Cloning And Functional Analysis Of Phosphoethanolamine Methyltransferase(PEAMT) Gene Promoter From Maize(Zea Mays L.)

Betaine,a non-toxic permeation protectant,could accumulate considerably under stress condition to maintain the osmotic pressure and promote plant growth.Phosphoethanolamine N-methyltransferase(PEAMT),a key enzyme for betaine synthesis,could be regulated by its upstream promoter.At present,there are few studies on endogenous PEAMT promoter promoters in maize(Zea mays L.).In this study,using PCR technology(polymerase chain reaction)to clone up-stream sequence of PEAMT according to the genome sequence of maize cv.B73.And the expression vectors with full-length promoter and different length promoter fragments fused with ?-glucuronidase gene(GUS)was constructed and then transformed to tobacco(Nicotiana benthamiana L.)using employing agrobacterium tumescence-mediated method.Through histrionically staining method and determination of GUS enzyme activity of transformed tobacco,to analyses the response ability of full length fragment and different deletion fragments of PEAMT gene promoter under different abiotic stresses conditions.The main results were as follows:1.According to the PEAMT gene sequence searching from NCBI database,the specific primers for PEAMT promoter was designed and cloned the up-stream sequence of PEAMT.The sequence length was 1048 bp and the details could be seen in appendix I.Using the on-line prediction software including Promoter Predication,PLACE and PlantCare to analysis of promoter predication and cis-element.The results showed that this up-stream sequence included 17 multiple stress response cis-acting elements,13 light-induced signal transduction elements,9 hormone response cis-acting elements,and 5 pollen-developing specific activation elements.2.A expression vector with full-length promoter fused with GUS gene was constructed and then was transformed to tobacco.PEAMT gene promoter induced GUS gene expression efficiently.Though GUS fluorescence quantitative analysis,PEAMT gene promoter activity showed a positive regulatory expression model.The activity of PEAMT gene promoter were 27.4,39.9,23.5 and 19.7 nmol 4-MU·min-1·mg-1 protein under drought,salt,cold and oxidative stress condition,which were higher than that in control by 1.64,2.84,1.47 and 3.59 times under different stress condition stress,respectively.3.Four kinds of deletion fragments of PEAMT gene promoter sequences were cloned and the sequence length were 826 bp(P2),642 bp(P3),428 bp(P4)and 245 bp(P5),respectively.Then the expression vectors of four deletion fragments with GUS gene were constructed and then transformed to tobacco.It was indicated that these four deletion promoter fragments had different activities under different types of abiotic stresses conditions using histrionically staining method and GUS enzyme activity analysis.The activity of P3 fragment promoter reached 37.18 and 19.76 nmol 4-MU·min-1·mg-1 protein and increased by 4.76-and 5.33-folds under drought and cold stress condition as compared with the control,respectively.The activity of P4 fragment promoter reached 36.46 nmol 4-MU·min-1·mg-1 protein and increased by 4.76-and 5.33-folds under salt stress condition as compared with the control.The activity of P2 fragment promoter reached 20.50 nmol 4-MU·min-1·mg-1 protein and increased by 7.51-folds under oxidative stress condition as compared with the control.It was concluded that the different deletions exhibited different response mode to stress and the P3 fragment promoter was more sensitive to these four abiotic stress.It was clarified the characteristics of sequence and expression of PEAMT promoter from maize in this study.And PEAMT gene romoter showed a positive regulatory pattern under different abiotic stress condition.These findings could provid a theoretical basis for the futher study of PEAMT gene expression regulation.