Cloning And Functional Analysis Of Two R2R3-MYB Genes In Flavonoid Metabolic Pathway Of Narcissus Tazetta Var.Chinensis

Chinese Narcissus(Narcissus tazetta var.chinensis),which belongs to the family of Amaryllidaceae is one of ten famous traditional flowers in China,city flower of Zhangzhou and provincial flower of Fujian.The development of narcissus industry had been limited by the absence of available resources and variety of colour.A series of structural genes in the Flavonoid metabolic pathway which are mainly involved in color formation of flower are mainly regulated by the R2R3-MYB transcription factor.Two R2R3-MYB transcript factors were selected from transcriptome of Narcissus 'Jinzhanyintai' floral and basal plate,and the full length sequence had been cloned by the use of RT-PCR and RACE.The relative expression level in different flowering stages and different organs of Chinese narcissus were analyzed by QPCR.The expression vectors were constructed and transformed into Agrobacterium for the following tobacco transient and stable transformation research.The expression level of structural genes in the Flavonoid metabolic pathway were analyzed by QPCR from plant materials of tobacco transient and stable transformation.The results provided a reference for the study of R2R3-MYB flavonoid metabolic pathway in Chinese narcissus.The main results were summarized as follows:1.NtMYB5 had been cloned by RT-PCR methods from cNDA of narcissus petal.The open reading frame of NtMYB5 681bp which was capable of encoding 226 amino acids.The blast result showed NtMYB5 has highest similarity with CsMYB4a?DkMYB13 and GmMYB5.Multiple alignments showed that NtMYB5 contain R2-domain and R3-domain,a pdLNLD/ELxiG/S motif was also found near c-terminal.Phylogenetic tree analysis showed NtMYB5 was closer to repressor genes of anthocyanin;The expression level of NtMYB5 was highest in perianth and increased during the process of flowering and there dont't have much differences in other organs;2NtMYB6 had been cloned by RT-PCR and RACE methods from cNDA of narcissus basal plate which the accession number is KY645961.The cDNA was 1008 bp in length with an open reading frame 750 bp which was capable of encoding 249 amino acids.The blast result showed NtMYB6 has highest similarity with FvMYB12.The Multiple alignments also showed it has R2-domain and R3-domain,Phylogenetic tree analysis showed NtMYB6 was closer to activator genes of proanthocyanin.The expression level of NtMYB6 was highest in basal plate.3.The expression vectors of MYB genes were constructed for transient.Several treatments been injected to tobacco leaves.The accumulation of anthocyanin activated by StMYB was suppressed by NtMYB5 in transient expression of tobacco,most structure genes of flavonoid pathway were down-regulated but FLS was up-regulated;The accumulation of anthocyanin activated by StMYB was inhibited slightly by NtMYB6;PAL and 4CL was up-regulated when NtMYB6 was injected alone.4.Transgenic tobacco plants were obtained and the colour of transgenic plant flowers become lighter.The QPCR results of petals in NtMYB5 transgenic plants were similar to those in transient expression.NtMYB6 up-regulated the expression of LAR gene in the stable transformed tobacco petals.5.These results indicate that NtMYB5 down-regulates the expression of some structural genes in the flavonoid pathway of Narcissus sativus L.and significantly inhibits the accumulation of anthocyanins.NtMYB6 up-regulates the expression of LAR,a key gene for proanthocyanidins synthesis.