Cloning And Comparative Genomic Study Of C4H Gene Families Of Brassica Napus And It's Parental Species

Phenylpropanoid pathway produces a large number of biologically important secondary metabolites through several important branch pathways. One of them synthesizes lignins, which play fundamental roles in mechanical support, solute conductance and disease resistance in higher plants. Another important branch pathway synthesizes various flavonoid compounds. In addition to attracting pollinators and protecting plants from UV irradiation and attacks by fungi and animals, flavonoids also possess anti-inflammatory, antiallergenic, antioxidant or cancer preventive functions in human. Phenylpropanoid pathway also synthesizes coumarins, salicylic acid, isoflavonoids (phytoalexins), chlorogenic acids and stilbenes to act as signaling molecules or antagonistic ingredients. Manipulation of phenylpropanoid pathway metabolites has long been a hotspot. Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) catalyzes the hydroxylation of trans-cinnamic acid to 4-hydroxycinnamate and is the second key enzyme of common phenylpropanoid pathway. It belongs to CYP73A subfamily of cytochrome P450-dependent monooxygenase superfamily. Core enzymes of phenylpropanoid metabolism are believed to form an enzyme complex, and C4H plays a pivotal role at the interface between cytosolic phenylpropanoid pathway and membrane-localized electron-transfer reactions.Brassica napus, B. rapa and B. oleracea are important oil or horticaultural crops worldwide. In them, many agronomically important traits related to phenylpropanoid pathway are focuses of genetic improvement pursued by researchers for many years. For example, the commonly occurred lodging problem calls for stronger stems and branches. Improvement of resistance to diseases needs quicker and enhanced cell wall lignification in response to pathogen invasion. Genetic engineering of lignin pathway flux, monolignol ratio and lignin composition provides a promising strategy to cope with these problems. In recent years yellow seed trait of oilseed rape has attracted many researchers due to its good quality. However, lacking of yellow-seeded genotypes together with instability of yellow seed phenotype has largely retarded breeding and application of yellow-seeded rapeseed. The mechanism of yellow seed trait formation of oilseed rape especially B. napus is still not clear. The most typical feature of yellow seed trait is the reduction of lignin and pigment contents in the seed coat. As has been revealed, plant seed coat pigments are polymers of proanthocyanidin, a metabolite of flavonoid pathway. Study on Brassica C4H genes will help dissect the mechanism of yellow seed trait formation and lay the base for transgenic creation of stable yellow-seeded B. napus. In family Brassicaceae, except the characterized C4H gene from Arabidopsis thaliana (AtC4H), no other full-length C4H gene has been cloned, though many important oilseed and vegetable crops are included in this family. This dissertation reports the cloning, molecular characterization, and comparative genomic analysis of C4H gene families from B. napus and its parental species B. rapa and B. oleracea.Using typical black-seeded B. napus inbred line 5B, full-length cDNAs of 18 members, genomic sequences of most members, together with conserved region of 1 member, of B. napus C4H gene family (BnC4H) were isolated and named from BnC4H-1 to BnC4H-19.BnC4H-1 gene: 2192bp, 5' untranslated region (UTR) of 93bp, 3' UTR of 131bp, intron 1 of 71bp (879-949), intron 2 of 379bp (1084-1462), open reading frame (ORF) of 1518bp, encoding a typical 505-aa C4H protein, molecular weight (Mw)=57.73KDa, isoelectric point (pI)=9.11.BnC4H-2 gene: 2108bp, 5' UTR of 95bp, 3' UTR of and 103bp, intron 1 of 85bp (881-945), intron 2 of 327bp (1080-1406), ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw= 57.74KDa, pI=9.13.BnC4H-3 mRNA: 1831bp, 5' UTR of 183bp, 3' UTR of and 130bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw= 57.83KDa, pI=9.04.BnC4H-4 gene: 2110bp, 5' UTR of 11bp, 3' UTR of and 190bp, intron 1 of 71bp (797-869), intron 2 of 320bp (1002-1321), ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw= 57.76KDa, pI=9.21.BnC4H-5 gene: 2430bp, 5' UTR of 124bp, 3' UTR of and 145bp, intron 1 of 75bp (910-984), intron 2 of 640bp (1113-1752), single-base deletion in coding region leading to ORF shifting, ORF of 1446bp, encoding a non-typical 481-aa C4H protein, Mw= 55.38KDa, pI=9.06.BnC4H-6 gene: 2557bp, 5' UTR of 45bp, 3' UTR of and 163bp, intron 1 of 90bp (831-920), intron 2 of 741bp (1055-1795), ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw= 57.60KDa, pI=9.16.BnC4H-7 mRNA: 1686bp, 5' UTR of 8bp, 3' UTR of and 1414bp, mutation in coding region leading to premature stop codon, ORF of 264bp, encoding a non-typical 87-aa C4H protein, Mw= 9.61KDa, pI=9.87. BnC4H-8 mRNA: 1588bp, 5' UTR of 57bp, 