| Chinese narcissus(Narcissus tazetta L.var.chinensis)is a perennial bulbous plant and belongs to the Amaryllidaceae family,which shows high ornamental value of traditional flowers.And it is a kind of highly sterile triploid plant,hence can only be propagated vegetatively with bulblet generally.Therefore,it is difficult to cultivate new varieties by traditional hybrid breeding methods,which limits the variety and color of Chinese narcissus.As a result,the development of Chinese narcissus has been restricted severely.Studying the molecular mechanism and transformation of Chinese narcissus will be an effective method for the innovation of Chinese narcissus colors.One b HLH transcript factor was selected from transcriptome of Narcissus ‘Jin Zhan Yin Tai' petals and corona,named Ntb HLH1,the full length sequence had been cloned by the use of 3?RACE.The relative expression level of basal plates,petals and corona of Chinese narcissus in bud stage,half opened stage and full stage were analyzed by q-PCR.The expression vectors were constructed for the stable transformation research.The expression level of structural genes in the flavonoid metabolic pathway from plant materials of stable transformation were analyzed by q-PCR.The contents of total flavonoids and anthocyanins in transgenic plants were determined.The interactionbetween Ntb HLH1 and 7 R2R3-MYBs transcription factors was verified by yeast two-hybrid experiments.The regulatory effect of Ntb HLH1 and7 R2R3-MYBs complexes on the structural genes of flavonoid synthesis pathway was analyzed by a dual luciferase reporter assay.The results understand the function of Ntb HLH1 transcription factor of the regulatory effect of the MYB-b HLH complex on the structural gene promoter of the flavonoid synthesis pathway,and provide a foundation for further understanding the regulatory mechanism of Chinese narcissus flavonoid synthesis pathway.The main results were summarized as following:1.The c DNA sequence of Ntb HLH1 gene was cloned from the bulb of Chinese daffodil using 3?RACE and RT-PCR.The sequence analysis showed that the open reading frame size of Ntb HLH1 gene was1896 bp,encoding 632 amino acids.Ntb HLH1's Gen Bank accession number is QDS02912.1.The Multiple alignments showed Ntb HLH1 contains two domains that are conserved in flavonoid-related b HLH-type TFs,including an N-terminal b HLH-MYC?N and C-terminal HLH domain.The phylogenetic analysis showed that Ntb HLH1 belongs to the IIIf subfamily,Ph JAF13,At GL3,and At EGL3 in the same subfamily with Ntb HLH1,which involved in regulating theflavonoid biosynthesis.q-PCR results showed that Ntb HLH1 transcript accumulated in the basal plate.The relative expression of Ntb HLH1 gene of petals and corona decreased at first and then increased with the opening of Chinesedaffodils,and the petals have the highest expression during the flower bud stage while the corona had the highest expression during the full flower stage.2.The expression vectors of p SAK277-Ntb HLH1 were constructed then transform into agrobacteria.Transgenic tobacco plants were obtained and the colour of transgenic flowers become redder.The results of q-PCR analysis indicated that over-expression of Ntb HLH1 affected the transcript level of the flavonoid biosynthesis pathways genes.Expression of CHS,DFR,and AN1 were significantly up-regulated in transgenic flowers compared with contrast tobacco.The anthocyanidins and flavonoids in transgenic plants showed a higher expression than that of wild types.The results of the study showed that Ntb HLH1 regulates the expression level of the transcription factor AN1 on the tobacco flavonoid synthesis pathway,thereby regulating the synthesis and accumulation of anthocyanins in tobacco.3.A yeast two-hybrid expression vector for Ntb HLH1 and 7R2R3-MYB proteins was constructed,and the interaction between Ntb HLH1 and seven MYB proteins was studied by using yeast two-hybrid.The results show that Ntb HLH1 can interact with Nt MYB4,Nt MYB6,Nt MYB7 and Nt MYB8 to form MBW complex.4.Results of double-luciferase assays show that Ntb HLH1 can activate the expression of the structural gene Nt FLS of the narcissusflavonoid synthesis pathway alone,and the combined transformation activation effect of Ntb HLH1 and Nt MYB4 is more significant.Nt MYB6 and Nt MYB7 can activate the expression of the structural gene Nt DFR promoter alone,and Ntb HLH1 transform with Nt MYB6 can activate the expression of the structural gene Nt DFR of the Chinese narcissus flavonoid synthesis pathway more significant.Single transformation of Ntb HLH1 or Nt MYB2 cannot activate the Nt LAR promoter activity,only Ntb HLH1 transform with Nt MYB2 can activate the expression of the structural gene Nt LAR of the Chinese narcissus flavonoid synthesis pathway. |
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