Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.)
文献类型： 外文期刊
作者： Priyadarshani, S. V. G. N. 1 ; Hu, Bingyan 1 ; Li, Weimin 1 ; Ali, Hina 1 ; Jia, Haifeng 1 ; Zhao, Lihua 1 ; Ojolo, Si 1 ;
作者机构： 1.Fujian Agr & Forestry Univ, Fujian Prov Key Lab Haixia Appl Plant Syst Biol, Ctr Genom & Biotechnol,State Key Lab Ecol Pest Co, Coll Crop Sci,Coll Resources & Environm,Minist Ed, Fuzhou 350002, Fujian, Peoples R China
2.Fujian Agr & Forestry Univ, Coll Life Sci, Fuzhou 350002, Fujian, Peoples R China
3.Chinese Acad Trop Agr Sci, South Subtrop Crops Res Inst, Zhanjiang 524091, Guangdong, Peoples R China
关键词： Protoplast; BAP; NAA; Transfection; Pineapple
期刊名称：PLANT METHODS （ 影响因子：4.993； 五年影响因子：5.312 ）
ISSN： 1746-4811
年卷期： 2018 年 14 卷
页码：
收录情况： SCI
摘要： BackgroundAn efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. This has limited the full exploration of the genetic repository of pineapple as well as the study of its genes, protein localization and protein interactions.ResultsTo address the above limitations, we have developed an efficient system for protoplast isolation and subcellular localization of desired proteins using pineapple plants derived from tissue culture. A cocktail of 1.5% (W/V) Cellulase R-10 and 0.5% (W/V) Macerozyme R-10 resulted in 51% viable protoplasts with 3h digestion. Compared to previously reported protocols, our protoplast isolation method is markedly faster (saving 4.5h), requires only a small quantity of tissue sample (1g of leaves) and has high yield (6.5x10(5)). The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Additionally, colocalization analysis of two pineapple Mg2+ transporter genes in pineapple protoplasts was consistent with the results in a tobacco transient expression system, confirming that the protoplast isolation method can be used to study subcellular localization. Further findings showed that the system is also suitable for protein-protein interaction studies.ConclusionBased on our findings, the presently described method is an efficient and effective strategy for pineapple protoplast isolation and transformation; it is convenient and time saving and provides a greater platform for transformation studies.
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