Primary Study On The Role Of ATCNGC2 In The Increase Of Cytoplasmic Free Ca²⁺ Triggered By Jasmonic Acid

Cytosolic free Ca2+([Ca2+]cyt) is a ubiquitous second messenger in plant cells.A number of external and internal stimuli trigger sustained or transient elevations of [Ca2+]cyt and consequntly evoke downstream stimulus-specific response.Generation of [Ca2+]cyt signals depended on Ca2+ influx into the cytosol from the extracellular and intracellular Ca2+ stores through Ca2+ channels.Thus,Ca2+ channels in the plasma membrane play an essential role in Ca2+-mediated signaling process.However,the direct recordings and analysis of channel-facilitated Ca2+ influx are very limited and upstream regulation mechanisms of the Ca2+-channels in plant cells remain unclear so far.As a plant hormone,jasmonic acid play a very important role in regulating plant defense reponses. It has been reported that jasmonic acid (JA) can trigger the physiological and biochemical reactions in the cell by the increase of [ Ca2+]cyt.Plant cyclic nucleotide gated channels(cngcs) are known to be involved in numerous signal transduction cascades as cell membrane,ligand gated nonselective cation(conducting Na+,Ca2+,and/or K+)channels. To evaluate the role of plant cngc in JA signaling,research work as followed:(1) Expression of ATCNGC2 in mutants of yeast(trk1,2) was tested for establishment of an experimental model of cAMP-ATCNGC2 interaction.The result showed that the addition of exogenous cAMP in the culture medium significantly influenced growth of expression of ATCNGC2 in mutants of yeast(trk1,2).(2) Base on pervious research result, guard cell protoplasts were isolated successfully by digesting with two-step enzyme digestion.0.8%(w/v)Cellulysin, 120 r/min,after 30 min of incubation at 25℃with gentle shaking; Epidermal strips were floated on medium containing 1.5%(w/v) Cellulase RS,0.02%(w/v)Pectolyase Y-23,after 40~60min of 60~70r/min,shaking at 22℃,guard cell protoplasts were isolated. The activity of isolated protoplasts were measured with FDA,the result showed that activated protoplasts were isolated and they could be used in patch clamp experiment.(3) Arabidopsis thaliana leaf,Arabidopsis thaliana guard cell protoplasts and yeast protoplasts were used for this reseach work. Load the Ca2+ sensitive fluorescent probe Fluo-3/AM into leaf and guard cell protoplasts by incubating in Fluo3/AM solution at 4℃for 2 h followed by 2 h incubation at 20℃. Load the Fluo3/AM into yeast protoplasts by incubating in Fluo3/AM solution at 28℃, followed by 2 h incubation in the dye-free solution at 20℃.(4) The DNA fragment of VSP2 gene was amplified by reverse transcription polymerase chain raction(RT-PCR) with mRNA from Arabidopsis thaliana mutant dnd1 as the tempalte, and ligated into PMD19-T cloning vector. After sequencing,the correct DNA fragment was identified. Bioinformatics technique was used to analyze the gene sequence homology of VSP2.The VSP2 gene fragment was highly homologous to VSP2. Arabidopsis thaliana VSP2 gene was cloned successfully.