipulating TTG1 genes. 2) Protein structural features of TTG1 families from B.napus and its parental speciesDeduced BnTTG1-1,BnTTG1-2,BoTTG1-1,BoTTG1-2,BrTTG1-1 and BrTTG1-2 proteins are 337 amino acids in length,with molecular weights(Mw) of 37.28,37.26,37.27,37.31,37.28 and 37.20 respectively and isoelectric points(pIs) of 4.66 for all.They all are typical acidic proteins. Serine is the richest one in their amino acid compositions,and acidic residues are higher than basic residues.They all have many potential phosphorylation sites predicted by NetPhos 2.0,26 for BnTTG1-1, BoTTG1-1 and BrTTG1-1,25 for BnTTG1-2 and BrTTG1-2,and 27 for BoTTG1-2,implying that phosphorylation might be involved in regulating their protein activity.They were predicted with no signal peptide and transmembrane domain,while they all were predicted to be located in cytoplasm.Predicted by SOPMA,BnTTG1-1,BnTTG1-2,BoTTG1-1,BoTTG1-2,BrTTG1-1 and BrTTG1-2 proteins share very similar secondary structures.Random coil is the most abundant proportion,followed by extended strand.The whole molecule is dominated by a large number of random coils.These features are similar to those of AtTTG1.Their tertiary structure couldn't be modeled in SWISS-MODEL.3) B.rapa and B.oleracea both are parental species of B.napusBnTTG1-1 and BrTTG1-2 share 97.2%and 99%of identities on whole genomic sequence and coding-region scales,and BnTTG1-2 and BoTTG1-1 have 98.7%and 98.3%of identities on mRNA sequence and coding-region scales,respectively.Furthermore,clues from position and length of the GA repeat structure in the 5' UTR,and featured mutation sites on both nucleotide and amino acid sequences,all point to the corresponding relationships of BnTTG1-1 to BrTTG1-2 and BnTTG1-2 to BoTTG1-1,suggesting that B.rapa and B.oleracea are donors of genetic substances of B.napus. This research provided straight and concrete evidence for revealing the evolutionary relationships among B.napus,B.rapa and B.oleracea,based on a profile of comparative cloning of full-length functional gene family.4) Several features of Brassica TTG1 gene families were revealedThe Brassica TTG1 genes all have alternative transcription iniation sites in the 5'UTR,In their 3' UTRs,altenative poly(A) tailing sites were detected.More studies are needed to confirm whether this poly(A) site polymorphism is a cis-regulatory pattern or just is a result of allowed errors in poly(A) tailing process.TTG1 have extreme protein-level conservation.Though the genomic sequences and their coding regions differ from each other,their amino acid similarities to each other are astonishingly high.Their amino acid similarities to AtTTG1 are also astonishingly high.TTG1 or AN11-type proteins from monocots even animals also share high similarities to Brassica TTG1 proteins, especially the absolute conservation in the middle and the carboxyl regions.TTG1/AN11-type genes in eukaryotes maintain highly conserved protein structures through coding region conservation and synonymous substitution during speciation exist.This tendency could be generally observed in housekeeping or important structural genes.Regulatory genes tend to diverge faster than structural genes.Brassica TTG1 proteins are even more conservative than most structural genes.TTG1 genes have directional evolution features especially genus-specific nucleotide preference. The first is fast degeneration of the start codon leader sequence into a stretch of GA-repeats in Brassica TTG1 genes.The second is opposite directional nucleotide substitutions observed in Brassica TTG1(T→C) and AtTTG1(C→T),which result in quite differed GC contents in their coding regions.The genus-specific nucleotide preference of Brassica TTG1 genes is like that of the Cytochrome OxidaseⅡgene in Earwigs.5) Simultaneous cloning of the full-length TT18 gene families from B.napus,B.oleracea and B.rapaFull-length cDNAs and corresponding genomic DNA sequences of TT18 gene famlies from B. napus,B.oleracea and B.rapa were isolated based on Rapid Amplification of cDNA Ends(RACE) technology.The full-length genomic sequences of BnTT18-1,BnTT18-2,BnTT18-3,BnTT18-4,BnTT18-5, are 1875 bp,1508 bp,1897 bp,1972 bp,1491 bp,and their full-length typical-type mRNAs are 1481 bp,1421 bp,1477 bp,1279 bp,1404 bp,respectively.The full-length genomic sequences of BoTT18-1 and BoTT18-2 are 1923 bp and 1491 bp,and the full-length typical-type mRNAs of BoTT18-1 and BoTT18-2 are 1482 bp and 1404 bp, respectively.The full-length genomic sequences of BrTT18-1,BrTT18-2 and BrTT18-3 are 1898 bp,1489 bp and 1491 bp,and the full-length typical-type mRNAs are 1478 bp,1399 bp and 1404 bp, respectively.The Brassica TT18 genes share an ORF of 1074～1077 bp,except that BnTT18-2 has an ORF of only 338 bp because of prestop mutation.Their 5'UTRs are 