Cloning And Structural Analysis Of The Glycosyltransferase Gene EuCGT1 Promoter In The Eucommia

The promoter is a DNA sequence located upstream of the 5'end of a gene that binds RNA polymerase to initiate transcription of the gene.The promoter contains a plurality of cis-acting elements which,by binding to a trans-acting factor,determine the initiation or absence of gene transcription and the efficiency of gene transcription.Eucommia ulmoides is a valuable Chinese herbal medicine in China.It has set off a research boom of researchers because of its pharmacological effects such as blood pressure reduction,blood lipid regulation and blood sugar lowering.Glycosyltransferase,as one of the important structural modification enzymes of plant secondary metabolism,has the function of recognizing glycosyl acceptors and catalyzing the glycosylation of specific sites to synthesize glycosides.Studying the Eu CGT1 promoter of the glycosyltransferase gene in Eucommia ulmoides can not only understand the function of this promoter but also help to reveal the promoter-mediated gene expression regulation pattern.In this study,the full length of the Eu CGT1 gene was cloned from the Eucommia ulmoides genome after sequencing in the laboratory.The 1671 bp fragment of the Eu CGT1gene was isolated from the Eucommia ulmoides genome.The Eucommia ulmoides was used as the experimental material for cloning the promoter of Eu CGT1 by conventional PCR cloning.The preliminary expression analysis of the Eu CGT1 has finished,the main results are as follows:1.The sequence of 1671 bp upstream of the glycosyltransferase gene Eu CGT1 was cloned in the genome of Eucommia ulmoides.The sequence was analyzed by Plant CARE and found that the promoter contains many core cis-acting elements such as TATA-box and CAAT-box.There are also many cis-acting elements associated with environmental stress.2.To analyze the structure of the promoter,four 5'-end deletion promoter fragments were obtained by using the 5'-end deletion cloning:p I(1671 bp),p II(1224 bp),p III(891 bp),p IV(268 bp).The expression of the promoter was analyzed by GUS as a reporter gene.These promoter fragments were inserted into the upstream of the GUS gene of the plant expression vector p CAMBIA1391z,and four recombinant expression vectors were constructed,which were named p CAMBIA1391z-I-GUS,p CAMBIA1391z-II-GUS,p CAMBIA1391z-III-GUS,p CAMBIA1391z-IV-GUS.3.Agrobacterium-mediated leaf disk transformation was used to transform Xanthi tobacco,and the GUS gene was used as a reporter gene for expression analysis.The results showed that:p CAMBIA1391z-I-GUS,p CAMBIA1391z-II-GUS,p CAMBIA1391z-III-GUS,p CAMBIA1391z-IV-GUS were all dyed blue,while the p CAMBIA1391z was not dyed blue.This indicates that p IV(268 bp)can initiate the GUS gene expression.In addition,GUS staining of roots,stems and leaves of transgenic tobacco showed that the roots and leaves could be stained blue,which proved that the promoter was not tissue specific.4.The Plant CARE analysis revealed that the Eu CGT1 promoter contained an auxin response element,a gibberellin response element,a methyl jasmonate response element,and a drought response element.Analysis the relative expression of Eu CGT1 with q RT-PCR after exogenous auxin(IAA),gibberellin(GA₃),methyl jasmonate(Me JA)spraying and drought and high salt stressing.It was found that exogenous hormones,drought and high salt stress can all affect the expression of Eu CGT1.After spraying with Me JA for 3 h,6 h and 12 h,the expression of the gene decreased to 0.25,0.21 and 0.25 times of 0 h,respectively.After spraying with IAA for 3 h,6 h,12 h and 24 h,the expression of the gene increased to 2.92,4.95,7.86,and 14.1 times than 0 h respectively.After spraying with GA₃for 12 h and 24 h,the expression of the gene increased to 3.54 and 3.57 times of 0 h respectively.Within 24 h of drought stress,there was no significant difference in the expression of genes.After 6 h and 12h of high salt stress,the gene expression increased to 3.1 and 5.57 times of 0 h respectively.