Generation Of A Set Of Organelle Markers In Arabidopsis Thaliana Pollen Tube

In flowering plants,pollen plays a vital role in plant fertility and crop production through the generation and delivery of the male gametes to the embryo sac for double fertilization.Pollen germination and pollen tube growth are the key step in the plant sexual reproduction.And also,pollen tube is the ideal system for studying the polar cell growth.It is well known that the intracellular localization of uncharacterized protein,which functions in pollen tube growth can be used to infer the likely functions of this protein.There are a varity of common technologies to study protein subcellular localization.It contains the orientation of bioinformatics prediction,immunohistochemical method,organelle proteomics,marker enzymes for cell fractionation studies,and fusions of protein-coding regions to genes of fluorescent proteins method.Fusion of fluorescent proteins method is to fuse target gene and reporter gene of fluorescent protein,and express fusion protein.It is the fluorescent protein to show the subcellular localization of the target protein.This approach reveals subcellular localization of the native protein under normal expression conditions and it is harmless to the living cell,which has been widely used in the protein subcellular localization.Fluorescent proteins usually contain green fluorescent protein,red fluorescent protein,yellow fluorescent protein and blue fluorescent protein,etc.Organelle markers are based on the confirmed specific target sequence to locate on the target organelles,which fuse with fluorescent protein.In terms of the colour of fluorescence,it is the GFP that is widely used in subcellular localization experiment.Although the fluorescence pattern can sometimes be identified as similar to a specific organelle,it is often necessary to compare the distribution of the uncharacterized protein to well-established organelle markers in the same cell.In general,Arabidopsis transgenic lines of pollen-specific organelle markers are made with only one fluorescent protein and in one binary vector.So a multi-colored set of organelle markers in pollen tube is needed.In order to express in the pollen tube,we used a pollen-specific promoter LAT52 to drive expressing.We generated a unified collection of fluorescent markers for five membrane-bounded organelles such as endoplasmic reticulum,Golgi apparatus,trans-Golgi network,prevacuolar compartment,and lytic vacuole.The organelle markers in this set are based on well-established targeting sequence.All markers were made with two different FPs(GFP and mCherry)and in two different binary vectors(pME18 and pBI121)to facilitate the free combination of these markers with each other and with other constructs.The former has the plant resistance to hygromycin,the latter has a plant kanamycin resistance.This multicolored set of organelle markers with two different plant resistances can be used for co-localization studies in pollen tube.We used laser confocal microscope to observe the expression of this set of organelle markers.The confocal micrographs showed that ER was evenly distributed throughout the cytoplasm of pollen tube.Golgi,TGN and PVC markers appeared as punctate florescence signals in the pollen tube cytoplasm.And LV markers labeled several circular structures in pollen tube.We generated a set of fluorescent organelle markers that can be used for visualizing organelle dynamics in pollen tube.These markers themselves that can also be used for co-localization studies with a wide range of other proteins in pollen tube,which has great significance to learn the function of these proteins.