Cloning Of FOMTs From Narcissus Tazetta Var. Chinensis And Screening Of Transcription Factors Of FOMT2

Plant O-methyltransferases(OMTs)are thought to be involved in the methylation of plant secondary metabolites,especially phenylpropanes and flavonoids.However,the enzymatic reaction in the biosynthesis of flavonoids is regulated by transcription factors.There are few reports on the interaction between transcription factors and flower color genes in Narcissus tazetta var.chinensis.This paper focuses on cloning and analyzing the FOMTs of N.tazetta,discusses the characteristics of FOMTs,and establishes a single hybrid library.The transcription factors with transcription regulation function are screened out by yeast single hybrid,and provided a new reference for the study of flower color genes in Narcissus tazetta var.chinensis.The main results are as follows:1.We isolated flavonoid biosynthetic pathway gene FOMTs full length cDNA:Three FOMT genes were selected from the RNA-seq database for differential expression.The FOMTs were isolated from N.tazetta.'Jinzhanyintai' by RACE and RT-PCR technipue,which were NtFOMTl,NtFOMT2?NtFOMT3.The full length of NtFOMT1 cDNA is 1341 bp,including 82 bp 5' UTR,162 bp 3,UTR and 1098 bp ORF,encoding 366 amino acids.The full length of NtFOMT2 cDNA is 1404 bp,including 33 bp 5' UTR,279 bp 3' UTR and 1092 bp ORF,,encoding 363 amino acids.The full length of NtFOMT3 cDNA is 1206 bp,including 117 bp 3' UTR and 1089 bp ORF,encoding 362 amino acids.2.The bioinformatics analysis,phylogenetic tree construction and fluorescence quantitative expression analysis of NtFOMT1,NtFOMT2 and NtFOMT3 were carried out.The results of quantitative PCR analysis showed that NtFOMTl varied greatly in different stages of Narcissus,and its expression pattern was ?>?>?>? in petals and secondary crown.The highest expression level of NtFOMT2 and NtFOMT3 was observed in the crown,and the expression pattern of NtFOMT2 and NtFOMT3 was different in different periods of the Narcissus.The expression pattern of NtFOMT2 and NtFOMT3 was ?>?>?>?.3.To analysis the regulating factors of pigment genes in Narcissus tazetta,regulation sequence of 5' end of NtFOMT2 gene was cloned with method of chromosome walking method:The cis-acting element of promoter was analyzed by on-line software.The results showed that the promoter sequence of NtFOMT2 gene also included cis-acting elements such as ACE,ATCT-motif,G-box,Sp1 and many light responsive elements,besides necessary transcription start site TATA-box and CAAT enhancer.Furthermore,this promoter sequence included binding site MBS element participating in drought inducement of MYB,and a MBS binding site of MYB transcription factor and so on.4.One-hybrid cDNA library is simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination.Constructing the Bait Plasmid and Bait Yeast Strain,test the Y1HGold bait strain for background AbAr expression,determining the Minimal Inhibitory Concentration of Aureobasidin A for Your Bait.Use SMART Technology and yeast biology to construct and screen the library,get 177 positive clones.Distinguishing Genuine Positive from False Positive Interactions.45 positive clones were finally obtained.After comparing with the local RNA-Seq database and NCBI online database of Narcissus tazetta var.chinensis,5 positive clones were determined.Perform point-to-point One-Hybrid verification.The verification results indicated that all were positive clones.5.Quantitative expression analysis of five clones obtained:Quantitative fluorescence analysis showed that the five genes were expressed in different developmental stages of Narcissus tazetta var.chinensis and their expression levels were different.The expression levels of NtGENE13 and NtGENE177 were the same and all of them were down-regulated in petals,the expression levels in different periods were I>II>III>IV;the overall expression levels in the sub-crown were down-regulated,and their expression levels were ?>?>?>?.Consistent with the NtFOMT2 expression pattern,it may be positively regulated.The expression levels of NtGENE31,NtGENE35,and NtGENE103 were the same,which was the upregulation mode.Contrary to the NtFOMT2 expression pattern,they may be negatively regulated.