槟榔原生质体分离及瞬时转化体系的建立  

Isolation of Protoplast and Establishment of Transient Expression System in Areca catechu

作　　者：曹春艳 王威[1] 杨新奇 李东栋[2] Cao Chunyan;Wang Wei;Yang Xinqi;Li Dongdong(College of Tropical Crops,Hainan University,Haikou,570228;Sanya Nanfan Research Institute,Hainan University,Sanya,572024)

机构地区：[1]海南大学热带作物学院,海口570228 [2]海南大学三亚南繁研究院,三亚572024

出　　处：《分子植物育种》2023年第17期5730-5737,共8页Molecular Plant Breeding

基　　金：海南省重点研发项目(ZDYF2022XDNY148);海南省自然科学基金创新团队项目(2019CXTD397);国家自然科学基金项目(31660222)共同资助。

摘　　要：为建立一套稳定高效的槟榔叶肉细胞原生质体分离及瞬时转化方法,本研究采用酶解法以槟榔嫩花苞、嫩叶片、胚状体为实验材料,对影响其原生质体产率、活力及瞬时转化效率的因素进行了研究。结果表明:槟榔花苞在酶解4 h产量和活力最高,产量为9.1×10~7个/mL,活力为92%。槟榔原生质体瞬时转化中原生质体/质粒与40%PEG-4000水浴(45℃)30 min、室温PEG孵育40 min的条件下,利用PEG介导法将表达绿色荧光蛋白的质粒P35-EGFP转入槟榔原生质体中,转化效率达到13%。通过共聚焦显微镜观察和荧光定量分析证明,EGFP与MYB108都成功被过表达。该研究建立了槟榔叶肉原生质体的分离及瞬时转化方法,有较好的应用价值。可以用作蛋白质亚细胞定位和基因调控作用的研究,后续还可以被用作验证蛋白质与蛋白质或蛋白质与DNA的互作等研究。In order to establish a stable and efficient method for protoplast isolation and transient transformation of Areca catechu mesophyl cells,the factors affecting the protoplast yield,viability and transient transformation efficiency of Areca catechu mesophyl cells were studied by enzymolysis using bud,tender leaf and embryoid body as experimental materials.The results showed that the yield and activity of Areca catechu buds were the highest at 4 h of enzymatic hydrolysis,with the yield of 9.1×10~7/mL and the activity of 92%.In the transient transformation of Areca catechu protoplasts,the plasmid P35-EGFP expressing green fluorescent protein was transferred into Areca catechu protoplasts by PEG-mediated method under the conditions that the protoplasts/plasmids were incubated with 40%PEG-4000 water bath(45℃)for 30 min and PEG at room temperature for 40 min.The transformation efficiency reached 13%.Both EGFP and MYB108 were successfully overexpressed by confocal microscopy and fluorescence quantitative analysis.The method of isolation and transient transformation of Areca catechu mesophyll protoplasts was established,which has good application value.It can be used for the study of protein subcellular localization and gene regulation.It can also be used to verify the interaction between protein and protein or protein and DNA.

关 键 词：槟榔(Areca catechu) 原生质体 瞬时表达系统 亚细胞定位 基因功能 

分 类 号：S792.91[农业科学—林木遗传育种]