The Establishment Of A Living Fluorescence Detection System For High-throughput Screening In The Ocimene Signaling Pathway

Recent research demonstrates:the marker gene PR1 of SA pathway and PDF1.2 of JA/ET pathway expressed at the same time in plants treated by ?-ocimene, which indicates that ?-ocimene signal transduction pathway is not same with JA and SA signaling exactly In order to efficiently find various signal transduction components mutants in ocimene signaling pathway, as well as shed light on the mechanism of defense responses induced by ocimene, The establishment of a living fluorescence detection system for high-throughput screening by forward genetic strategy in the ocimene signaling pathway. The results are as follows:1. Putative cis-elements were forecasted for defense responses pathway marker genes PR1, PDF1.2, VSP2 promoters, results showed:The core elements of Eukaryotic promoters CAAT-box and TATA-box appeared several times in PR1?PDF1.2? VSP2 sequences. Moreover, there are many specific cis-elements are related to defense pathway:LS5?LS7? GCC-box?TGACG-motif. CGTCA-motif and so on.2. PR1? PDF1.2?VSP2 promoter fluorescence expressing vector PR1pro::Luc? PDF 1.2pro::Luc? VSP2pro::Luc were constructed successfully.3. The PR1pro::Luc? PDF1.2pro::Luc?VSP2pro::Luc was transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation, and through Hyg antibiotic selection PR1pro?PDF1.2pro T3 transgenic homozygous plants were obtained, VSP2pro have not transgenic plants.4. A suitable high sensitivity CCD camera, camera obscura and software were purchased,through assembling and debugging, a self-made, affordable in vivo fluorescence detector was successfully generated. After treated by jasmonic acid, salicylic acid or ocimene alone followed by fluorescent substrate spraying, homozygous transgenic plants were submitted to fluorescence detector, high intensity of induced-fluorescence signal in transgenic plants was detected, indicating that in vivo fluorescence imaging detection system was successfully constructed. These results provided a powerful forward genetic methodology and made it possible to use high-throughput selection of plants carrying mutations in ocimene signaling pathway components, as well as shed light on the mechanism of defense responses ocimene induced by ocimene.