| Chinese narcissus(Naricissus tazetta var.Chinensis)belongs to perennial herb of Amaryllidaceae family,is one of the most famous traditional bulbous flower in China.On base of attractive flowers pattern and good fragrances,Chinese narcissus has very important ornamental and economic values in global floriculture market.Chinese Narcissus has been reported as a triploid species,mostly propagated by a vegetative method,as genetically advance improvement using traditional cross breeding is complicated.Therefore,only a few cultivars have been used for commercial production in China and many agronomic characters have to be improved such as flower colors.The alteration of petal color through genetic engineering may assist to solve this problem.The compositions of the flavonoid compounds in narcissus cultivars were found to be flavonols,with no anthocyanins detected.Therefore,the investigation on molecular mechanisms why narcissus has no anthocyanins biosynthesis would provide benefit for developing new cultivars with diverse color patterns.Flavonoids are a group of diverse secondary metabolites which are broadly distributed in plants consisting of the flavones,isoflavones,flavonols,anthocyanins and proanthocyanidins.Several transcription factors play crucial role in plant growth regulation and development.R2R3 MYB transcription factors play key functions in the regulation of flavonoid metabolites.In present study,three novel R2R3 MYB transcriptional factors NtMYB2,NtMYB3 and NtMYB4 were identified from Chinese Narcissus and were functionally characterized.Results are listed below.1.The 3' end of NtMYB2 ORF was isolated by 3' RACE-PCR.NtMYB2 contained 783 bp nucleotides encoding 251 amino acids residues.The NtMYB3 gene had a 606 bp ORF encoding 201 amino acids.Sequence alignment of amino acids indicated that NtMYB2 and NtMYB3 have bHLH-interaction motif[D/E]Lx2[R/K]x3Lx6Lx3R and DNEI motif in R3 repeat.NtMYB2 and NtMYB3 belongs to subgroup 4 R2R3 MYB transcriptional factors that are related to repressor MYBs group involved in the regulation of anthocyanin and flavonoids.NtMYB2 and NtMYB3 contained C2 repressor motif at C-terminal like other members of subgroup 4.Phylogenetic tree analysis showed that the NtMYB2 and NtMYB3 classified into the MYB4-like clade.NtMYB4 is also a member of R2R3 MYB gene family in Chinese narcissus.It belongs to members of subgroup 19,which are mostly flowers specific genes.In addition to R2-R3 region,NtMYB4 display conserved motif "NF/YWSV/MEDF/LW" at the end of C-terminal.Phylogenetic tree analysis revealed that NtMYB4 group with MYB transcriptional factors which are flower specific.Subcellular localization assay indicated that NtMYB2,NtMYB3,NtMYB4 were all nuclear proteins.2.Three MYB genes have different expression patterns in Chinese narcissus.NtMYB2 gene is expressed in all examined Chinese narcissus tissues including petals,corona,and basal plate;however the levels of transcription in petals and corona at bud stage was significantly higher than that at full opened stage of petals and corona and in leaves.NtMYB3 displayed more expression levels in basal plates.NtMYB4 was found mainly expressed in flowers,especially in the full open stage of petals and corona.3.Transient expression in agroinfiltrated tobacco leaves showed that NtMYB2 and NtMYB3 strongly reduced the red pigmentation induced by the positive control StMYB.NtMYB4 also reduced the red pigmentation that was induced by an activator StMYB,but the repress effect was weaker than NtMYB2 and NTMYB3.The presence of bHLH didn't reduce the repress effects of NtMYB2 and NtMYB3,but reduce the repress effect of NtMYB4.qRT-PCR results indicated that NtMYB2,NtMYB3 and NtMYB4 significantly reduced the transcript levels of all structural gene involved in anthocyanin biosynthetic pathway.4.Ectopic overexpression of NtMYB2 in tobacco significantly alters the floral phenotype and reduced the pigmentation in transgenic tobacco flower.Total anthocyanin contents were lower in transgenic flowers than those of wild type.In addition to flower colour,flowers size and length were smaller that wild type.qRT-PCR analysis suggested that NtMYB2 repressed the transcript levels of key genes of flavonoid biosynthesis pathway such as CHS,CHI,F3H,DFR,ANS,UFGT,ANR and FLS,especially UFGT gene.Ectopic overexpression of NtMYB3 alters the flowers pigmentation from deep pink to almost white in transgenic tobacco.The anthocyanin contents were lower in the transgenic flowers carrying the NtMYB3 gene as compared to wild type.In addition to flower colors,some other phonotypical changes were also observed in transgenic flowers.The flowers size and length were small in transgenic tobacco flowers than those of wild type.Moreover,the position of stigma was above the anther in transgenic flowers.QPCR results indicated that the expression levels of structural genes including CHS,CHI,F3H,DFR,ANS and UFGT encoding for anthocyanin biosynthesis pathway were strongly decreased in transgenic flowers.The expression level of FLS was also significantly lower in transgenic flowers than wild type.While the expression levels of genes including ANR and LAR were up-regulated in transgenic flowers,both these two genes are involved in the regulation of proanthocyanin biosynthetic.Overexpression of NtMYB4 in tobacco changes the flower color by reducing the pigmentation and anthocyanin contents in transgenic flowers.Elongations of stigma were observed in transgenic flowers.Furthermore,NtMYB4 overexpression lines showed dwarfism with small flower size and narrow leaves.QPCR analysis showed that expression levels of anthocyanin/flavonoid biosynthesis genes were down-regulated in transgenic flowers carrying NtMYB4 gene.5.These results revealed that NtMYB2,NtMYB3 and NtMYB4 are all repressors of flavonoid biosynthesis.NtMYB2 is involved in regulation of flavonoid biosynthesis pathway which may play a repressive role in down-regulating anthocyanin levels in flowers.NtMYB3 may be a negatively regulator of anthocyanin and flvonol but an activator of PA biosynthesis in basal plates.NtMYB4 is also a negatively regulator of flavonoid biosynthesis and may play a role by competing the combination of bHLH factors with MYB activators in flowers of Chinese narcissus. |
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