Cloning And Functional Analysis Of Ugt79b1 Gene Promoter From Arabidopsis Thaliana

The biosynthetic pathway of anthocyanin is basically well studied,but the research on its regulatory mechanism is still unclear.Ugt79b1 encodes an anthocyanin glycosyltransferase,which catalyzes the attachment of UDP-xylose to the 3'-glucosyl 2'' position of anthocyanin and produces the xylose-based modification of anthocyanin.Glycosides have an important role for the stability of anthocyanins.Ugt79b1 gene was synergistic with the expression of biosynthetic gene in the mid and late stage of anthocyanin pathway,indicating that ugt79b1 gene and anthocyanin LBG gene should have the same expression regulation mechanism.This work hopes to provide the basic data for further revealing the transcriptional regulation mechanism of anthocyanins by studying the transcriptional regulation mechanism of the promoter of the ugt79b1 gene.1.In this work,the promoter region of the ugt79b1 gene was first cloned from the Arabidopsis genome,which only has 548 bp in length.The shorter promoter region length is also useful to the study of the transcription function of promoter.The promoter region was subscribed on the website for predictive analysis of transcription factor binding sites.The results showed that the promoter region contained several light-related signal sites,one auxin signal control site,two light and abscisic acid shared signal control sites,endosperm-related signal sites,and two cell cycle regulation related signal sites.2.The promoter was divided into three parts according to the distribution of the regulatory signal sites on the promoter region of the ugt79b1 gene.The first segment is 211 bp in length at the 5 'end,which contains multiple light control sites and a growth cycle regulatory site.The second segment is from the 212 bp to 411 bp,a total length of 200 bp,which contains two abscisic acid-related sites.The two abscisic acid-related sites and light control sites overlaps.The other binding site is a MYB regulatory factor associated with endosperm expression.The third segment is from 412 bp to 548 bp,with a total length of137 bp.This region contains one growth cycle-related regulatory signal site and one high expression-related site.3.The promoter region of the ugt79b1 gene was deleted from the 5 ' end,and the GUS reporter vector pBU23 containing the second and third promoter regions was constructed.The GUS reporter vector pBU3 containing only the third promoter region was constructed,and also a GUS expression vector pBU123 containing a full length of 548 bp promoter region.The three plasmids were transformed into Arabidopsis thaliana,and three transgenic Arabidopsis thaliana were obtained by kanamycin screening and PCR verification.4.The expression of GUS reporter in transgenic Arabidopsis thaliana was analyzed.Anthocyanins are a synthetic product of stress in Arabidopsis thaliana.The anthocyanins in the Arabidopsis thaliana leaves on the normal growth state are essentially non-synthetic.Therefore,in order to allow the exogenous promoter to play a transcriptional activity in the transgenic Arabidopsis thaliana,the transgenic Arabidopsis should be treated with stresses.In this work,transgenic Arabidopsis thaliana seedlings were induced by abscisic acid hormones.The expression of GUS reporter in the leaves of Arabidopsis thaliana seedlings transformed with pBU123 plasmid and pBU23 plasmid was significantly higher than that in transgenic Arabidopsis seedlings with pBU3 induced by abscisic acid.The activity analysis showed that the expression level was similar in seedlings of pBU123 and pBU23.And GUS was not expressed in the leaves of Arabidopsis thaliana seedlings transformed with pBU3 plasmid.As the resulsts showed that the fragment from 212 bp to 411 bp is the central regulator region of the ugt79b1 promoter when treated by ABA.