文献类型：期刊文献

英文题名：Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

作者：Shao, Yingying[1] Mu, Detian[1] Pan, Limei[2] Wilson, Iain W.[3] Zheng, Yajie[1] Zhu, Lina[1] Lu, Zhiguo[1] Wan, Lingyun[2] Fu, Jine[2] Wei, Shugen[2] Song, Lisha[2] Qiu, Deyou[4] Tang, Qi[1]

第一作者：Shao, Yingying

通信作者：Tang, Q[1];Song, LS[2]

机构：[1]Hunan Agr Univ, Coll Hort, Changsha 410128, Peoples R China;[2]Guangxi Bot Garden Med Plants, Nanning 530023, Peoples R China;[3]CSIRO Agr & Food, Canberra, ACT 2601, Australia;[4]Chinese Acad Forestry, Res Inst Forestry, State Key Lab Tree Genet & Breeding, Beijing 100091, Peoples R China

年份：2023

卷号：24

期号：4

外文期刊名：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

收录：;Scopus(收录号：2-s2.0-85149017693);WOS:【SCI-EXPANDED(收录号:WOS:000945067000001)】；

基金：This work was supported by the Horticulture's Open program of Hunan Agricultural University (2021YYXK002), the Key Scientific Research Fund of Hunan Provincial Education Department (21A0138), the National Natural Science Foundation of China (No.81903752, No.31600238), the Project of Hunan Natural Science Foundation (2022JJ30305), and the Project of Guangxi Natural Science Foundation (2019GXNSFBA185026).

语种：英文

外文关键词：Uncaria rhynchophylla; protoplast isolation; PEG-mediated transfection; subcellular localization; dual-luciferase (Dual-LUC) assays

摘要：Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 degrees C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 x 10(7) protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 degrees C with the 40 mu g of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.