菠萝叶基原生质体分离与瞬时转化体系的建立  

Establishment of an isolation and transient transformation system forleaf base protoplast in pineapple(Ananas comosus)

作　　者：贺涵 阳习鹏 赵婉欣 邵雪花[1] 杨凤玺 刘传和[1] HE Han;YANG Xipeng;ZHAO Wanxin;SHAO Xuehua;YANG Fengxi;LIU Chuanhe(Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs,Guangdong Provincial Key Laboratory of Tropical and Subtropical Fruit Tree Research,Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences,Guangzhou,Guangdong 510640,China;Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization,Environmental Horticulture Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou,Guangdong 510640,China)

机构地区：[1]广东省农业科学院果树研究所,农业农村部南亚热带果树生物学与遗传资源利用重点实验室,广东省热带亚热带果树研究重点实验室,广东广州510640 [2]广东省农业科学院环境园艺研究所,广东省园林花卉种质创新综合利用实验室,广东广州510640

出　　处：《西北农林科技大学学报（自然科学版）》2024年第7期81-88,98,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：广东省农业科学院协同创新中心项目(XTXM202203);国家自然科学基金青年基金项目(32002018);广东省现代农业产业技术体系创新团队建设专项(2023KJ109)。

摘　　要：【目的】研究菠萝叶基原生质体的高效分离方法,并对制备的原生质体进行瞬时转化,为利用菠萝原生质体进行菠萝基因功能研究提供技术支持。【方法】以‘巴厘’菠萝(Ananas comosus,‘Comtede Paris’)幼嫩叶片的叶基为材料,采用3因素3水平正交试验L _(9)(3^(3))分析纤维素酶质量浓度(6,12,20 g/L)、离析酶质量浓度(3,6,9 g/L)和甘露醇浓度(0.4,0.5,0.6 mol/L)对分离原生质体产量与活力的影响;同时,利用单因素试验(设置酶解时间2,4,6 h)筛选制备原生质体的最适酶解时间,确定分离菠萝叶基原生质体的最适条件;在最适条件下制备原生质体,采用聚乙二醇(PEG)介导法转化质粒,建立菠萝原生质体瞬时转化体系。【结果】L 9(33)正交试验结果显示,不同处理分离的原生质体产量为1.01×10^(6)~1.93×10^(6)g^(-1),纤维素酶质量浓度是决定原生质体产量的关键因素;原生质体活力为65.55%~87.00%,甘露醇浓度对原生质体活力的贡献最大。最适酶解条件为:菠萝叶基在含12 g/L纤维素酶、6 g/L离析酶、0.6 mol/L甘露醇的酶液中避光酶解4 h,产量可达1.96×10^(6)g^(-1),活力为85.54%。菠萝叶基原生质体通过PEG介导法转化pAN580-WRKY75质粒后,可在激光共聚焦显微镜下观察到核内的绿色荧光信号。【结论】确定了菠萝叶基原生质体的最适分离条件与瞬时转化方法,建立了适用于菠萝基因功能分析的原生质体瞬时表达体系。【Objective】This study investigated efficient methods for separation and transient transformation of pineapple leaf base protoplasts to provide technical support for pineapple gene function analysis.【Method】Young leaf bases from‘Comtede Paris’cultivar were used for a three-factor and three-level orthogonal test to analyze the effects of cellulase concentration(6,12 and 20 g/L),macerozyme concentration(3,6 and 9 g/L)and mannitol concentration(0.4,0.5 and 0.6 mol/L)on protoplast yields and vitalities.A single-factor test of digestion time(2,4 and 6 h)was also used to determine the optimal digestion time for protoplast isolation.Protoplasts were prepared under optimal conditions and transformed with plasmids using the polyethylene glycol(PEG)mediated transformation method to establish an efficient protoplast transformation system in pineapple.【Result】The orthogonal test revealed that the protoplast yield of‘Comtede Paris’leaf bases ranged from 1.01×10^(6)g^(-1)to 1.93×10^(6)g^(-1)under different treatments,and cellulase concentration was the primary factor influencing protoplast yield.Protoplast vitality ranged from 65.55%to 87.00%,and mannitol concentration was the most influential factor.Optimal conditions for pineapple leaf base protoplast isolation were determined as 12 g/L cellulase,6 g/L macerozyme and 0.6 mol/L mannitol,resulting in 1.96×10^(6)g^(-1)yield of protoplasts with 85.54%vitality after 4 hours of digestion in dark.The obtained protoplasts were successfully transformed with the pAN580-WRKY75 plasmid,and green fluorescence signal was observed in the nucleus of transformed protoplasts under a confocal microscope.【Conclusion】This study determined the optimal conditions for isolating and transforming pineapple leaf base protoplasts and established a transient expression system for pineapple gene function analysis.

关 键 词：菠萝叶基 原生质体 瞬时转化 绿色荧光蛋白 

分 类 号：S668.304.3[农业科学—果树学]