Screening And Phenotype Analysis Of Arabidopsis Mutants Deficient In Intracellular Calcium Elevation Under Salt Stress

At present,the global saline-alkali land area is about 800 million hectares,accounting for 6%of the world's total land area.At the same time,due to improper irrigation and fertilization and industrial pollution,the global salinized soil area shows a continuous increase.Plants in high-concentration saline soils are subject to plasma and osmotic stress,which hinders the absorption and utilization of water and nutrients and inhibits plant growth and development.Therefore,in the long evolutionary process of fixed plants,a variety of mechanisms to resist high salt stress have been produced to ensure the normal growth and development of plants under salt stress.Plant response to external salt stress can be divided into four stages:sensing salt stress signals,transmitting signals,cellular responses,and cell adaptation.However,most of the current researches focus on the last three processes,and there are few studies on how plants convert external salt stress signals into intracellular chemical signals.Existing research shows that when plants are subjected to salt stress,the intracellular calcium ion concentration increases rapidly within 15 seconds,and different environmental stress stimuli can induce cells to produce different forms of calcium oscillations,which is called"characterization of calcium."After receiving the calcium signal,the downstream components in the cell will further reduce the damage of plants by salt stress by regulating the expression of related genes and protein activity.However,there is very little research on genes involved in the plant's ability to sense salt stress.In order to solve the scientific problem of how plants sense salt stress,we use forward genetic screening to explore the elements in plant cells that specifically sense the stimulation of external salt stress.In this experiment,the model plant Arabidopsis thaliana is used as the material.The principle of aequorin?AQ?can specifically bind calcium ions and oxidize coelenterazine to generate fluorescence?fluorescence is stronger when the concentration of intracellular calcium ions increases,otherwise the fluorescence Weak?,combined with the overall plant imaging system,screened Arabidopsis mutants that were deficient in increased intracellular calcium concentration under salt stress.The findings are as follows:1.Through EMS chemical mutagenesis,an EMS mutant library expressing Aequorin plant materials was obtained;plants with weak fluorescence after NaCl treatment were selected as candidate mutants using the plant overall imaging system,and a total of 6000 T₁ generation single plants were screened.Among them,there are1358 strains with weak fluorescence.2.After screening the 1358 candidate mutant lines for three consecutive generations,three mutant strains with genetically stable phenotypes were finally obtained,numbered 81#,186#,1123#?81/186/1123?.3.It was found that the 81/186/1123 mutant strain specifically lacks an increase in intracellular calcium ion concentration under NaCl stress,while other stresses?5mM H₂O₂,600mM Sorbitol,4°CH₂O,45°CH₂O?can induce cell defects.Calcium ion concentration rises rapidly.4.It was found that the 81/186/1123 mutant strain has a strong tolerance to high-concentration NaCl.The mortality of the mutant strain in the MS medium containing NaCl and the soil containing 400mM NaCl was significantly lower than that of the wild-type AQ strain.5.By resequencing the 81/186/1123 mutant genomic DNA,it was found that a common SNP site exists in all three mutants,and the transcription of the 720 gene was terminated prematurely.6.The phenotype of growth and development?e.g.,fresh weight,leaf area,plant height?was identified in 81/186/1123 mutant at each stage,and no significant difference was found between the mutant strain and wild-type AQ at each stage.In summary,we screened and obtained mutants of Arabidopsis thaliana that were deficient in the intracellular calcium ion concentration under NaCl-specific stress.This scientific question provides new research materials and ideas.