Application Of OsMSL5 Gene To Improve The Ability Of Rice To Cope With High Salinity And Drought

Rice(Oryza sativa L.)plays an important role in solving the problem of food and clothing for human beings.Plant cells can sense osmotic pressure changes in mechanical stimulation and respond.Mechanically sensitive ion channel proteins regulate osmotic pressure by conducting ions.The Msc S-like(Mechanosensitive channel of small conductance-like,MSL)protein family is a typical anion channel family activated by tension,which can not only improve the drought and salt resistance of plants,but also respond to low temperature stress.OsMSL5 is a member of rice MSL protein family,but its function in rice has not been reported.Therefore,further study on the biological function of OsMSL5 gene can further enrich the function of MSL protein family.In this study,the T-DNA insertion mutant(osmsl5)and overexpression strain(OsMSL5-OE)of OsMSL5 were used as the materials to explore the roles of OsMSL5 as osmotic safety valve to directly sense the change of membrane tension,conduct ions to regulate the water and osmotic balance of plants,participate in the growth and development of rice and respond to stress.The main research work and results are as follows:(1)Analysis of the spatio-temporal differential expression pattern of OsMSL5 with the software GENEVESTIGATOR.We found that OsMSL5 was highly expressed especially at seedling and mature stage of rice,but was lower at tillering stage and heading stage;The expression level was higher in callus,cell culture/primary cells,seedlings,inflorescences,caryopsis and rhizomes,but lower in panicle cells and buds.The response of OsMSL5 to different plant hormones and abiotic stresses was analyzed by q RT-PCR.OsMSL5 was up-regulated by ABA and abiotic stress signals such as Na Cl and PEG.The results of subcellular localization showed that OsMSL5,as a membrane protein,was mainly localized Golgi apparatus,endoplasmic reticulum and cell membrane.(2)The functional deficiency of OsMSL5 resulted in the decrease of main root length,lateral root number,lateral root density,adventitious root number and average adventitious root length of rice.In osmsl5 mutants,the plant was reduced but the tiller number increased at mature stage.Thus,OsMSL5 played an important role in the growth and development of rice.(3)OsMSL5 played crucial roles in maintaining the cell size under low osmotic pressure.The response of rice and tobacco protoplast cell to osmotic pressure showed that there was no significant difference between wild type(DJ)and osmsl5 rice protoplast cells under isotonic condition,while under hypotonic condition,the protoplast cells of wild type(DJ)and osmsl5 mutant rice became larger,but the protoplast size of osmsl5 was increased more obviously.Under isotonic conditions,there was no significant difference in the cell size of tobacco protoplasts transiently expressing GFP or OsMSL5-GFP fusion protein;while under hypotonic conditions,the cell size of tobacco protoplasts expressing GFP or OsMSL5-GFP was increased,but the cells expressing GFP was much larger.(4)The loss of OsMSL5 reduced the tolerance of rice to high salt and drought.Under Na Cl or PEG conditions,the growth and development of wild type(DJ)and osmsl5 plants were inhibited,but the inhibition degree of osmsl5 was more significant.The results of physiological parameters showed that the content of malondialdehyde(MDA)and relative conductivity in osmsl5 were higher,while the content of proline was lower after high salt and drought treatment,compared with wild type.After high salt and drought treatment,compared with wild type(DJ),the expression level of reactive oxygen species(ROS)in the leaves of osmsl5 was higher after DAB and NBT staining.The q RT-PCR analysis showed that,after Na Cl and PEG stress treatment,compared with wild type,the expression level of ROS-synthesis genes in osmsl5 mutant plants was higher,while the expression level of ROS-scavenging genes was lower.(5)The loss of OsMSL5 reduced the sensitivity of rice to ABA.Compared with wild type,the water loss rate of leaves in vitro of osmsl5 mutant plants was faster after drought treatment The assays of stomatal moving showed that,after ABA treatment,the number of completely closed columns in wild type(DJ)was increased more greatly than that of osmsl5.The results of q RT-PCR analysis showed that after high salt osmotic stress and drought stress,compared with wild type,the expression level of ABA metabolic gene was more significantly up-regulated in osmsl5 mutant plants.In addition,after stress treatment,the expression levels of stress response genes Os HKT1 and Os RAB11,which depend on ABA pathway,were significantly lower than those of wild type(DJ)in osmsl5 mutant plants.(6)The expression of OsMSL5 will affect the pollen development of rice.OsMSL5-overexpressing transgenic plants(OsMSL5-OE)were obtained.Compared with wild type,the stamens of osmsl5 mutants are abnormal,and the anthers were deformed and shriveled.However,the stamen development of in OsMSL5-OE lines was not affected.Taken together,our results indicated that OsMSL5 can be used as osmotic safety valve to sense extreme osmotic environment,and also be used as membrane tension sensor to respond to the increased membrane tension,maintain cell size and shape.Thus it can be applied in enhancing the adaptability to various abiotic stress in genetic engineering breeding.