马尾松原生质体制备及瞬时转化体系的建立  

Establishment of a System for Preparation and Transient Transformation of Protoplasts of Pinus massoniana

作　　者：李岩硕 王民炎 刘殿宽 李伟[1] 殷恒福 LI Yanshuo;WANG Minyan;LIU Diankuan;LI Wei;YIN Hengfu(State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin 150040;Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Hangzhou 311400)

机构地区：[1]林木遗传育种全国重点实验室(东北林业大学),哈尔滨150040 [2]中国林业科学研究院亚热带林业研究所,杭州311400

出　　处：《植物研究》2025年第2期202-210,共9页Bulletin of Botanical Research

基　　金：国家科技创新2030重大项目(2022ZD04016)。

摘　　要：该研究旨在通过建立马尾松(Pinus massoniana)原生质体的分离和制备系统,实现基因的瞬时转化,从而推动马尾松基因功能研究和遗传转化体系的发展。研究选取了马尾松不同发育时期的外植体材料,包括针叶、下胚轴和愈伤组织,探讨了不同酶解条件对原生质体分离效率的影响,并通过试验优化了马尾松原生质体的制备流程,涉及不同类型的组织、不同的渗透压和酶解时间。研究结果表明:在0.5 mol⋅L-1甘露醇渗透液中,使用马尾松针叶组织进行6 h酶解处理是提取原生质体的最佳条件。基于此,该研究构建了一个适合马尾松的瞬时转化体系,并通过对比不同转化条件,实现了外源基因在马尾松原生质体中的瞬时表达。研究还发现,经过优化的原生质体制备体系能够显著提升原生质体的质量和转化效率,在使用聚乙二醇(PEG)介导的转化方法时,转化效率高达47.83%。综上,该研究成功构建了马尾松原生质体高效分离体系,为针叶树种基因功能解析提供了可靠的瞬时表达系统。This study aimed to establish a system of the protoplast preparation in Pinus massoniana for the transient transformation of genes,so as to promote the study of gene function and the development of genetic transformation system in P.massoniana.The explants were selected from different developmental periods of P.massoniana,including needle leaf,hypocotyls and callus tissues.The effects of different enzymatic conditions on the isolation efficiency of protoplasts were explored.The protoplasts preparation process involving different types of tissues,different osmotic pressures and enzymatic hydrolysis time were optimized.The results showed that pine needle tissue in 0.5 mol⋅L-1 mannitol osmotic solution for 6 h was the optimal condition for protoplasts extraction.Based on this,a transient transformation system of exogenous genes expression in P.massoniana protoplasts was constructed by comparing different transformation conditions.The optimized protoplast preparation and transformation system could significantly improve the quality and transformation efficiency of protoplasts,and the transformation efficiency of protoplasts was as high as 47.83%when using a polyethylene glycol(PEG)-mediated transformation method in P.massoniana.In summary,this study successfully constructed an effective protoplast isolation system for P.massoniana,which provides a reliable transient expression platform for analyzing gene functions in coniferous species.

关 键 词：马尾松 原生质体 瞬时转化 基因功能 

分 类 号：Q813[生物学—生物工程]