3' UTR of and 13bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.78KDa, pI=9.19.BnC4H-9 mRNA: 1659bp, 5' UTR of 11bp, 3' UTR of and 130bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.86KDa, pI=8.90.BnC4H-10 mRNA: 1594bp, 5' UTR of 6.5bp, 3' UTR of and 11bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.74KDa, pI=9.21.BnC4H-11: 538-bp conserved fragment.BnC4H-12 mRNA: 1706bp, 5' UTR of 124bp, 3' UTR of and 394bp, mutation in coding region leading to premature stop codon, ORF of 1197bp, encoding a non-typical 398-aa C4H protein, Mw=45.47KDa, pI=9.16.BnC4H-13 gene: 1957bp, 5' UTR of 60bp, 3' UTR of and 11bp, intron 1 of 83bp (846-928), intron 2 of 285bp (1063-1347), ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw= 57.70KDa, pI=9.20.BnC4H-14 mRNA: 1589bp, 5' UTR of 60bp, 3' UTR of and 11bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.60KDa, pI=9.20.BnC4H-15 mRNA: 1704bp, 5' UTR of 65bp, 3' UTR of and 121bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.73KDa, pI=8.95.BnC4H-16 mRNA: 1722bp, 5' UTR of 45bp, 3' UTR of and 159bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.45KDa, pI=9.11.BnC4H-17 mRNA: 1737bp, 5' UTR of 65bp, 3' UTR of and 988bp, direct tandem repeat in coding region leading to premature stop codon, ORF of 684bp, encoding a non-typical 227-aa C4H protein, Mw=26.22KDa, pI=9.79.BnC4H-18 mRNA: 1594bp, 5' UTR of 65bp, 3' UTR of and 323bp, mutation in coding region leading to premature stop codon, ORF of 1206bp, encoding a non-typical 401-aa C4H protein, Mw=45.92KDa, pI=9.23.BnC4H-19 mRNA: 1723bp, 5' UTR of 45bp, 3' UTR of and 160bp, ORF of 1518bp, encoding a typical 505-aa C4H protein, Mw=57.67KDa, pI=8.93.Quite a few members of BnC4H family show coding region mutations that affect protein coding ability. Because of point mutation, base deletion, or insertion of direct tandem repeat, BnC4H-5, BnC4H-7, BnC4H-12, BnC4H-17 and BnC4H-18 do not encode typical C4H proteins. The 745-887bp region of BnC4H-17 is a 143-bp direct repeat of the 602-744bp region, which results in premature mutation in coding region. A single base C was deleted in BnC4H-5 at 2240bp, also leading to premature mutation in 3' coding region。The same single base C deletion exists after 1525bp of BnC4H-12 mRNA, and a G to A mutation occurred at 1321bp leading to earlier premature mutation than BnC4H-5. BnC4H7 shows the earliest premature mutation at 270bp of A to T, leading to drastic ORF shifting, and encodes a polypeptide of only 87 aa.. BnC4H18 mRNA also has a premature mutation at 1270bp of G to A, encoding a polypeptide of only 401 aa.Except above 5 members and the fragment BnC4H-11, all other 13 members of BnC4H family encode standard C4H proteins and show typical C4H features, such as the P450 conserved domain, CypX conserved domain, enzyme active site residues I₁₀₉, K₁₁₃, V₁₁₈, F₂₂₀, E₃₀₁, N₃₀₂,I₃₀₃, V₃₀₅, A₃₀₆, T₃₁₀, R₃₆₆, R₃₆₈, A₃₇₀, I₃₇₁, P₃₇₂, L₃₇₄, V₃₇₅, P₃₇₆, H₃₇₇, K₄₈₄, F₄₈₈ and L₄₉₀, haeme binding motif PFGVGRRSCPG, T-containing motif A₃₀₆AIETT₃₁₁, and substrate binding site SRSs. Besides, they also contain one signal peptide, 2 transmembrame regions, 22 to 24 potential phospharylation sites, 1 to 3 potential N-glycosylation sites, 4 to 6 potential amidation sites, and 5 to 8 potential sites. Their predicted secondary and tertiary structures are also similar to each other. BnC4H-3 and BnC4H-4 also possess a potential cAMP-cGMP dependent protein kinase phosphorylation site R₂₄₈-R₂₅₁. BnC4H-5, BnC4H-7, BnC4H-12, BnC4H-17 and BnC4H-18 show quite differences from other members, since they lack short or long a C-terminal region resulted of premature mutation.Using Brassica oleracea L. var. acephala DC, full-length cDNAs and corresponding genomic sequences of 3 members of C4H family (BoC4H) were isolated. Similarly, 3 members of Brassica rapa C4H gene family (BrC4H) were also isolated. BoC4H-1 is almost identical to BnC4H-5 in sequence aligment, and they also show the same single base premature mutation. Other BoC4H and BrC4H members encode standard C4H proteins and share similar protein features, such as conserved domains, predicted enzyme active sites residues, haeme domain, substrate binding motif, with BnC4H family members.Members of BnC4H, BoC4H and BrC4H gene families show high homologies to each other, and they also show high homologies to AtC4H. On the phylogenetic tree, AtC4H lie among the various BnC4H members and divide them into 2 distinct large groups. AtC4H first group with BnC4H-6, BnC4H-7, BnC4H-16 and BnC4H-19, and then group with other Brassica C4H members. BnC4H-5, BnC4H-7, BnC4H-12, BnC4H-16, BnC4H-19, BnC4H-6, BoC4H-1, BoC4H-2 and BoC4H-3 constitute a distinct large group, which keeps considerable distance with AtC4H and is more distant to other Brassica C4H members. BnC4H-5 and BoC4H-1 are slmost identical to each other with the 2.4kb full-gene region, with only one base of difference at 1608-bp oligoT site, and share