Development And Application Of Stable Transgenic Rice Lines Expressing Sublocalization Markers

Sub-cellular localization refers to the specific presence of a protein or a gene expression product in specific organelles.Sub-cellular localization studies can provide important supporting information for determining the function of an unknown protein.In a previous study,our lab has constructed a set of plant expression vectors containing GFP/RFP fusion markers for different organelles.Our lab has also produced T0 transgenic rice plants.The sub-cellular localization types include cell membrane,nuclear,endoplasmic reticulum,plastid,mitochondria,Golgi apparatus,vacuole,peroxisome,and cytoskeleton.This study further screened stable transgenic progeny plants by hygromycin-resistance testing,PCR sequencing,fluorescence observation of protoplasts and of leaf sheath tissues.Proof-of-concept evaluations were also performed for co-localization and Magnaporthe oryzae-rice interaction by using the developed organelle marker lines.The results are as follows:1.After hygromycin-resistance screening,PCR verification,protoplasts and rice sheath fluorescence observation,it is verified that most of the previously cultured marker fusions can stably express the fluorescence signal and correctly locate the predicted organelle.2.For detection of a weak or no fluorescence signal materials such as pCXSN-RFPAtCCASP、pCXSN-GFPmTn、pCXSN-RFPH2B、pCXSN-RFP,the vector was reconstructed and verified.The reconstituted vector replaces the 35S promoter with a strong Ubiquitin promoter for driving the expression of the fluorescent protein fusion structure.3.As an example,a rice transcription factor WRKY45-RFP fusion construct was transformed into GFP-H2B rice protoplasts.Fluorescence observation showed that WRKY45-RFP and GFP-H2B were co-located in the nucleus,showing that the fluorescent marker lines could be applied for sub-cellular co-localization study of rice proteins.4.The sheath tissue of GFP-H2B rice was infected with a PWL2-mCherry-NLS-transfomed blast isolate pBV591.The result showed that PWL2-mCherry-NLS and GFP-H2B were co-located in the nucleus of rice,consistent with previous studies that PWL2-mCherry-NLS was translocated to rice nuclei after expression in M.oryzae.This example showed that the rice fluorescence marker lines has a good application prospect in M.oryzae-rice interaction research.