Functional Analysis Of Three Drought-related Genes In Rice And Research On Its Drought Resistance Mechanism

Rice is one of the most important food crops in the world and feeds 60%of the world’s population.However,rice production is restricted by many factors,among which droμght is the most important abiotic factor that limits rice production.In recent years,with global climate changes and a large increase in population,food security has become a major issue that needs to be resolved.Using molecular biology methods to clarify the drought resistance mechanism of rice,and cultivating drought-resistant and high-yielding rice varieties is of great significance for solving food security problems.The DUF1644 family is induced by various abiotic stresses such as high salt,drought,and ABA,and plays an important role in the stress resistance of rice.In this study,two protein coding genes Os16 and Os28 which interacted with the protein coded by the DUC1644 family member Os05 and the gene Os32,a class Ⅲ member of the plant peroxidase superfamily,were mainly studied.The main results are as follows:The results of bioinformatics analysis showed that the protein encoded by Os16 contained Fesl domain.The results of stress physiology experiments showed that overexpression of Os16 in rice resulted in increased tolerance of the transgenic rice to drought stress,mannitol simulated drought stress and oxidative stress induced by methyl viologen and Os16 knockout plants showed a super sensitive phenotype to drought and oxidative stress.In addition,overexpression of Os16 can enhance the sensitivity of rice to ABA,and knocking out this gene will make rice less sensitive to ABA.Real-time quantitative PCR(qRT-PCR)results showed that some ABA synthesis genes,ABA response genes and ROS clearance genes were up-regulated in Os16-overexpressing plants,and down-regulated in Os16 knockout plants.The ABA-induced stomatal closure experiment showed that the proportion of open stomatals of Os16 transgenic plants and wild-type plants before ABA treatment was basically the same.The closed stomatal ratio of Os16-overexpressing plants after ABA treatment was significantly higher than that of wild type and Os16 knockout plants.These results indicate that Os 16 can mediate stomata closure through ABA-dependent pathways,thereby positively regulating rice drought resistance.The results of ROS scavenging gene expression and MDA content measurement showed that under mannitol stress,ROS levels in Os16-overexpressing plants were significantly lower than those in wild-type and Os 16 knockout plants.These results suggest that Os 16 can also regulate drought resistance in rice by adjusting ROS homeostasis.The results of bioinformatics analysis showed that the protein encoded by Os28 contains a conserved UspA domain,which is involved in the general stress response of plants.Stress treatment experiments and related physiological data measurement results showed that Os28-overexpressing plants had reduced resistance to drought and mannitol simulated drought,and Os28 knockout plants had increased tolerance to drought.In addition,Os28 knockout plants showed a hypersensitive phenotype to ABA,suggesting that Os28 may regulate drought tolerance in rice through the ABA pathway.DAB staining and MDA content determination results showed that under mnnitol stress,the accumulation of H2O2 in Os28-overexpressing plants was significantly higher than that of wild-type and knockout plants.These results indicate that Os28 can also negatively regulate rice drought tolerance by participating in the regulation of ROS homeostasis.The results of bioinformatics analysis showed that the protein encoded by Os32 belongs to the plant peroxidase Ⅲ family and contains heme and Ca2+binding sites.Tissue expression analysis results show that the protein is mainly distributed in the leaf apoplast space,vascular tissue and stem and young roots.The qRT-PCR results showed that Os32 was induced by high salt,PEG,chilling injury(4℃)and ABA.Stress physiology experiments showed that Os32-overexpressing plants had increased resistance to drought stress,and knocking out this gene resulted in a drought-sensitive phenotype in rice.The measured results of the water loss rate of detached leaves showed that the water loss rate of Os32-overexpressing plants was lower than that of wild type plants.These results indicate that Os32 positively regulates the drought resistance of rice.