Ffunctional Identification Of OsPPCK2 In Regulating Drought Resistance Of Rice

Rice(Oryza sativa L.)is one of the main food crops in the world,the cultivation and production of rice are severely affected by a variety of abiotic stresses such as drought,high temperature,and chilling injury.Besides,the continuous population growth and environmental damage caused by human activities make the problem of food security more serious.Therefore,it is necessary to identify more important genes related to stress resistance and explore their genetic variations for application in genetic improvement.In this study,based on the 34 association loci identified by Dr.Zilong Guo via genome-wide association study(GWAS)based on image-based traits and traditional drought resistance traits by drought experiment of a natural population containing 533 rice germplasm resources,four loci were chosen for candidate gene analysis.By combining analyses of GWAS,RNA-seq,gene chip,metabolome,and other data,a total of 23 candidate genes in the four loci were selected for genetic analysis.The mutants by CRISPR technique and the over-expression rice lines for these candidate genes were generated,and then the genotypes and expression levels of these genes in the transgenic lines were examine.After drought stress treatment,the mutant plants of genes PK17,PK20,and PK22 displayed a drought-sensitive phenotype compared to the wild-type plants.PK17 encodes phosphoenolpyruvate carboxylase kinase 2(OsPPCK2).The function of OsPPCK2 was further investigated.The main results are as follows:1.The mutant lines of OsPPCK2 were more sensitive to drought than wild-type at seedling and adult stages,and mutation of OsPPCK2 inhibited the growth and development of plants.2.There was a large number of natural variations in the OsPPCK2 promoter region.A few SNPs in the promoter may be related to the gene expression of this gene based on analyses of the phenotypic association of the variations in the natural population,drought-responsive expression levels,and promoter activity assay in protoplasts.3.Subcellular localization assay showed that OsPPCK2 was located in both the nucleus and the cytoplasm.4.It was found that OsPPCK2 could affect the proline biosynthesis,PEPC activity,and the transpiration rate of rice.5.OsPPC2 b,a phosphoenolpyruvate carboxylase,was identified as a potential substrate of OsPPCK2 via in vitro phosphorylation assay and mass spectrometry.