The Study Of Calcium Signal Transduction Induced By Oxidative Stress?H₂O₂?

In the process of growth,plants are limited by all kinds of biotic stress such as infection of pathogenic bacteria and insect pest,or abiotic stress including drought stress,cold stress,thermal stress,salt stress,etc..In the process of resisting external stimulus,plants have evolved a sophisticated and effective defense mechanism.Moreover they have evolved a series of signal transduction pathways that respond to different stresses.When the plants are under stress,stimulus-induced[Ca²⁺]_cytyt increases in plant cells occur as repetitive oscillations or spiking of[Ca²⁺]_cyt,plasma membrane and organelles membrane sensors feel stress signal,the extracellular or vacuole endoplasmic reticulum intracellular calcium ions are released of into the cell,inducing transient increases of free Ca²⁺in the cytoplasm(cytosolic free Ca²⁺,[Ca²⁺]_cyt),breaking the calcium homeostasis,resulting in calcium oscillation,which is encoded as calcium signature and decoded by Ca²⁺-binding proteins.The signals activate downstream pathways by modulating protein kinase and so on,causing a series of stress response and physiological processes.With this,calcium signals activate NADPH oxidase to produce reactive oxygen species?ROS?.As an important second messenger,ROS is involved in plant growth and development and cell signal transduction,its accumulation can damage plant cell activity and influence the metabolism of gene expression?physiological growth and development.At present,there has been a lot of research on the downstream pathways of oxidative stress and lack the studies of the upstream perception mechanism of calcium signal induced by oxidative stress.This experiment detects intracellular calcium concentration indirectly by detecting the intensity of fluorescence produced by the combination of jellyfish fluorescent protein?AEQUORIN?and calcium ions by using calcium imaging system and fluorescence indicator method.And the mutants of early calcium signal deletion were screened under H₂O₂ treatment with the AEQUORIN-expressing transgenic Arabidopsis and its EMS mutant library,and the molecular mechanism of oxidative stress induced calcium signaling is going to be further explored.At present,the results we have achieved are as follows:1.With treatment of arabidopsis mutants library of EMS mutagenesis,combined with imaging phenotypic screening of specific validation of signaling pathways,we obtained some mutants under H₂O₂ stress,the loss of intracellular calcium signal of hic1?hydrogen induced calcium increase?shows more significance.2.The screening of the imaging phenotype of the candidate mutant hic1 and the third generation?Backcorss F₃ generation,BF₃?has been completed.We has established the mutant return group,so as to prepare for the next genome resequencing3.The genome resequencing of the mutant hic1 was completed,and the mutation interval was mapped to the specific site of the mutated gene,so as to prepare the functional verification of the gene.4.Hic1 was treated with different concentrations of H₂O₂,and the salt stress was selected to deal with the growth of hic1,and it was found that hic1 was more sensitive to salt stress,indicating that there was a lack of early upstream signal in hic1Through the above studies,we hope to reveal the mechanism of the relevant molecular mechanisms of sensing and response of plants to upstream signal of the stress,and to make a comprehensive explanation of the the way the plant sense oxidative stress.