Cloning And Comparative Genomic Study Of TT12 Gene Family Of Brassica Napus And Its Parental Species

In Brassicaceae, Arabidopsis thaliana is an important model plant, and Brassica contains many important oilseed, vegetable and ornamental species. Comparative genomic study both within Brassica and between Brassica and Arabidopsis is a hotspot in theoretical study, and is a powerful tool to bring the research results on functional genes achieved in Arabidopsis into effect in genetic improvement of Brassica crops. B. napus is an amphidiploid of B. oleracea and B. rapa. Comparative genomic study has revealed that the genome of diploid species of Brassica has experienced a triplication process as compared with that of Arabidopsis, accompanied by loss, reduplication or rearrangement of local regions. Mainly using molecular marker means, present comparative genomic studies mainly focus on revealing the degree Of collinearity between mega regions of genomes, while comparative genomic study and comparative biological study based on comparative cloning of functional genes has both theoretical and applicable implications for Brassica crops.With outstanding comprehensive quality advantages and prospective marketability, yellow seed trait is a hotspot, but also a nodus, of rapeseed research worldwide. Both B. oleracea and B. rapa have stable-phenotyped natural yellow seed genotypes, but B. napus has not. And creating yellow seed stocks through means like 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 genomic and comparative biological 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 major constitutents of seed coat pigments of plants like Arabidopsis are polymers of proanthocyanidin monomer, which is synthesized via phenylpropanoid-flavonoid pathway. Among the many functional genes of this pathway, A. thaliana TT12 (AtTT12) is specifically expressed in the seed coat, and it functions to transport seed coat pigments into the vacule by encoding a Multidrug transporter protein. Single gene mutation of AtTT12 leads to the turning of the seed color from the dark brown wild-type to transparent testa (tt, yellow seed) type. Since B. napus and Arabidopsis both belong to the same family Brassicaceae and share similar seed color trait, this research performed isolation of full-length TT12 genes from B. napus and its parental species, and carried out comparative genomic analysis of these genes.1) Cloning of TT12 genes from B. napus and its parental speciesUsing rapid amplification of cDNA ends (RACE) technology, full-length cDNAs and corresponding genomic sequences of TT12 genes from B. napus and its parental species were isolated. B. napus BnTT12-1 gene is 2712 bp with an mRNA of 1747bp (not including poly A tail), and BnTT12-2 gene is 3000 bp with an mRNA of 1678 bp. B. rapa BrTT12 gene is 2711 bp with an mRNA of 1747 bp. B. oleracea BoTT12 gene is 3062 bp with an mRNA of 1739 bp. This result lary the base for study of function, evolution, and regulatory mode of TT12 genes of Brassica, and for creation of novel yellow seed stocks by suppression of intrinsic TT12 gene expression (via antisense or RNA interference) or by LILLING mutants screening.2) BnTT12-1, BnTT12-2, BrTT12 and Bo TT12 conform to typical features of TT12They all have 7 introns with the same positions as those of AtTT12. All the introns conform to canonical intron splicing boundary sequence "GT...AG". At 62-1585, 17-1540, 60-1583 and 78-1601 bp of BnTT12-1, BnTT12-2, BrTT12 and BoTT12 mRNAs, there are an open reading frame (ORF) of 1524 bp (including stop codon). Their 5' UTRs are 61, 16, 59 and 77 bp, and 162, 138, 164 and 138 bp for 3' UTR, respectively. Their poly A tailing signal AAATAAA is located upstream the latest poly A tailing site with an interval of 96, 83, 98 and 83 bp respecitvely.Like AtTT12, the deduced BnTT12-1, BnTT12-2, BrTT12 and BoTT12 proteins all are 507 aa in length. BnTT12-1 has an Mw of 55.080 kDa and a pI of 3.24, while 55.168 kDa and 3.24 for BnTT12-2, 55.199 kDa and 4.24 for BrTT12, and 55.128 kDa and 3.24 for BoTT12, respectively. They are typical acidic proteins. Leucine is the richest (15.11%) one in their amino acid compositions. BnTT12-1, BnTT12-2 and BoTT12 have 21 potential phosphorylation sites, and