Study On The Components Of Low Temperature Stress Signal Pathway In Arabidopsis Thaliana

In recent decades,studies have shown that low temperatures and various other stimuli can cause different forms of intracellular free calcium concentration?[Ca2⁺]_i?to increase.Considering that animals use Ca²⁺channels as temperature receptors,low-temperature-induced([Ca²⁺]i)increase has been widely speculated to participate in plant low-temperature stress However,the molecular nature of the corresponding Ca²⁺channel has not yet been fully explained.As a second messenger Ca²⁺is a very important regulatory factor in the whole physiological process of plant cells and response to external stimuliwhich is located at the core of the complex signaling network.Plant cells can awareness and response to different environmental signals through complex calcium signaling pathways,but due to the redundant function of the protein in the long-term evolution process,or a particular receptor may be necessary for the plant growth and development,and the lack of function leads to the mutant not survive or other reasons,resulting in only a few of plant stress sensors.was cloned so farOur laboratory has mature technology and advanced complete system,We have identified and cloned osmotic receptors OSCA1in the model plant Arabidopsis thaliana Arabidopsis,,and on this basis,we continue to use the method of forward genetic screening,we hope that we can identify the key components of plant low temperature stress signal pathway with a high probability.The calcium ion probe AQ?Aequorea?was expressed in Arabidopsis thaliana to detect real-time changes in cytoplasmic calcium concentration caused by low temperature.We found that the we found that the calcium signal induced by low temperature is very rapid.The calcium signal induced by low temperature reached the peak within a few seconds.considering the processing time of the influence of the calcium signal collection,we will screen the calcium signal collection time from 10s-5min,,preliminary as 1 minute;We are from 0?to shrinking temperature range between 20?,it was found that the wild-type under 10?cold stress had moderate calcium signal intensity and could distinguish potential mutants,so we preliminary screening of the low temperature calcium signal defects mutant conditions at room temperature 10?low temperature stimulation,we use 10?low temperature conditions,collection of calcium signal 1 min screened 81600 per plant seedlings.Considering the functional redundancy and diversification of proteins,we conducted specific detection on the candidate mutants after determining the low-temperature phenotype,and selected the candidate mutants that were only specific to the low-temperature defects,but had normal responses to hyperosmosis,hydrogen peroxide and salt ions.Finally,we obtained 28 candidate mutants with stable phenotype and obvious specificity at low temperature.After four generations of normal self-cross-breeding,we found their stable inheritance of calcium signaling phenotype.We selected m17,a mutant with obvious calcium signal defect and excellent specificity,to construct a mapping population,and established a mapping clone population of 720 single strains of a low-temperature calcium-ion-responsive defective mutant.Furthermore,the linkage chromosome of the mutant gene was obtained by second-generation sequencing.Subsequently,the SNP mutation site was precisely located by map-based cloning technology and primers designed by CAPs/d CAPs.The candidate gene was initially determined at c-chromosome,5-8M.Candidate genes are expected to be obtained from candidate mutants.Studies have confirmed that plants can respond to stress through the perception of short-term signals and the construction of long-term signaling pathways.So we used short-and long-term phenotypes to determine the quality of candidate mutants.Short-term physiological experiments showed that the mutant we screened,m1 and m17,showed physiological phenotypes such as short roots,yellowing and short roots,and petiole elongation,respectively,when they grew at low temperature.Long-term physiological experiments showed that the candidate mutant had fewer rosettes and flowers earlier than the mutant.This indicates that the mutant we screened has problems in some signaling pathways at low temperature,leading to plants'inability to fully cope with low temperature and escape from adversity.This project is expected to open up a new way to study the low-temperature stimulus stress signal pathway and calcium channel mechanism,and provide a potential molecular genetic target for engineering low-temperature resistant crops.Up to now,the genetic verification of candidate mutants has been partially completed in this project.After 4 generations of natural reproduction,the low-temperature related phenotype of the candidate mutants remains unchanged,indicating that the calcium signal defects are heritable.On the other hand,we conducted short-term and long-term physiological phenotype experiments on the two candidate mutants,and found that the mutants had physiological phenotypes such as short roots and early flowers at low temperature.This dissertation focuses on how to use advanced calcium imaging technology to screen related mutants induced by low temperature stress,and then to obtain genes related to low temperature signaling pathway through map cloning.And carried out preliminary study on some physiological aspects of mutants...