Studies On The Function Of Flavonoids 3'-hydroxylase Gene (CpF3'H) And Its Promoter In Wintersweet

Wintersweet(Chimonanthus praecox(L.)Link),the unique winter flowering woody plants in China,with elegant fragrant,and its color is mainly yellow,some varieties of flowers can be presented in varying degrees of red.So,to explore the causes and differences in the color of wax plume has become an urgent problem to be solved in wintersweet production and academic studies.In this study,we used the wintersweet labelled H29 with red inner petals in Huazhong Agricultural University as research material.By analyzing transcriptome library data of H29,the key enzymes of flavonoid3'-hydroxylase(F3'H)was selected.Then this function gene was cloned with full lenth named CpF3'H.To detect the function of this gene in wintersweets' color formation,pCAMBIA1302CpF3'H expression vector was constructed by homologous recombination method.And this functional gene was tranferred into the mutant Arabidopsis thaliana N799662 to detect its function.Moreover,the expression pattern of CpF3'H of different organs and color related periods was analyzed by real-time quantitative(qRT-PCR).In addition,the promoter fragment of the gene was cloned by combining the methods of FPNI-PCR and Takara Genome Walking Kit,named CpF3'HP.Bioinformatics analysis showed that this sequence has some typical promoter cis-components,and then the regulatory mechanisms of this gene could be further analyzed connected with the cis-components factors.And also lay the foundation for further subsequent related experiments.For further study,promoter CpF3'HP expression vector was build,and then associate CpF3'HP with MYB transcription factors our lab has isolated in wintersweet(CpTT2,Cp111,CpANT,CpMM)in arabidopsis protoplast,then according to the expression activity to determine the interaction between them.In order to investigate the effects of light,low temperature and hormone on the gene,we investigated shading and NAA treatments,and the expression of this gene was detected by qRT-PCR.The main results of this experiment are as follows:1.Relaying on the library of H29 transcripts and the flavonoid 3'-hydroxylase sequence obtained from other species was cloned,Unigene3579 in H29 transcript library was screened and cloned to the full length,named CpF3'H.By analyzing the full sequence on the ORF Finding,ORF region is 1560 bp,encoding 519 amino acids,the isoelectric point pI is 6.47 and the molecular weight is 56.997 kD.2.The expression vector pCAMBIA1302CpF3'H was constructed by homologous recombination method,which was transferred into Arabidopsis thaliana mutant N799662,and positive Arabidopsis plants were obtained.The results showed that the transferredArabidopsis' s stem and the base of petiole turned dark purper,deepen than the mutant and wild type plants.3.The result of qRTPCR of the CpF3'H in different organs and different periods of flower color formation showed that it expresses in roots,stems,leaves,flowers and fruits but had the highest expression in flowers.According that,it was also confirmed it plays an important role in the wintersweet flower's color formation.At the same time,in different flower stages,the expression level of the gene was the highest in the flower full open stage.Meanwhile,the expression level of CpF3'H in inner petals was lower than that of in the medium.all the above shows that this gene CpF3'H plays a very important role in the anthocyanin biosynthesis pathway of the red wintersweets.4.A 653 bp promoter fragment,named CpF3'HP,was cloned using the nine fusion primers of FPNI-PCR and the cloning PCR procedure of the Takara Genome Walking Kit.By PlantCare software analysis,the sequence has typical promoter cis-components,expect the generic TATAbox and CAATbox,the sequence includes light regulate components chs-CMA2 a,MNF1;CCGTCC-boxes related to tissue-specific differentiation;Bz2 gene expression and MYB binding site are related to the G-box;and endosperm specific expression is related to Skn-1_motif,etc.,in addition,there are still a few other components.Bioinformatics analysis showed that the promoter fragment might be related to the regulation of some treatments on the wintersweet like stress,light and auxin.Thereby,CpF3'HP plays a pivotal role in transcriptional regulation.5.Build CpF3'HP expression vector CpF3'HP-pCAMBIA-1391 Z,and associate CpF3'HP with MYB transcription factors our lab has isolated in wintersweet(CpTT2,Cp111,CpANT,CpMM)in arabidopsis protoplast,the result shows that the MYB transcription factors CpANT,Cp111 and CpMM can activate the expression level of the promoter of CpF3'H,it proved the interaction between the promoter and MYB transcription factors.6.The results showed that shading and cold treatment at 5? 24 h could enhance the expression of CpF3'H slightly,but NAA and 5?48h could significantly up-regulate the gene expression.This result is also consistent with the analysis of the cis-components of the promoter.