RESUMO
Molecular dissection of the Brassica yellow seed trait has been the subject of intense investigation. Arabidopsis thaliana TRANSPARENT TESTA 12 (AtTT12) encodes a multidrug and toxic compound extrusion (MATE) transporter involved in seed coat pigmentation. Two, one, and one full-length TT12 genes were isolated from B. napus, B. oleracea, and B. rapa, respectively, and Southern hybridization confirmed these gene numbers, implying loss of some of the triplicated TT12 genes in Brassica. BnTT12-1, BnTT12-2, BoTT12, and BrTT12 are 2,714, 3,062, 4,760, and 2,716 bp, with the longest mRNAs of 1,749, 1,711, 1,739, and 1,752 bp, respectively. All genes contained alternative transcriptional start and polyadenylation sites. BrTT12 and BoTT12 are the progenitors of BnTT12-1 and BnTT12-2, respectively, validating B. napus as an amphidiploid. All Brassica TT12 proteins displayed high levels of identity (>99%) to each other and to AtTT12 (>92%). Brassica TT12 genes resembled AtTT12 in such basic features as MatE/NorM CDs, subcellular localization, transmembrane helices, and phosphorylation sites. Plant TT12 orthologs differ from other MATE proteins by two specific motifs. Like AtTT12, all Brassica TT12 genes are most highly expressed in developing seeds. However, a range of organ specificity was observed with BnTT12 genes being less organ-specific. TT12 expression is absent in B. rapa yellow-seeded line 06K124, but not downregulated in B. oleracea yellow-seeded line 06K165. In B. napus yellow-seeded line L2, BnTT12-2 expression is absent, whereas BnTT12-1 is expressed normally. Among Brassica species, TT12 genes are differentially related to the yellow seed trait. The molecular basis for the yellow seed trait, in Brassica, and the theoretical and practical implications of the highly variable intron 1 of these TT12 genes are discussed.
Assuntos
Brassica napus/genética , Genes de Plantas , Proteínas de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Southern Blotting , Brassica/genética , Brassica rapa/genética , Clonagem Molecular , Sequência Conservada , Primers do DNA/genética , DNA de Plantas/genética , Evolução Molecular , Duplicação Gênica , Íntrons , Dados de Sequência Molecular , Especificidade de Órgãos , Fenótipo , Filogenia , Pigmentação/genética , RNA Mensageiro/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
Three members of Brassica napus TRANSPARENT TESTA 2 (BnTT2) gene family encoding potential R2R3-MYB regulatory proteins of proanthocyanidin biosynthesis were isolated. BnTT2-1, BnTT2-2, and BnTT2-3 are 1102 bp with two introns, and have a 938-bp full-length cDNA with a 260 amino acid open reading frame. They share 98.2-99.3% nucleotide and 96.5-98.5% amino acid identities to each other, and are orthologous to Arabidopsis thaliana TT2 (AtTT2) with 74.1-74.8% nucleotide and 71.1-71.8% amino acid identities. An mRNA type of BnTT2-2 was found to contain unspliced intron 2 and encode a premature protein. They all have an alternative polyadenylation site. BnTT2-1 and BnTT2-3 also have an alternative transcription initiation site. Aligned with AtTT2, their 5' untranslated regions (UTRs) are astonishingly conserved, and two conserved regions were also found in their 3' UTRs. Oligonucleotide deletion leads to double-start codons of them. Resembling AtTT2, BnTT2 proteins are nuclear-located R2R3-MYB proteins containing predicted DNA-binding sites, bHLH interaction residues, and transcription activation domains. Southern blot indicated that there might be three BnTT2 members in B. napus, lower than triplication-based prediction. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that the expression of BnTT2-2 is mostly like AtTT2 with intensive expression in young seeds, but it is also expressed in root in which AtTT2 has no expression. BnTT2-1 shows lower tissue specificity and transcription levels, whereas BnTT2-3 is the lowest. Comparative cloning and RT-PCR indicated that seed color near-isogenic lines L1 and L2 have equivalent BnTT2 genes, and the yellow seed color in L2 might be caused by locus/loci other than BnTT2. Our results lay the basis for further investigating the regulatory mechanism of BnTT2 genes in flavonoid pathway and for transgenic creation of novel yellow-seeded B. napus stocks.
Assuntos
Brassica napus/genética , Família Multigênica , Proteínas de Plantas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Sequência de Aminoácidos , Sequência de Bases , Brassica napus/metabolismo , Clonagem Molecular , Genoma de Planta , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Proantocianidinas/biossíntese , Proteínas Proto-Oncogênicas c-myb/classificação , Proteínas Proto-Oncogênicas c-myb/metabolismo , Alinhamento de Sequência , Transcrição GênicaRESUMO
The development of yellow-seeded varieties of Brassica napus for improving the oilseed quality characteristics of lower fiber content and higher protein and oil content has been a major focus of breeding researches worldwide in recent years. With the black-seeded 'Youyan 2' as male and the yellow-seeded GH06 as female parents respectively, F2 population of 132 individuals were obtained. A linkage map was constructed with 164 markers including 125 AFLP, 37 SSR, 1 RAPD and 1 SCAR markers distributed over 19 linkage groups covering approximately 2 549.8 cM with an average spacing of 15.55 cM. Two loci located on the 5th and 19th group were detected for the trait of seed coat color based on the linkage group using multiple interval mapping method and explained 46% and 30.9% of the phenotypic variation, respectively.
