Specialized metabolites in tea(Camellia sinensis)are fundamental quality factors.It is important to characterize gene function in vivo to identify key enzymes and reactions involved in the biosynthesis of such metabol...Specialized metabolites in tea(Camellia sinensis)are fundamental quality factors.It is important to characterize gene function in vivo to identify key enzymes and reactions involved in the biosynthesis of such metabolites.Here we report a transient expression method to analyze gene function in isolated tea mesophyll protoplasts.This method is an alternative approach to traditional genetic transformation for studies on gene function in vivo.We screened several tea cultivars and different digestion conditions to optimize protoplast isolation.Digestion of newly emerged leaves of C.sinensis‘Zhongbai 4’with 3%cellulase R-10 and 0.3%macerozyme R-10 for about 12 h yielded approximately 107 mesophyll protoplasts.Genes encoding enzymes involved in secondary metabolite synthesis were transiently expressed in the protoplasts,and their subcellular locations were determined.With further improvements in the transfection efficiency,this transient expression system will contribute greatly to the analyses of in vivo gene function in tea.展开更多
基金the National Natural Science Foundation of China(31870684)the Pearl River Science and Technology New Star Fund of Guangzhou(201806010018)+3 种基金the Youth Innovation Promotion Association of Chinese Academy of Sciences(Y821131001)the Regional Key Project of Science and Technology Service Network Plan of Chinese Academy of Sciences(KFJ-STS-QYZX-093)the National Key Research and Development Program of China(2018YFD1000601)the Guangdong Provincial Special Fund For Modern Agriculture Industry Technology Innovation Teams(2020KJ120).
文摘Specialized metabolites in tea(Camellia sinensis)are fundamental quality factors.It is important to characterize gene function in vivo to identify key enzymes and reactions involved in the biosynthesis of such metabolites.Here we report a transient expression method to analyze gene function in isolated tea mesophyll protoplasts.This method is an alternative approach to traditional genetic transformation for studies on gene function in vivo.We screened several tea cultivars and different digestion conditions to optimize protoplast isolation.Digestion of newly emerged leaves of C.sinensis‘Zhongbai 4’with 3%cellulase R-10 and 0.3%macerozyme R-10 for about 12 h yielded approximately 107 mesophyll protoplasts.Genes encoding enzymes involved in secondary metabolite synthesis were transiently expressed in the protoplasts,and their subcellular locations were determined.With further improvements in the transfection efficiency,this transient expression system will contribute greatly to the analyses of in vivo gene function in tea.
