Cloning And Functional Analysis Of OsABCC10 In Rice

Salt stress is one of the major abiotic stresses which inhibit crop yield.Currently,approximately 20%of the arable lands and nearly half of the irrigated lands globally have been affected by soil salinity,with the area of salt affected soils up to 9.O×108 hm2,which poses a great threat to agricultural productivity and sustainability.Rice is one of the main food crops in China and even in the whole world.More than half of Chinese people take rice as the staple food.As a moderately sensitive crop to salt stress,rice yield will be seriously affected under salt stress,which will seriously threaten the human food security.Therefore,it is very important to study the molecular mechanisms of rice salt tolerance,which provide theoretical basis for breeding excellent salt-tolerant rice varieties,so as to solve the increasingly serious food problems.ABC transporter is one of the known superfamily proteins,and they are found in all living organisms.Plant ABC transporter is an important family of transmembrane transport proteins,not only participates in the transport of lipids,hormones,metal ions,secondary metabolites and foreign substances in plants,but also participates in the interaction between plants and pathogens and the important physiological processes such as ion channel regulation in plants.ABCC protein is one subfamily of the ABC transporter superfamily,which is mainly involved in the export of various molecules from the cytosol to the external medium or the matrix of organelles.At present,structurale and functional analysis of plant MRP family mainly focus on Arabidopsis,but rarely on rice.In this study,we obtained two knockout lines of OsABCC10 by the CRISPR/Cas9 genome editing technology and analysis of tissue expression pattern of OsABCC 10 and its expression under salt stress,subcellular localization,transport activity,and physiological phenotypes under salt stress condition in rice.The main results of this study are as follows:(1)The expression pattern of OsABCC10 in rice different tissues was analyzed,and we found that OsABCC10 was expressed in rice root,stem and leaf,but its expression level in root was the highest,which was 4 and 5 times higher than that in stem and leaf,respectively.The expression of OsABCC10 in rice roots was induced by salt stress in a dose-dependent manner.A time-course experiment showed that the expression of OsABCC10 was upregulated with times under salt stress.These results suggested that OsABCC10 is involved in the salt-related processes.(2)OsABCC10 was successfully knockout by CRISPR/Cas9 genome editing technology,and two homozygous osabcclO mutants,namely osabcc10-2 and osabcc10-3,respectively,were obtained.Under salt stress,compared with the wild type,the osabcc10-2 and osabcc10-3 showed growth sensitivity,shorter main roots,fewer lateral roots and more wilted leaves.After salt stress,normal rice nutrient solution was used for recovery.The osabccl0-2 and osabcc10-were found to recover more slowly than the wild type.Difference in the growth between wild type and knockout lines after 150 mmol/L NaCl treatment was more significant.Under the condition of 50 mmol/L NaCl,the Na+ concentrations in the xylem sap of the osabccl0-2 and osabccl0-3 were higher than the wild type.These results indicated that OsABCC10 is required for rice salt tolerace by regulating Na+ transport from root to shoot in rice.(3)Subcellular localization of OsABCC10 was conducted in rice protoplasts,Green fluorescence signals was observed at the cytoplasm and nucleus in the cells transformed with pYL322-P35s-GFP,while the cells transformed with pYL322-P35s-OsABCC10-GFP showed green fluorescent signal in the vacuole membrane,indicating that OsABCC10 was localized in the tonoplast.when OsABCC10 was expressed in yeast cells,the growth of the yeast strain carrying pYES2-OsABCC10 was significantly inhibited on SD-Ura medium containing different NaCl concentrations compared with the the yeast strain carrying empty vector pYES2.It indicated that OsABCC10 may have Na+ transport activity in yeast and that,the yeast cells carrying pYES2-OsABCC10 may accumulates more Na+which has a toxic effect.This result was contrary to the results of physiological phenotypes under salt stress condition,which may be caused by the different subcellular localization of OsABCC10 in yeast and rice.