Cloning And Functional Identification Of Viviparous-1 And W73 Promoters In Wheat

Improvement of the PHS and environment stress tolerance is one of the most important objectives in wheat breeding. The study on the promoter cloning and its functional investigation contributes to understand the signal transduction pathways and gene expression regulation model. In this study, promoters of the Vp-1 gene and DREB transcription factors genes W73, W42 and W6 had been isolated. At the same time, the further study of the function of the Vp-1 and W73 gene promoters had been made. This study presents the relational theoretical basis for wheat molecule breeding. The main details of the result are as follows:1. The promoter of Vp-1 had been isolated and it includs the endosperm special expression cis-elements: SKn-1 and RYREPE. Otherwise, there are some stress-related and hormone induced cis-elements in it, such as drought responsive element (DRE), ABRE (ABA) and so on.2. W73, W42 and W6 gene promoters were cloned. There are some stress induced cis-elements in them: drought and low-temperature responsive element (DRE, LTRE) and hormone responsive elements, as ABRE (ABA), ERE (ethylen), GARE (GA) and CARE (GA). Furthermore, W6 promoter has some disease and wounding induced cis-elements, as BIHD1 and W-BOX.3. Using PCR method, different fragments of Vp-1 promoter were amplified and inserted upstream of GUS reporter gene. Then, these vectors containg different promoter fragments were transferred into wheat calli using particle bombardment. Then, the transgenic wheat calli were treated under different stress conditions. Activation of GUS protein was analyzed to identify the function of the putative cis-elements in vivo by histochemical staining. The result suggested that Vp-1 promoter is more induced by ABA, GA and low-temperature than PEG and NaCl. We also found that the 319 bp promoter fragment also has the activity, and it can drive the GUS gene in low level expression under the different stress conditions.4. The W73 promoter was fused into upstream of the GUS gene to construct a plant expression vector. The histochemical staining indicated that the W73 promoter is a typical stress inducible promoter. It could drive GUS gene expression under drought, NaCl, low-temperature, ABA and SA, and the expression level is relatively higher under ABA, SA and low-temperature induced.5. The transgenic Arabidopsis plants transformed with Vp-1.P:GUS and W73.P:GUS were obtained.