菊花花瓣原生质体分离与瞬时转化体系的建立  

Establishment of isolation and transient expression system for protoplasts from chrysanthemum petals

作　　者：李志美 张碧佩 伍青 吕金 赵鑫 潘燕 郭炜珊 周厚高[1] 王凤兰[1] 罗红辉 LI Zhimei;ZHANG Bipei;WU Qing;LU Jin;ZHAO Xin;PAN Yan;GUO Weishan;ZHOU Hougao;WANG Fenglan;LUO Honghui(Zhongkai University of Agriculture and Engineering,Key Laboratory of Tropical&Subtropical Flowers and Landscape Plantsof Guangdong Higher Education Institutions,Guangzhou 510225,China)

机构地区：[1]仲恺农业工程学院/广东省普通高校热带亚热带花卉与园林植物重点实验室,广州510225

出　　处：《植物生理学报》2023年第10期1951-1963,共13页Plant Physiology Journal

基　　金：广东省重点领域研发计划项目(2020B020220009);贵州省科技计划项目(黔科合支撑[2021]一般225);2022年广东省级乡村振兴战略专项资金种业振兴项目(2022-NPY-00-043)。

摘　　要：本研究以3个菊花(Chrysanthemum morifolium)品种‘帝王水晶’(‘Chrystal Regal’)、‘紫红托桂’(‘Zihong Tuogui’)和‘紫风车’(‘Zi Fengche’)为实验材料,通过轻摇酶解法及酶解前真空处理制备优质的菊花原生质体,并初步以改良的聚乙二醇4000(PEG 4000)介导转化结合热激处理技术建立了菊花花瓣原生质体瞬时转化体系。结果表明,在菊花花瓣组织中可获得完整并有活力的原生质体数量比叶片中多;其中,采用含10 g·L^(-1)纤维素酶、6 g·L^(-1)离析酶、2 g·L^(-1)崩溃酶和400 mmol·L^(-1)甘露醇的酶解液体系,酶解前抽真空处理20~25 min,并置于摇床中22℃、30 r·min^(-1)轻摇酶解12~14 h,‘帝王水晶’和‘紫风车’花瓣原生质体产量分别可达2.8×10^(5)和7.0×10^(5)个·g^(-1)(FW),活力分别为63.9%和76.9%,能满足瞬时表达实验要求。用等体积的含100 mmol·L^(-1)CaCl_(2)、400 mmol·L^(-1)甘露醇和300 g·L^(-1)PEG 4000的缓冲液体系分别介导3个菊花品种的花瓣原生质体(200μL,2×10^(5)个·mL^(-1))转化,并于28℃热激处理60 s,35S启动的外源基因(OsVPE1-GFP)在花瓣原生质体中瞬时表达的绿色荧光信号较强,瞬时转化效率为30%以上。In this study, three cultivars of chrysanthemum(Chrysanthemum morifolium), ‘Chrystal Regal',‘Zihong Tuogui' and ‘Zi Fengche', were used as experimental materials. Chrysanthemum protoplasts with good quality were isolated by the improved enzymolysis condition with vacuum pretreatment. Furthermore, transient expression system for chrysanthemum petal protoplasts was preliminarily established by the polyethylene glycol 4000(PEG 4000)–mediated transformation with heat shock treatment. The results show that the number of intact, viable protoplasts isolated from petal tissues is more than that from leaf tissues. Specifically, yields of petal protoplasts isolated from ‘Chrystal Regal' and ‘Zi Fengche' could reached 2.8×10^(5) and 7×10^(5) protoplasts·g^(-1)(FW) with the viability of 63.9% and 76.9%, respectively, when the cell enzymolysis solution contains 10 g·L^(-1) cellulase, 6 g·L^(-1) macerozyme, 2 g·L^(-1) driselase and 400mmol·L^(-1) mannitol. During isolation of the above protoplasts, the vacuum pretreatment last for 20–25 min before enzymolysis, and the enzymolysis reaction was performed by gently shaking for 12–14 h in a shaker at 30 r·min^(-1) and 22℃. Thus, these petal protoplasts could be used in the transient expression system.Transient expression of an exogenous gene(Os VPE1-GFP) with 35S promoter in petal protoplasts(200 μL,2×10^(5) protoplasts·m L^(-1)) isolated from the three chrysanthemum cultivars was mediated by adding equal volume of 300 g·L^(-1) PEG 4000 buffer containing 100 mmol·L^(-1) Ca Cl_(2) and 400 mmol·L^(-1) mannitol. And when the transient expression system was heat-shocked at 28℃ for 60 s, the GFP signal of green fluorescence in the transformed petal protoplasts was strong and the transient transformation efficiency was more than 30%.

关 键 词：菊花 花瓣 叶片 原生质体分离 瞬时转化 

分 类 号：S682.11[农业科学—观赏园艺]