Subcellular Localization And Prokaryotic Expression Of Two PPR Proteins In Rice

The PPR protein is a family of proteins with a pentatricopeptide repeat(PPR),one of the largest families of proteins in terrestrial plants.Most PPR proteins can specifically edit the RNA of mitochondria and chloroplast genes,and participate in the process of plant growth,development and regulation of embryogenesis.However,the function of the PPR genes involved in biotic and abiotic stress in rice remains largely unknown.The main contents of this research include the following three aspects:(1)The expression levels of multiple PPR family genes in Nipponbare rice were analyzed with salt stress,and the expression of Os01g0815900 and Os01g0913400 genes was up-regulated.And the two genes were cloned and identified.And their sequences were basically analyzed by several bioinformatics software.(2)This study constructed these PPR genes' prokaryotic expression vector for prokaryotic expression in vitro.The full-length CDS sequences of Os01g0815900 and Os01g0913400 were constructed into the prokaryotic expression vector pET-30a(+),and the expression strain BL21 was transformed.The Os01g0815900 protein was not expressed in E.coli,and another 17.5 kD protein was identified as Os01g0913400 protein,which provided an experimental basis for subsequent large-scale expression and purification,antibody preparation and functional study of PPR protein.(3)In order to clarify the subcellular localization of these two PPR proteins,this study constructed the fluorescent-tag vectors pM999-Os01g0815900-GFP and pM999-Os01g0913400-GFP,respectively.The protoplasts of rice yellow seedlings were transformed and subcellular localization was observed by laser confocal microscopy.The results showed that Os01g0815900 was located in the cytoplasm;while Os01g0913400 was located in mitochondria of rice protoplasts.This study will lay the foundation for the interpretation of the subcellular localization mechanism and preliminary function of the PPR protein family of rice,and will also promote the further study of PPR protein function in rice growth and development.