BrTT12 has 19, implying that phosphorylation might be involved in regulating their protein activity. They were predicted with no signal peptide or signal anchor, while they were predicted with the higheset possibility to be located on cytoplasmic membrane since each of them possesses 9 predicted significant transmembrane domains. These features imitate the membranen-associated transporter AtTT12.The 4 proteins share very similar secondary structures,αhelix is the most abundant proportion (64.69%, 62.52%, 64.89% and 64.10%), followed by random coil (20.71%, 21.10%, 19.72% and 18.93%). The whole molecule is dominated by a large number ofαhelices, especially largeαhelices exist at near-N-terminus, near-C-terminus and C-terminus regions. Extended strands,β-turns and random coils distribute nearly evenly along the whole protein. These features is similar to those of TT12-type MATE family transmembrane domain proteins. Their tertiary structure could not be predicted currently.BLAST at both nucleotide and amino acid levels, pairwise-and multi-alignment of sequences, and phylogenetic anylysis all indicate that these 4 genes share the highest homologies with AtTT12. Clues from gene structure, protein structure and sequence identities all suggest that BnTT12-1, BnTT12-2, BrTT12 and BoTT12 are orthologous genes of AtTT12.3) In Brassica basic (diploid) species, the TT12 gene was not triplicated as compared with AtTT12Through exhaustive cloning, this research only isolated 2, 1 and 1 TT12 gene(s) from B. napus, B. rapa and B. oleracea respectively. Southem blot results coincided with this result. Hence, it is concluded that, after divergence with Arabidopsis, the TT12 gene in Brassiceae has not been triplicated, or most of the triplicated members were lost right after the triplication event, leading to the single-copy situation in current Brassica diploid species.4) B. rapa and B. oleracea both really are parental species of B. napusBnTT12-1 and BrTT12 share 96.5% of identities on whole genomic sequence scale, and 99.4% between BnTT12-2 and BoTT12. These homologies are much higher than that between BnTT12-1 and BnTT12-2, which are intra-species paralogs. Ananlysis with amino acid sequences also gave the same trends. On phylogenetic trees of both nucleotide and amino acid sequences, BnTT12-1 groups with BrTT12 first, and BnTT12-2 groups with BoTT12 first. Furhermore, clues from intron similarities, position and length of the oligo A structure in the 5' UTR, and featured mutation sites on both nucleotide and amino acid sequences, all point to the corresponding relationships of BnTT12-1 to BrTT12 and BnTT12-2 to BoTT12, 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.5) Some new structural features of TT12 are revealedThe 4 Brassica TT12 genes all have alternative transcription iniation sites in the 5' UTR, BnTT12-1 at A₁ and A₆, BnTT12-2 at A₁ and A₂, BrTT12 at A₁ and G₃, and BoTT12 at A₁, A₅, A₃₀, A₃₅ and G₅₉, respectively. In their 3' UTRs, altenative poly A tailing sites were detected, BnTT12-1 at C₂₆₂₇, T₂₆₉₄, C₂₆₉₄ and T₂₇₁₂, BnTT12-2 at T₂₉₂₉, C₂₉₃₉ and C₃₀₀₀, BrTT12 at C₂₆₂₄, C₂₆₈₈, C₂₆₉₁ and T₂₇₁₁, and BoTT12 at T₂₉₉₄, T₃₀₄₀ and C₃₀₆₂, respectively. They perhaps represent one kind of cis-regulation, or just allowable deviations in transcription initiation and poly A tailing processes.Oligo A stretch just upstream the start codon ATG is a conserved common feature of Brassicaceae TT12 genes. In BnTT12-1, it is 22 bp, with 7 bp of interval upstream ATG, while 10 bp / 6 bp for BnTT12-2, 25 bp / 7 bp for BrTT12, and 10 bp / 6 bp for BoTT12, respectively. Interestingly, AtTT12 also has such a structure, which is 10 bp and 21 bp upstream ATG. This oligo A structure might play a role in recognition or binding of the ribosome in translation. So it deserves further study.6) Construction of an antisense plant expression vector for suppression of BnTT12 family, BrTT12 and Bo TT12 A 589-bp antisense fragment (drived by CaMV 35S promoter) conserved in BnTT12 family, BrTT12 and BoTT12 was integrated into intermediate vector pCambia2301G to replace the GUS gene, then an antisense plant expression vector p2301G-TT12A was constructed for transgenic suppression of all intrinsic BnTT12, BrTT12 and BoTT12 transcripts.