Development Of A Set Of Fluorescent Protein Fusion Markers For Protein Sub-localization Studies In Rice

About tens of thousands of proteins are synthesized in eukaryotic cells.And these proteins are translocated to particular subcellular space or organelles so that they can carry out their functions normally.Therefore,to determine the subcellular localization of the proteins will be of great significance to the study of their functions.Fluorescent protein tagging is one of the most common approaches to study subcellular localization of unknown proteins.Observation of fluorescence by co-localization with organelle-specific protein of known localization is an effective strategy to determine the localization in a special organelle of the unknown protein.In this study,we have searched and cloned the coding sequence of proteins or protein motifs which locate conservatively in major organelles of plant including endoplasmic reticulum,plastid,mitochondria,Golgi apparatus,tonoplast,peroxisome,plasma membrane,nucleus and cytoskeleton.These coding sequences were fused respectively with the coding sequences of green fluorescent protein(GFP)or red fluorescent protein(RFP)in-frame.A set of plant expression vectors carrying the fluorescent protein fusion cassettes were developed and were preliminarily assessed by Agro-infiltration in Nicotina benthamiana leaves.Furthermore,we developed transgenic rice lines expressing the fluorescent protein fusion cassettes via Agro-mediated transformation,and determined the subcellular localization of the fusion fluorescent proteins in rice cells by fluorescence assay.The plant expression vectors and transgenic rice lines we developed will be very helpful for subcellular localization of unknown proteins and functional genomics in rice.