Subcellular Localization Mechanism Of AtSUC4 And AtPsaL In Arabidopsis Thaliana Chloroplast

Sucrose transporters(SUTs)which have 12 transmembrane areas are typical-membrane protein in higher plant.They are responsible for sucrose transport across membranes and play main role in plant growth and development.As a major photosynthetic product,sucrose is used for long-distance carbon transporting in plant.Meanwhile,as a signaling molecule,sucrose can affect extensively the plant especially in the metabolic activity and developmental stage of fruit.And sucrose metabolism also plays pivotal roles in development,stress response,and yield formation,mainly by generating a range of sugars metabolites used for energy metabolism and synthesizing essential compounds(including protein,cellulose,and starch).Additionally,as signals,they regulate expression of microRNAs,transcription factors,and other genes and crosstalk with hormonal,oxidative,and defense signaling.The process of sucrose synthesis,transport,utilization and storage is strictly regulated by multiple signal networks,being subjected to cellular developmental stage and environmental adaptation.Sucrose transporters are the key elements or regulators in the complex signaling networks.However,little is known about the cellular signaling regulation of sucrose transporters in plants.The previous available studies have indicated that AtSUC4 interacts with AtPsaL,a subunit of photosystem I(PSI)localized in thylakoid membrane of chloroplast in A.thaliana.So the main research of this reasearch is to study the localization of At SUT4 and AtPsaL in A.thaliana chloroplast.The main research results are as follows:1.At SUT4 was localized in chloroplast inner,outer membrane and thylakoid.Construct AtSUC4-gDNA-GFP vector,transform 35S::gSUC4-GFP into Col-0 successfully,and screening out positive colnes.The fluorescence of GFP was observed in the chloroplasts by laser scanning confocal microscope.Then,we separated the components of chloroplast andfound AtSUC4 in chloroplast inner,outer membrane and thylakoid through western blot method.2.At PsaL was localized in the periphery of chloroplasts and intrachloroplast compartment.Construct 35S-AtPsaL-cDNA-GFP vector,transiently transformed the A.thaliana protoplasts.The fluorescence of GFP was observed in periphery of chloroplasts and intrachloroplast compartment.3.The N-and C-terminal of AtSUC4 was exposured to the cytoplasmic side.Construct truncated 35S::gSUC4-GFP vector,and transiently transform the A.thaliana protoplasts.The fluorescence of GFP was observed in the chloroplasts to confirm that the N-terminal and C-terminal of AtSUC4 exposured to the cytoplasmic side.4.Successfully complement the phenotype of suc4 p.The constructed complement lines of the mutant suc4 p,showed that the phenotypes,gene expression and protein levels of transgenic lines got a recovery to certain extent.5.Using Affymetrix A.thaliana ATH1 Genome Array,we analyzed differential expressed genes between the mutants and screened out candidate genes(IAS,BAM,SUS,HXK,SPS,TCP,ERT,and WRKY)related to sucrose-starch metabolism and sucrose signal transduction.6.Using real-time PCR to detect expression of AtSUC4,AtPSAL and some related genes in Col-0 and mutants.Knocking out AtSUC4 and AtPSAL leaded to the down-regulation of AtSUC2,AtPsaC,AtPsaD and up-regulation of AtPsaB and At PsaH.7.Analysis the protein abundance in Col-0 and suc4 p,suc4.1,psal.1,psal.2 and double mutants.The results have showed that the protein abundance of SUC4 was down regulated in suc4 p,suc4.1 and double mutants.8.Analysis the codon usage bias of sucrose transporter family genes in A.thaliana.The codon usage bias of AtSUC3 and At SUC4 is the lowest,and have the highest similarity with SNF3,RGT2.