The Indentification Of Element In The Promoter Sequence Of OsWRKY12Responses To SA、MeJA And ET

Plant hormone is an important regulatory material of plant growth in adversity stresses, it can regulate the physiological and biochemical responses of plants through some important signal pathways. In the process of regulatory response to the stress, normally more than one hormone involving in, the synergistic or antagonistic action among hormones and the cross function of signaling pathways can both make plants achieve a state of balance, in order to help plants facilitate the growth and resistance to the external environment, In signaling pathways of plant hormones regulates stress reaction, related resistance genes get expressed and require the participation of transcription factors. In order to identifying the reason of transcription factor genes specifically expressed in a specific time, specific tissues and stress conditions, studying its promoter region regulatory elements is necessary. This research focuses on the response of the transcription factor OsWRKYl2to hormones by transient expression experiment, in order to find out the critical areas and cis-elements of response to related hormones in promote of OsWRKYl2. The main results were as the follows:1-. OsWRKY12promoter region named OsWRKY12P from Japanese rice was cloned.According to the rice genome sequence of chromosome1in GeneBank, we identifyed the OsWRKY12upstream promoter sequence. Then the specific primers were designed to amplify OsWRKY12promoter regions from Japan rice, we named it as OsWRKY12P. After the Blastn in NCBI, the results showed that the fragment is highly consistent with the original sequence.2、Transient expression experiments proved that OsWRKY12P responed to various hormonal response and environmental stresses such as drought, high salt, low temperatures.Further design primers with restriction enzyme cutting site EcoR I and Pst I, the amplification of the fragment EOsWRKY12P length of981bp was inserted to the Multiple cloning sites of the promoter expression vector pCAMBIA1381Z with a GUS reporter gene. Through chemical method transfer the expression vector p1381Z-EOsWRKY12P::GUS into Agrobacterium EHA105competent cells, Wild type tobacco NC89was used as experimental material through the Agrobacterium-mediated method for transient expression. Deal with tobacco seedlings with hormone(MeJA, ET, SA, ABA, IAA, GA), high salt, low temperature, drought and so on. GUS staining results showed that OsWRKY12P responded to MeJA, ET and SA, but did not to ABA, IAA, GA and high salt, low temperature, drought and so on.3、Constructing deletion mutation expression vector and process transient expression experiments, further to determine the key cis-element of response ethylene, salicylic acid, jasmonic acidWe used the PLACE website to analyze the element of OsWRKY12P, results showed that the related components was as follows:one GCCbox associated with ET and MeJA, eight WBOXATNPR1related to SA, basic function element is two TATABOX and five CAATBOXs, and some other components. According to the position of the element design four specific forward primers with restriction enzyme cutting site EcoR I:F1(-840bp)、F2(-575bp)、F3(-344bp)、 F4(-202bp), reverse primer consistent with the reverse primer of amplificating EOsWRKY12P, amplify corresponding fragment P1/2/3/4and build on pCAMBIA1381Z, transferred into agrobacterium EHA105competent cells respectively, and through Agrobacterium-mediated experiment for transient expression, results showed that critical areas of response to the SA, MeJA were from-575bp to-344bp in promoter, critical areas of response the ET were from-344bp to-202bp in promoter.