Genetic Screening For Arabidopsis Mutants Defective In Cold-induced[Ca²⁺]_i

Many environmental factors constrain the traditional agriculture,especially the recent changes in global temperature and the emergence of extreme weather,and the decrease in temperature will affect the growth and development of plants and reduce productivity.When the external environment is changed,plants will feel these changes and produce certain defense mechanisms.Ca²⁺,as a second messenger,is an important factor in regulating plant response to external stimuli.Under normal circumstances,the intracellular calcium ion level will be maintained at a very low level（about 100 n M）.Ca²⁺is released into cytoplasm from extracellular or intracellular calcium pools,perceiving the external stimulus,thus generating calcium signals,which are transmitted to downstream signal pathways,causing cells to make corresponding adjustments to the stimulus.Many components of the downstream signal pathways induced by cold stimulation have been discovered,but it is still unknow how plants perceive environment stimuli through calcium signals and convert calcium signals into biological signals to trigger downstream signal pathways.We used forward genetics method to screen Arabidopsis mutants defective in cold-induced[Ca²⁺]_iin stable transgenic EMS population expressing aequorin by calcium imaging.Map-based cloning is used to identify the underlying genetic cause of the mutant phenotype.The results achieved are as follows:1、We optimized the screening conditions for M₂ EMS population:seeding age,recording time,treatment temperature and so on.2、After three rounds of screening more than 50000 M₃ EMS seeds,we got more than 200 candidate mutants,reduced to cold-induced[Ca²⁺]_i,and kept breeding multiple generations to rescreening for stable and heritable mutants.3、To screen the cold specific mutants,we used several kinds of stimulus to treat more than 200 mutants while recording calcium image,and finally obtained 27 cold-specific mutants.4、we also did some physiological experiments,including sorbitol,sodium chloride and hydrogen peroxide treatments to rank these mutants for further physical mapping.5、For map-based cloning,we crossed the mutant with wild-type AQ,screened F₂ and F₃,selected mutant lines to extract DNA for second-generation whole genome sequencing,and further realize gene localization to obtain the target gene.