Cloning Of DFR、FLS Promoters And RNAi Vector Construction Of LYCE And LYCB In Narcissus Tazetta Var.Chinensis

Narcissus tazetta var.chinensis is one of the traditional famous flowers in China and an important ornamental flower.But the color of Chinese Narcissus is limited to yellow and white at present.Carotenoid metabolic pathway and flavonoid metabolic pathway are mainly involved in color pattern formation in Chinese narcissus.With the cloning of related key genes,the mechanism of flower color regulation is understood gradually.In this study,Zhangzhou narcissus was used as the experimental material.The 962 bp and 968 bp promoter fragment of Nt DFR and Nt FLS gene respectively were amplified from genomic DNA by genome walking method.Sequence bioinformaticsanalysis,vector construction,GUS staining and q PCR analysis of the promoters were performed and the regulation of R2R3-MYB transcription factor to these two promoters was studied.The binary gene expression vector p TCK-LYCE/LYCB-FT RNAi was constructed.The results will provide more understanding of color regualtion in Chinese narcissus.Following are the main results:1.The 962 bp and 968 bp promoter fragment of Nt DFR and Nt FLS gene were cloned from genomic DNA by genome walking method,assession number is JX310698 and MH472580,respectively.The results of promoter sequence analysis show that the promoter contain a number of TATA-box,CAAT-box elements and other promoter elements,such as light responsive elements,hormone response elements,stress-response elements etc.Moreover,the promoters also contains the MYB transcription factor combination elements.2.The plant expression vector p BI121 was used to construct the p BI121-p Nt DFR::GUS vector and p BI121-p Nt FLS::GUS vector.The activity of promoter was determined by GUS histochemical staining with agro-infiltrated tobacco leaves.The GUS color of tobacco leaves treated with Nt DFR and Nt FLS promoters turned blue,indicating that Nt DFR and Nt FLS promoters had promoter activity.3.R2R3-MYB genes of Chinese narcissus,NtMYB2、NtMYB3、NtMYB4、NtMYB5、NtMYB6、NtMYB7 and NtMYB8 were transient expressed with p BI121-p Nt DFR::GUS in tobacco leaves seperately.The results of real-time quantitative PCR and GUS histochemical staining analysis indicated that NtMYB2、NtMYB3、NtMYB4、NtMYB5、NtMYB6 and NtMYB7 repressed the activity of the Nt DFR promoter.NtMYB8 positively regulated the promoters of Nt DFR.4.NtMYB2、NtMYB3、NtMYB4、NtMYB5、NtMYB6、NtMYB7 and NtMYB8 were transient expression with p BI121-p Nt FLS::GUS in tobacco leaves seperately.The results of real-time quantitative PCR and GUS histochemical staining analysis indicated that NtMYB2、NtMYB3、NtMYB5 and NtMYB8 repressed the activity of the Nt FLS promoter.NtMYB4 、 NtMYB6 and NtMYB7 positively regulated the promoter of Nt FLS.5.The total RNA from flowers of Zhangzhou daffodils was extracted and reverse transcribed into c DNA.The 204 bp of LYCE、188bp of LYCB and 531 bp of FT gene conservative fragment were isolated.Based on the plant expression vector p KANNEL and p TCK303,the binary the binary gene expression vector harboring LYCE/LYCB RNAi was constructed.