41～144 bp,with 3'UTRs of 161～322 bp.This is the first time of systemic isolating full-length TT18 genes from Brassica.It will lay a base for further studying the functional,evolutionary and regulatory patterns of TT18 genes and for artificial modification of seed color of Brassica speices by manipulating TT18 genes.7) Protein structural features of TT18 families from B.napus and its parental speciesDeduced BnTT18-1,BnTT18-2,BnTT18-3,BnTT18-4,BnTT18-5,BoTT18-1 and BoTT18-2 proteins are 337～338 amino acids in length,with molecular weights(Mw) of 40.86,40.86,40.83, 40.89,40.86,40.86,40.84,40.87,40.77,40.86 kD,and isoelectric points(pIs) of 5.13～5.30.They all are typical acidic proteins.Glutamic acid is the richest one in their amino acid compositions,and acidic residues are higher than basic residues.They all have many potential phosphorylation sites predicted by NetPhos 2.0,with 10,13,12,7, 10,7,13,7 and 7 respectively,implying that phosphorylation might be involved in regulating their protein activity.They were predicted with no signal peptide and transmembrane domain.However, they all are predicted to be located in cytoplasm.We predicted Conserved Domain of TT18 protein from Brassica.They all have two conserved domains,cl01206 and COG3491.Both of them are typical domains of 2-oxoglutarate-dependent dioxygenases.Predicted by SOPMA,TT18 proteins from Brassica share very similar secondary structures. Random coil is the most abundant proportion,followed byαhelix.The whole molecule is dominated by 15-16αhelices,and the positions of theseαhelices are the same as those in AtTT18.Theseαhelices are connected by random coils or extended strands.These features are similar to those of AtTT18.Predicted by SWISS-MODEL,TT18 proteins from Brassica share very similar tertiary structures. After automatic alignment with PDB database,we found the highest similar protein is AtTT18 (AtANS).TT18 proteins from Brassica have 83.5%-92.0%of similarity to AtTT18(1gp6A.pdb model).The predicted models of TT18 proteins are the same as AtTT18.The conserved amino acids ligating ferrous iron(H-X-D) and residues participating in 2-oxoglutarate binding(R-X-S) were found in TT18 proteins at the corresponding positions like other proteins from different species. TT18 proteins from Brassica have a jellyroll motif in the enzyme core consisted ofβ-sheet,a typical structure shared by all 2-oxoglutarate-dependent dioxygenases.BLAST on both nucleotide and amino acid levels,pairwise and multi alignment of sequences and phylogenetic anylysis all indicate that these 10 genes share the highest homologies with AtTT18. Clues from gene structure,protein structure and sequence identities all suggest that they are orthologous genes of AtTT18.8) B.rapa and B.oleracea both are parental species of B.napusBnTT18-1 and BoTT18-1 share 97.5%and 99%of identities on whole genomic sequence and Coding-region scales,BnTT18-2 and BrTT18-2 share 95.3%and 95.6%of identities on whole genomic sequence and coding-region scales,BnTT18-3 and BrTT18-1 share 99.3%and 99.0%of identities on whole genomic sequence and coding-region scales,BnTT18-5 and BrTT18-3 share 100%of identities on whole genomic sequence and coding-region scales,respectively.These homologies are much higher than those among TT18 genes from B.napus which are intra-species paralogs.Furthermore,clues from featured mutation sites on both nucleotide and amino acid sequences also point to the corresponding relationships of BnTT18-1 to BoTT18-1,BnTT18-2 to BrTT18-2,BnTT18-3 to BrTT18-1,BnTT18-5 to BrTT18-3,respectively.Since the couterpart genes of BnTT18-4 and BoTT18-2 haven't been cloned,we downloaded the GSS and EST sequences for TT18 gene family from NCBI,and aligned them.The results show that BnTT18-4 has high similarities to GSS DU984686.1 and DU984271.1 and EST EX080049.1 from B.rapa.BoTT18-2 has high similarities to EST CD828811.1,EG020755.1,EV0.39607.1,EL62498.1,EE471700.1 and EV016901.1 from B.napus.The results suggest that B.rapa and B.oleracea are donors of genetic substances of B.napus.Therefore,BnTT18-2,BnTT18-3,BnTT18-4 and BnTT18-5 came from B. rapa(AA group),while BnTT18-1 and BnTT18-6 came from B.oleracea(CC group).This research provided direct and concrete evidence for revealing the evolutionary relationships among B.napus,B.rapa and B.oleracea,based on a profile of comparative cloning of full-length gene families.It should be pointed out that on the TTG1 locus the gene number in B.napus is not the total set of those from B.rapa and B.oleracea,some genes from the parental species do not exist in B.napus; on the TT18 locus the gene numbers from the diploid basic species B.rapa and B.oleracea are obviously different.These put forward some complexities to the evolution of Brassica species, which need further elucidation.