Study On Calcium Signaling Pathway Induced By Osmotic Stress And Function Exploration Of OSCA Gene Family

Drought environment will cause irreversible damage to plants and seriously restrict the development of agricultural economy.However,the mechanism of plant sensing and responsing to drought stress still is not clear.As a second messenger,calcium signal is used to convert environment external signal to biological internal signal,transmit signals to downstream and induce resistant response in plants when plants are exposed to external stimuli.Aequorin is an indicator that can detect calcium concentration in Arabidopsis thaliana,which makes it possible tocan record calcium signals of living plants in real time.In the first part of this study,17 candidates of osmotic insensitive mutants were rescreened and identified on the basis of precursor studies in our laboratory,and a mutant 1b3 which intracellular calcium concentration was lower than wild-type under osmotic stress.This mutant did not respond to hyperosmotic stress which was stable and heritable.A variety of physiological experiments and experiments of calcium specific response to different stresses were designed for 1b3.The root growth of the mutant 1b3 was inhibited by the addition of osmotic stress on 1/2MS medium compared with that wild-type.In the experiments of the calcium signal specificity of the mutant to different stresses,the mutant 1b3 did not respond to osmotic stress but respond to oxidative stress,indicating that the mutant 1b3 did not respond specifically to osmotic stress.Then,the 1b3 linkage chromosome was obtained by using Arabidopsis whole genome resequencing and map-based cloning techniques,and one candidate gene was found by designing molecular markers.OSCA1 is known to be the first osmotic sensor discovered in plants.The second part of this study is to explore whether the two OSCA family member genes X and Y have a functional complement the phenotype of osca1 mutant.First,construct an expression vector containing the genes OSCAX and OSCAY with the promoter from OSCA1 gene,then transfer the target gene into the osca1 background by the Agrobacterium inflorescence infection method.After obtaining the homozygous transgenic material,the calcium signal of the transgenic materials treated by osmotic stress in vivo were recorded through calcium imaging system to verify whether the gene OSCAX and OSCAY can rescue the defective hyperosmotic response phenotype in osca1.The results showed that OSCAY can rescue this phenotype of osca1 but not OSCAX,indicating that OSCAY is another hyperosmotic sensor in OSCA family,as well as OSCA1.In conclusion,the first part of this study is to explore the key components of Arabidopsis osmotic stress signaling pathway by using whole-genome resequencing and map-based cloning techniques,thus providing a theoretical basis and molecular target for drought resistance research in agriculture.The second part of this study is to explore whether the OSCA family genes OSCAX and OSCAY have a functional redundant relationship with OSCA1,which is helpful to reveal the response mechanism of OSCA1 to osmotic stress.