the same premature mutation. Many BoC4H and BrC4H gene family members are much similar to BnC4H members than to intraspecies members.Sequence multi-alignments also indicated that many Brassica C4H members share the same regional fragments. In fact, many members are just tandem repeats of local regions from other genes. This phenomenon can only be explained as intra-allelic or inter-allelic fragment exchanges. This result directly proves that homologous fragment exchange is very active in the genome of the cross pollinated Brassica species like B. napus.Southern blot hybridization of C4H gene families of these 3 species resulted in 9, 4 and 5 bands respectively, further indicating that C4H in these species are encoded by multiple genes, and the copy numbers in B. napus are just the sum of those from B. oleracea and B. rapa.Using total RNA from root, stem, hypocotyl, cotyledon, leaf, flower, bud, seed of 10 d after flowering (10DAF seed), 20DAF seed, 20DAF seed and silique pericarp of typical black-seeded line 5B, the transcription of representative members BnC4H-1, BnC4H-2, BnC4H-6, BnC4H-10 and BnC4H-13 of BnC4H gene family was detected by member-specific primer directed semi-quantitative RT-PCR. BnC4H-1 is expressed highest in flower, followed by pericarp, with the lowest in 20DAF seed, and little difference among other organs. BnC4H-2 is also expressed highest in flower, followed by bud, hypocotyl, leaf, 10DAF seed and 20DAF seed, weak in cotyledon and pericarp, and no expression in root. BnC4H-6 is intensively expressed in all tissue organs except for 20DAF seed. BnC4H-10 is expressed highest in flower, followed by bud, root, 20DAF seed and 30DAF seed, while weak in other organs. BnC4H-13 is intensively expressed in hypocotyl, stem, flower, 10DAF seed, and pericarp, but weak in cotyledon, leaf, 20DAF seed and 30 DAF seed.Using member-specific primers of above 5 members, their expression in major reproductive organ tissues of near-isogenic black-seeded line L1 and yellow-seeded line L2 was detected and compared by semi-quantitative RT-PCR. In main stages of seed development especially in 30DAF seed, L2 showed obviously lower levels of transcription of BnC4H-1, BnC4H-2, BnC4H-6, BnC4H-10 and BnC4H-13 than L1.Summarily, the following conclusions and findings can be inferred:1. Cloning of gene family members and Southern blot detection indicated that in B. napus and its parental species C4H is encoded by muti-gene family members.2. In the ancestor species of family Crucifereae, C4H is mutagenic, and the single copy phenomenon is A. thaliana is caused by loss of some members during evolution. AtC4H corresponds to BnC4H-1-type members, but the member(s) orthologous to BnC4H-5-type members is lost.3. The basic reasons leading to muti-gene family in Brassica are inheritance of the mutagenic feature of Crucifereae ancestor and the Brassica genome "triplication" as compared with A. thaliana. Different large groups of BnC4H family members may represent multigenic members inherited from Crucifereae ancestor, while the highly homologous intra-group members are the result of the Brassica genome "triplication". This result further proves the facticity Brassica genome "triplication" on gene family cloning level.4. Sequence alignments and Southern detection indicated that B. oleracea and B. rapa are actually the parental species of B. napus, or at least they are direct providers of genetic substances of B. napus. After the formation of amphidiploid, some C4H members are basically non-changed and their corresponding relationships with parental members are quite obvious (BnC4H-5 and BoC4H-1), while other members are not so obvious possibly due to fragment exchange, base mutation etc.5. Some members of C4H gene families of Brassica species have experienced fast mutation and sequence evolution/deterioration, such as allelic or non-allelic fragment exchange, premature mutation, direct tandem repeat insertion etc., which result in fast divergence of the encoded proteins.6. Another important evolution of C4H gene family members of Brassica species is the differentiation, division and complementation of some members in tissue specicificity of transcription.7. In major seed development stages of tested yellow-seeded stock line of B. napus, transcription of representative C4H members shows obviously lower levels as compared with black-seeded lines, indicating that suppression of C4H expression is actually an important reason of the formation of yellow seed trait. But the very most reason should be the mutation of upstream regulatory signals or the pivotal gene(s) defining seed coat development.This work lays the base for further investigating the roles C4H genes play in determining many important traits, dissecting the molecular mechanisms and evolution of C4H genes, and manipulating C4H-related important traits of Brassica species through regulating the expression levels of C4H genes.