Mutant Screening And Preliminary Analysis In Arabidovsis Thaliana That Was Insensitive To Salt Stress

Soil salt injury affected about three-quarters of the land in the world, directly led to decrease of the agricultural production and affect the livehood of people. Salt produced two kinds of stress, osmotic stress and ion stress. As the second messenger, calcium signal has played a vital role in response to the way of plant response salt stress. It can also regulate and control the downstream gene expression, so as to cope with stress.AQ means aequorin protein stable expression in Arabidopsis thaliana. AQ as material, we construct T-DNA insertion mutant library. Because of the jellyfish fluorescent protein fluoresce after combining with calcium, we screen mutant that can’t induce intracellular calcium increase under salt stress by sodium chloride, which is the most representative salt. We get the following results:(1) We have built 199 T-DNA insertion mutant libraries, and select 500~600 seeds to sterile cultivation for each library. When seedlings grow two euphyllas, record of the Arabidopsis thaliana seedlings under salt stress treatment by the Aequorin flurescence imaging photographic, and select that can’t fluoresce or fluorescent weaker candidate mutants. Each mutant library choose 40 seedlings to culture, and get F2 generation. We selected a total of 8000 candidate mutants.(2) We select the seeds about 20~30 grains every candidate, and point them linearly in the sterile culture medium. Using fluorescence imaging to verify this mutant. Finally, we get 16 mutants.(3) Every mutant, we select 4~5 seeds for cultivating to get F3 generation seeds as 4 or 5 lines of the mutant. We do the parallel experiments and determine the stability of the mutant for the next experiment.(4) We select sim2(salt-stress insensitive mutant 2) mutant for preliminary analysis. Using the Adapter Ligation to sim2 gene cloning technology. Through a seme-RT PCR, we find sim2 gene is not expressing in the mutant. We construct the GFP vector for subcellular location, GUS vector to study the tissue specificity expression of sim2, sim2 promoter with sim2 gene vector for complementation and over-expression vector, then get the transgenic plant by Arabidopsis flowers leaching method. And we have got the F3 generation seeds.(5) We do the physiological experiment comparing between sim2, control(AQ) and the sim2 gene of different locus mutation strain(order). Found under salt stress, the sim2 mutant growth similar to contrast; under drought stress, sim2 grows weaker than AQ. That sim2 mutant is not sensitive to osmotic stress caused by salt stress, cannot make intracellular calcium concentration increases, cannot cause downstream of the calcium signal transduction and regulation of the downstream gene expression. Plant can’t respond to salt stress and growth potential get weaken.(6) We put the three strains on the medium with H2O2 for culture. They grow similarly to each other, but sim2 show the resistance phenotype, aging slower than AQ and order.(7) Sim2 gene expression higher in root, leaf, filaments, calyx, and lower in the pod, petals, anthers and stems.After the research, we make a preliminary result of calcium signal transduction in Arabidopsis resistance to salt stress.