拟南芥种子的胚与胚乳中pOp6/LhG4AtO表达系统的构建与验证OA

[Objective]To construct the pOp6/LhG4AtO expression system for studying gene function in the embryo and endosperm of Arabidopsis seeds,and to validate its feasibility to provide a reliable tool for tissue-specific gene expression.[Method]Based on the pOp6/LhG4AtO binary transcriptional activation system,previously reported embryo-specific(TWS1)and endosperm-specific(FWA,ESH1)promoters were selected to construct the driver vectors.Meanwhile,a fluorescent reporter gene with enhanced nuclear localization(H2B-Clover)was employed to generate the effector vector.Genetic transformation was performed via Agrobacterium-mediated floral dip method to obtain positive transgenic lines.By crossing the driver lines with the effector line,the tissue-specific expression of the reporter gene was observed in F1 seeds at 2-5 d after pollination,and the expression patterns were compared with publicly available microarray transcriptome data(NCBI GEO;accession number GSE12404).[Result]The pOp6/LhG4AtO expression system was successfully constructed,and the required independent transgenic lines were obtained,including driver lines(pTWS1::LhG4AtO,pFWA::LhG4AtO,pESH1::LhG4AtO)and an effector line(pOp6::H2B-Clover).After crossing the driver line with the effector line,green fluorescence signals driven by pTWS1::LhG4AtO,were specifically and continuously observed at high levels in the nuclei of embryos cells only at 3-5 d after pollination(DAP).Similarly,green fluorescence signals driven by pFWA::LhG4AtO and pESH1::LhG4AtO were observed specifically and continuously at high levels in the nuclei of endosperm cells at 2-5 DAP after pollination.These results demonstrate that the fluorescence expression patterns driven by the pOp6/LhG4AtO system are highly consistent with published microarray transcriptome data.[Conclusion]This study established and validated the pOp6/LhG4AtO system for tissue-specific driver gene expression in the embryo and endosperm of Arabidopsis seeds,providing a reliable tool for functional studies of key genes during seed development.