Cloning And Functional Analysis Of The Inducible Soybean NAC Transcription Factor Promoters

Research on the response mechanism in environment stress of plant, and improvement of plant stress tolerance by gene engineering has become one of the efficient approaches of obtaining stress tolerance crops. Promoter is the switch of gene expression, and regulates gene expression pattern at transcription level. Stress-induced promoter is able to avoid the adverse effects on plant development caused by ectopic expression of exogenous genes. Their application has become one the hot area of stress tolerance plant transgenic engineering.In our previous study, NAC transcription factor genes GmST1 and GmST2 isolated from salt tolerance soybean, Shengdou No.9, have been proved to be genes response to abiotic stress. Their expressions were induced by salt and drought etc. On this basis, in this study, we cloned the promoters of the two genes, analyzed their transcription activation activity; determinated the their induction characteristics by abiotic stress and their tissue expression patterns; comfirmed the key fragment involved in the induction by analyzing the activity of different fragments; preliminarily investigated the function of the key cis-acting element; provide the fundamentals for the application of inducible promoter and its key cis-acting element in stress tolerance plant transgenic engineering.Main processes and results are as follows:1 Function study of the promoter of NAC transcription factor gene GmSTl from Shengdou No.9.1.1 Sequcene of GmSTl promoter and cis-acting elementsThe GmSTl promoter was cloned from Shengdou No.9 by using homology-based cloning, and its sequence contains 2000 bp nucleotides upstream initiation codon ATG. Analysis of the promoter by using PLACE and PlantCARE indicated that it contains TATA box, ABRE, W-Box and MYC etc. cis-acting elements.1.2 Expression patterns of full length GmSTl promoterWe constructed expression vector in which GUS was driven by full length GmST1 promoter, which was then transformed into the Arabidopsis. The homozygous T3 seeds were finally obtained.GUS staining analysis showed that:(1) GUS was expressed at cotyledon vein, basis of true leaves, root tip, lateral root primordial, anther and vascular tissue of mature sepal. (2) GUS expression was remarkably induced in root vascular tissue and leaves by NaCl treatment and ABA.1.3 Determination of salt-induced key fragment of GmST1 promoterWe constructed expression vectors in which GUS was driven by different GmST1 promoter fragments, which were then transformed into the Arabidopsis. The homozygous T3 seeds of fragment P1 and P2 were finally obtained.GUS staining analysis showed that P1 and P2 have the same activity as full length promoter, while the expression patterns changed, the density and range of P1::GUS and P2::GUS in root tip was weaker than GmSTl::GUS, and they were not induced by NaC1 stress.We constructed expression vectors in which LUC reporter gene was driven by different GmST1 promoter fragments, and analyze their activity by using Arabidopsis protoplast transfection assay.Analysis of LUC reporter gene activity indicated that four fragment can strongly promote LUC expression, the GmST1::LUC was about 4-fold increase by salt stress, P4 (-300 bp)::GUS was about 2-fold increase by salt stress, while others was barely induced by salt.These results suggested that the fragment (-2133 bp ～-1400 bp) between full length and P1 was the key one in root involved in salt stress response.2 Function study of the promoter of NAC transcription factor gene GmST2 from Shengdou No.9.2.1 Sequcene of GmST2 promoter and cis-acting elementsThe GmST2 promoter was cloned from Shengdou No.9 by using homology-based cloning, and its sequence contains 2000 bp nucleotides upstream initiation codon ATG. Analysis of the promoter by using PLACE and PlantCARE indicated that it contains TATA box, ABRE, W-Box and MYC etc. cis-acting elements.2.2 Expression patterns of full length GmST2 promoterWe constructed expression vector in which GUS was driven by full length GmSTl promoter, which was then transformed into the Arabidopsis. The homozygous T3 seeds were finally obtained.GUS staining analysis showed that:(1) GUS was expressed at cotyledon vein, primary true leaves, root tip, sepal, petal, filament, stigma, peel and carpopodium. (2) GUS expression was remarkably induced cotyledon by ABA.2.3 Determination of ABA-induced key fragment of GmST2 promoterWe constructed expression vectors in which GUS was driven by different GmST2 promoter fragments, which were then transformed into the Arabidopsis. The homozygous T3 seeds of fragment P2-P7 were finally obtained.GUS staining analysis showed that P2 has the same activity as full length promoter, activity of P3-P5 decreased obviously, while activity of P7 was partially restored, suggesting that fragment between P2 and P3(-1410bp～-1299bp) would enhance GUS expression, and fragment between P5 and P7 (-1053bp ～-350bp) contains key elements which inhibit transcription. P7::GUS was induced by ABA and mainly induced in cotyledon and true leaves. These results suggested that fragment P7 were the key fragments in leaves involved in ABA response.