林业科学2018，Vol.54Issue(1)：46-53,8.DOI:10.11707/j.1001-7488.20180105
油桐叶肉细胞原生质体分离及瞬时转化体系的建立
The Establishment of Isolation and Transient Transformation Methods of Protoplasts of Vernicia fordii Mesophyll Cells
摘要
Abstract
[Objective] This study tries to explore the optimal conditions of separation of mesophyll cell protoplasts of Vernicia fordii and to establish its genetic transformation system,making it possible to study gene functions of V.fordii in vivo.[Method] This research firstly obtained mesophyll cell protoplasts of V.fordii through enzyme hydrolysis method using mature leaves and test tube plantlet leaves respectively.Gene transformation method of V.fordii protoplast mediated by PEG was established with protoplast receptor system.[Result] Protoplast isolation revealed that the most significant effect on the yield and activity of protoplast was enzymatic hydrolysis time followed by the concentration of cellulase.The concentrations of macerozyme and mannitol showed less influence on the yield and activity of protoplast.The optimal conditions for the isolation of protoplasts from mature leaves were 1.5％ cellulase concentration,1％ macerozyme concentration,0.6 mol· L-1 mannitol concentration and 12 hours of enzymolysis.The optimal conditions for the isolation of protoplasts from young leaves of plants propagated by tissue culture were 2％ cellulase concentration,1％ macerozyme concentration,0.7 mol · L-1 mannitol concentration and 6 hours of enzymolysis.In order to establish transient transformation system of V.fordii protoplast,this research transformed Arobidopsis thaliana MGT6 gene into V.fordii protoplast using PEG-mediated method.The result showed that MGT6 was located in the plasma membrane of protoplast,indicating that gene transformation method of V.fordii protoplast can successfully introduce exogenous genes into protoplasts and make it express.[Conclusion] This research established isolation and transient transformation methods of protoplasts of V.fordii mesophyll cells obtained from mature leaves and young leaves of plants propagated by tissue culture,respectively.Considering the convenience of sampling materials and the influence on the subsequent protoplast culture,we suggest that young leaves of tissue-cultured plants should be used to isolate protoplasts with conditions of 2％ cellulase concentration,1％ macerozyme concentration,0.7 mol· L-1 mannitol concentration and 6 hours of enzymolysis.On the basis of isolation of mesophyll cell protoplasts of V.fordii,the PEG-mediated genetic transformation method used in this study can be used to efficiently introduce exogenous genes into protoplasts with successful expression.In summary,the results not only promote the development of the basic researches on V.fordii,but also have important significance in germplasm innovation through cell fusion and gene engineering.关键词
油桐/原生质体制备/遗传转化/亚细胞定位Key words
Vernicia fordii/protoplast preparation/genetic transformation/subcellular localization分类
农业科技引用本文复制引用
谷战英,杨若楠,陈昊..油桐叶肉细胞原生质体分离及瞬时转化体系的建立[J].林业科学,2018,54(1):46-53,8.基金项目
国家自然科学基金项目(31700600) （31700600）
“十三五”国家重点研发计划(2017YFD0601304) （2017YFD0601304）
湖南省教育厅资助科研项目(17C1658) （17C1658）
中南林业科技大学青年科学研究基金(QJ2017002Z) （QJ2017002Z）
中南林业科技大学林学重点学科开放基金项目(2016YB01). （2016YB01）
