摘要
近年来,茶树基因组测序的完成为茶树在分子和基因水平的研究奠定了基础。但由于转基因技术尚不成熟且茶树生长周期较长,茶树的基因功能研究依然不能有效开展。采用铁观音(Camellia sinensis var.sinensis cv.‘Tieguanyin’)实生幼苗叶片,通过筛选多种纤维素酶、果胶酶、离析酶和甘露醇的浓度组合,并结合原生质体的数量、活性和杂质含量综合确定了最佳配方,成功建立了铁观音茶苗叶片原生质体提取和PEG介导的高效瞬时转化体系,转化率达56.25%。利用该系统探索了茶氨酸代谢通路中2个重要合成酶(茶氨酸合成酶(TSI)和谷氨酰胺合成酶(GSII-1.1))的亚细胞定位。研究发现,这2种酶均定位于铁观音原生质体细胞质中。茶苗叶片原生质体提取和瞬时转化体系的建立为茶树基因组功能研究奠定了技术基础。
Recent advances in the genomic sequencing of tea plants have laid the foundation for tea research at the molecular and gene levels.However,the transgenic technologies are immature and the life cycles are long for tea trees,it is still difficult to conduct functional analyses of tea genes.This study used young leaves of Camellia sinensis var.sinensis cv.‘Tieguanyin’,established a useful formula by testing multiple concentration combinations of cellulase,pectinase,macerozyme and mannitol.By evaluating the quantity,viability and debris of resulted protoplast,we successfully established a highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in Tieguanyin seedling leaves,with a transformation rate reaching 56.25%.Using this system,the subcellular localization of two pivotal enzymes in the theanine metabolism pathway(the theanine synthetase(TSI)and the glutamine synthetase(GSII-1.1))were explored.Results show that,these two enzymes are both localized in the cytosol of the Tieguanyin protoplasts.Together,the establishment of this tea mesophyll protoplast extraction and transient expression system would lay a technological foundation for studying the function of tea genome.
作者
张玉琴
吴嘉诚
何萌
刘仁义
朱晓玥
Yuqin Zhang;Jiacheng Wu;Meng He;Renyi Liu;Xiaoyue Zhu(College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China;FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Center for Agroforestry Mega Data Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《植物学报》
CAS
CSCD
北大核心
2022年第3期340-349,共10页
Chinese Bulletin of Botany
基金
福建农林大学导师研究经费(2018)
国家自然科学基金(No.31970336)
福建农林大学科技创新专项基金(No.CXZX2020094A)
福建省自然科学基金(No.2021J01140)。
