Expression Analysis Of NtGCN2 Promoter In Nicotiana Tabacum

Complex changes in the natural environment make the growth of plants subject to a variety of adverse factors.When plants are affected by adverse factors,they will mobilize their own energy for defense response.In plants,the phosphorylation of eIF2α mediated by GCN2 can be activated by a variety of biotic and abiotic stresses,but how its promoter responds to stress remains unclear.In order to investigate the response mechanism of GCN2 to stress,the promoter of tobacco NtGCN2 was cloned and analyzed by bioinformatics.The promoter and its 5’ end deletion mutant were studied.The treatment and stress(low temperature,light,light,and low temperature,light,etc)of the promoter were studied the expression patterns of two types of promoters under drought stress were analyzed.The results mainly include the following aspects:firstly,NtGCN2 promoter was cloned,and two types of promoters,NtGCN-Nsy-pro2000 and NtGCN2-Nto-pro1987,were found in tobacco.Sequence analysis showed that NtGCN2-Nsy-pro2000 and NtGCN2-Nto-pro1987 may be derived from two parental and villous tobacco cultivated tobacco,respectively.The results of this study are consistent with the evolutionary analysis results of NtGCN2 gene.The prediction results of cis acting elements show that there are many cis acting elements on two types of full-length promoters,including abscisic acid response element(ABRE),drought response element(MBS),optical response element(AE box,G-box and TCT motif),ethylene(ER)and auxin response element(TGA),and two kinds of them are distributed There were differences in the distribution of cis acting elements in the promoter.Both types of promoters contain light responsive element,abscisic acid responsive element and methyl jasmonate responsive element.Among them,NtGCN2-Nsy-pro2000 contains an ethylene responsive element and NtGCN2-Nto-pro1987 auxin responsive element.In addition,NtGCN2-Nsy-pro2000 contains one photoresponsive element,while NtGCN2-Nto-pro1987 contains three photoresponsive elements.Secondly,according to the prediction results of cis acting elements of promoter,two types of 5 terminal deletion mutants of NtGCN2-Nto-pro2000 and NtGCN2-Nto-pro1987 were constructed,and the transgenic plants were obtained and propagated to T2 generation.Among them,the missing mutants of NtGCN2 are NtGCN2-Nto-pro1703::GUS,NtGCN2-Nto-pro1377::GUS,NtGCN2-Nto-pro1248::GUS,NtGCN2-Nto-pro1184::GUS and NtGCN2-Nto-pro860::GUS.The deletion mutants of NtGCN2-Nto-pro were NtGCN2-Nto-pro1987::GUS,NtGCN2-Nto-pro1923::GUS,NtGCN2-Nto-pro1811::GUS,NtGCN2-Nto-pro1318::GUS,NtGCN2-Nto-pro1195::GUS.Gus staining showed that the 5 ’deletion mutants of NtGCN2 promoter had promoter activity.Finally,in order to further explore the function of NtGCN2 promoter,the transgenic lines of NtGCN2 promoter 5 ’deletion mutant were treated with hormone(ABA,AZA,IAA,ET,)and stress(low temperature,light,drought)respectively.The results of GUS staining and NtGCN2 promoter driven GUS gene expression under different light intensity showed that the expression of NtGCN2 promoter could be activated rapidly by high light treatment,and GUS gene expression reached the maximum after 24 h treatment,while that of GUS gene in low light treatment reached the maximum at 48 h.In addition,different numbers of photoresponse elements have great influence on the expression activity of the two types of promoters.The GUS gene expression and enzyme activity driven by NtGCN2-Nto-pro type promoter(containing three photoresponse elements)are higher than those driven by NtGCN2-Nsy-pro type promoter(containing only one photoresponse element).Auxin treatment could activate the expression of NtGCN2-Nto-pro and NtGCN2-Nsy-pro promoters,and the GUS gene expression and GUS enzyme activity in the two types of 5 ’terminal deletion mutants were significantly increased.Ethephon inhibited the expression of NtGCN2-Nsy-pro1248::GUS promoter,but activated NtGCN2-Nsy-pro1703::GUS,NtGCN2-Nsy-pro1377::GUS,NtGCN2-Nsy-pro1184::GUS and NtGCN2-Nsy-pro860::GUS promoter.It is suggested that there might be DNA sequence between-1248～-1184 bp of NtGCN2 promoter to inhibit ethylene response.Drought treatment can activate the GUS gene expression driven by NtGCN2-Nsy-pro1703::GUS,NtGCN2-Nsy-pro13 77::GUS,NtGCN2-Nsy-pro1184::GUS and NtGCN2-Nsy-pro860::GUS.Cold stress and nonanoic acid treatment activated the expression of NtGCN2-Nto-pro and NtGCN2 promoters,and the 5 ’terminal deletion mutant driven GUS gene and GUS enzyme activity of both types of promoters increased significantly.GCN2 is widely involved in both biotic and abiotic stress responses,and plays an important role in plant stress response.In this study,we constructed a 5 ’deletion mutant of NtGCN2 promoter,and analyzed the function and response elements of the promoter under different stress treatments.The results laid a foundation for further analysis of GCN2 regulation mechanism.