Cloning And Expression Of A Calmodulin Interacting Protein TaCAMTA4Gene In The Interaction Process Between Wheat And Puccinia Triticina

Ca2+/CaM signaling transduction pathway has been improved to play animportant role in wheat defense response to leaf rust fungus (Puccinia triticina). Inorder to enrich the study of the signaling transduction system, we have conduct yeasttwo-hybrid using wheat TaCaM4-1as bait to screen a cDNA library of wheat infectedby leaf rust fungus and obtained7positive CaM-binding protein genes, one of whichwas TaCAMTA4gene. This study was based on the results of yeast two-hybrid.CAMTA (calmodulin-binding transcription activator) family is a transcription factorfamily which has CaM-binding activity. CAMTA proteins have four highly conserveddomains, as CG-1domain, TIG domain, ANK repeat domain and CaM-bindingdomain. Studies have showed that CAMTAs play important role in growth anddevelopment process and stress defense responses (for: pathogen, cold, hot, drought,etc.) in plants. In this study, we studied on the TaCAMTA4gene and results wereshowed below:(1)7positive plasmids obtained by yeast two-hybrid were co-transferred intoyeast cell with bait plasmid by one-to-one yeast co-transformation analysis to test thepositive clones.(2) The full-length cDNA of TaCAMTA4was obtained by RACE as2704bp.(3) Bioinformation analysis showed TaCAMTA4had the four domains ofCAMTA family and the protein was highly homologous with CAMTAs in manyplants. This indicated TaCAMTA4gene was a member of CAMTA family. The genewas named as TaCAMTA4.(4) Bimolecular fluorescence complementation (BiFC) analysis indicatedTaCAMTA4and TaCaM4-1could interact in the cytoplasm and nucleus in tobaccoleaf epidermal cells.(5) Electrophoresis mobility shift assay (EMSA) analysis indicated theCaM-binding sites of TaCAMTA4were located at C-terminal of the protein.(6) A prokaryotic expression vector pET28a-CG-1was constructed to express theDNA-binding region of TaCAMTA4gene. The optimum induced condition wasconfirmed as0.8mmol/L IPTG and20hours at37℃. (7) The transcription level of TaCAMTA4was detected by qRT-PCR in bothcompatible combination and incompatible combination. The results indicated thatTaCAMTA4may negatively regulate the wheat defense response to leaf rust fungus.The study laid a foundation of the further study of the function and mechanismof TaCAMTA4in wheat defense response to leaf rust fungus and the signalingtransduction system in the process.