Temperature Response Defective Mutant Screening And The Gene Identification In Arabidopsis

Plant growth and development are affected by constantly changing environments.Environmental factors include abiotic stresses,limit plant productivity in agriculture.Among the various abiotic stresses,temperature is a major environmental factor that threaten food security,variety of stimulis include mechanical stimulation,hormones,pathogens,and abiotic stresses triggers a transient increase in cytosolic free Ca²⁺concentration([Ca²⁺]_i),include cold stress,calcium as the important secondary messenger,involvedinsignaltransductionintheresponsetolow temperatures,especially ethyl methanesulfonate?EMS?,has become a successful forward genetic approach to identify new participants in specific biological processes or signaling pathways.In the past decades,the mechanism of plant tolerance temperature stress and genes involved in the cold stress signaling network have been investigated intensively.Several signal pathways are involved in temperature stress signaling.However,it's still unclear how plants convert environmental temperature change signals into biological signal pathways through calcium-mediated pathways.In this study,by using calcium-sensitive aequorin,we have combined calcium imaging technology with forward genetics,and used Arabidopsis thaliana EMS mutagenesis library as the material to screen the mutants,which increase of intracellular calcium concentration in plants under temperature stress.We want to find specific Ca²⁺-related ion channels or related regulatory genes in plant-induced temperature stress signaling pathways,and to reveal the molecular mechanism of calcium signaling in Arabidopsis response to temperature stress.The results achieved so far are as follows:1.The temperature signal-induced calcium signal deletion mutant was obtained from four rounds of screening in the EMS library,and the mutant gene was found by map based cloning.The mutant was hybridized with AQ,and the F2 generation were gradually screened,and then sent to sequencing,and the target gene was obtained according to the sequencing result.2.The physiological characteristics were studied,and the hypocotyl length of the mutant was inhibited at a higher temperature,and the flowering time of the mutant was later than that of the wild type at a lower temperature,which proved that the mutant was a temperature-sensitive mutant.3.We cloned the target gene,the mutant gene vector was constructed,transformed it into mutant plants by Agrobacterium infection,and screened the harvested T0 seeds with Hyg-resistant medium.After two generations of screening,T2 seeds were obtained.Complementary identification was carried out,and it was further concluded that the target gene is likely to be a related gene with low temperature response.In conclusion,it is expected to reveal calcium-mediated environmental temperature receptors in plants and their mechanism of action,provide strong theoretical basis for improving and establishing the molecular mechanism of plants responding to environmental temperature changes,and in practice,using genetic engineering methods to improve crops.Provide a theoretical basis for tolerating global climate change.