文献类型：期刊文献

中文题名：‘渤丰3号’杨叶片高效原生质体提取体系的建立与优化

英文题名：Establishment and Optimization of an Efficient Protoplast Extraction System for Populus‘Bofeng 3 hao’Leaves

作者：钟姗辰[1] 张伟溪[1] 张冰玉[1] 苏晓华[1,2] 唐芳[1,2]

第一作者：钟姗辰

机构：[1]中国林业科学研究院林业研究所,林木遗传育种全国重点实验室,国家林业和草原局林木培育重点实验室,北京100091;[2]南京林业大学,南方现代林业协同创新中心,江苏南京210037

年份：2025

卷号：38

期号：2

起止页码：116-126

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：;北大核心:【北大核心2023】；

基金：林木良种高效基因编辑技术研究(2021YFD2200103)。

语种：中文

中文关键词：欧美杨;‘渤丰3号’杨;原生质体酶解;原生质体纯化

外文关键词：Populus×euramericana;'Bofeng 3 hao'poplar;protoplast enzymolysis;Protoplast purification

分类号：S725.3

摘要：[目的]建立欧美杨‘渤丰3号’叶片高效的原生质体提取体系,为基因功能研究和基因编辑分子育种提供技术支撑。[方法]利用两种组分的酶解液和不同的预处理方法检测‘渤丰3号’杨叶片原生质体提取的效率和质量,优化原生质体纯化条件,以建立最适合‘渤丰3号’杨叶片高效的原生质体提取方法。[结果]结果表明,酶解液1(1.5%纤维素酶+0.75%离析酶+20 mmol·L^(-1)MES+10 mmol·L^(-1)KCl+0.6 mol·L^(-1)甘露醇+10 mmol·L^(-1)CaCl_(2)+1%BSA)酶解原生质体的效果优于酶解液2(0.8%纤维素酶+0.2%离析酶+5 mmol·L^(-1)MES+0.6 mol·L^(-1)甘露醇),酶解液1酶解得到的原生质体数量较多,且细胞较为完整,杂质少。酶解前的抽真空处理能略微提高原生质体的数量,但得到的原生质体与未抽真空处理的相比,细胞易碎、杂质更多;而换用更小空隙细胞筛对原生质体过滤的效果提升不明显。采用W5溶液直接洗涤可以获得最多的原生质体但有少许的杂质,在样品量有限的情况下可以使用该纯化方式。0.5 mol·L^(-1)蔗糖溶液纯化后的原生质体中杂质最少,但会损失较多的原生质体细胞;而0.75 mol·L^(-1)蔗糖溶液的纯化效果最好,既能减少杂质,又能获得较多的原生质体,适用于对原生质体纯度要求较高的情况。[结论]通过优化酶解条件、预处理步骤和纯化方法,本研究建立了欧美杨‘渤丰3号’叶片高效的原生质体提取体系。这一体系能够显著提高原生质体的提取效率和纯度,为后续的基因功能研究和分子育种实验提供了可靠的基础。
[Objective]To establish an efficient protoplast extraction system for Populus×euramericana'Bofeng 3 hao',providing a technical support for gene function research and gene editing molecular breeding.[Method]The efficiency and quality of protoplast extraction from'Bofeng 3 hao'were detected by two kinds of enzymatic combinations and different pretreatment methods.The protoplast purification conditions were optimized to establish the most suitable and efficient protoplast extraction method for'Bofeng 3 hao'.[Result]The results showed that enzyme solution 1(1.5%Cellulase+0.75%Macerozyme R-10+20 mmol·L^(?1)MES+10 mmol·L^(?1)KCl+0.6 mol·L^(?1)mannitol+10 mmol·L^(?1)CaCl_(2)+1%BSA)was more effective than enzyme solution2(0.8%cellulase+0.2%Macerozyme R-10+5 mmol·L^(?1)MES+0.6 mol·L^(?1)mannitol),and enzymatic solution 1 produced a larger number of protoplasts,with more intact cells and fewer impurities.Vacuum infiltration prior to enzymatic digestion slightly increased the number of protoplasts,but the protoplast cells were more fragile and contained more impurities compared to those without vacuum treatment;however,the effect of using smaller pore size filters on protoplast filtration was not significantly improved.Direct washing with W5 solution produced the most protoplasts with a small amount of impurities,which could be used when the sample was limited.Protoplasts purified with 0.5 mol·L^(?1)sucrose solution showed the least impurities,though significant cell loss was observed.The purification with 0.75 mol·L^(?1)sucrose solution provided the best results,reducing impurities while yielding a high number of viable protoplasts,making it ideal for applications required high purity.[Conclusion]By optimizing enzyme digestion conditions,pretreatment methods,and purification steps,this study established an efficient protoplast extraction system for the leaves of Populus×euramericana‘Bofeng 3 hao’.This system significantly enhances both the efficiency and purity of protoplast extraction,providing a reliable foundation for subsequent gene function research and molecular breeding experiments.