棉花真叶原生质体分离及瞬时表达体系的优化

doi:10.3864/j.issn.0578-1752.2021.21.003

摘要/Abstract

摘要：

【目的】真叶细胞能模拟植物内源条件,建立基于棉花真叶原生质体的高效瞬时表达体系,为快速有效研究棉花基因功能提供方法。【方法】以陆地棉TM-1真叶为材料,采用常用的纤维素酶和离析酶组合分离原生质体,探究影响原生质体分离的叶龄、渗透压、酶解液成分和酶解时间,及渗透压和酶解时间对原生质体活力的影响,并分析渗透压、PEG浓度和培养液种类对原生质体瞬时转化的影响,进而优化棉花真叶原生质体瞬时表达体系。构建GhLTP-GFP表达载体,对比融合蛋白在拟南芥、棉花原生质体和烟草表皮细胞中的亚细胞定位,验证该体系。【结果】不同于棉花子叶,酶解液中高浓度CaCl₂会显著抑制真叶细胞壁的酶解,含10 mmol·L^-1 CaCl₂的酶解液能有效分离棉花真叶原生质体。甘露醇显著影响原生质体得率,含0.5 mol·L^-1甘露醇的酶解液分离原生质体得率最高,且细胞形态维持较好,而0.4 mol·L^-1甘露醇条件下原生质体活力降低一倍,表明0.5 mol·L^-1甘露醇能较好维持棉花真叶原生质体渗透压。陆地棉刚展平的真叶分离所得原生质体大小合适,而展平后的嫩叶分离得到的原生质体细胞较大,得率降低一倍。酶解处理7 h前,原生质体游离缓慢,酶解9 h原生质体产量达到高峰,继续酶解原生质体将破裂,得率降低。在等渗条件下用40% PEG4000转化所得原生质体,转化效率最高,而普遍采用的低渗条件不利于棉花真叶原生质体的转化。转化后,用WI溶液继续培养原生质体,会引起原生质体的大量破裂,用含0.5 mol·L^-1甘露醇的W5溶液继续培养,有利于原生质体形态的维持,转化率提高到90%。表达载体35S:GhLTP-GFP分别转入棉花、拟南芥原生质体和烟草表皮细胞,GFP信号在细胞中的定位结果一致。【结论】建立的棉花真叶原生质体瞬时表达体系,可获得8.10×10⁶个/mL活力在95%以上的高质量原生质体,原生质体得率提高8倍,转化效率达到90%,可用于亚细胞定位、蛋白互作,以及代谢调控网络研究等。

关键词: 棉花, TM-1, 原生质体分离, 瞬时表达

Abstract:

【Objective】Cells of true leaves can well mimic the plant endogenous situation. It is an efficient way for expediting cotton functional study to establish an effective transient expression system using cotton protoplasts obtained from true leaves.【Method】The enzyme combination of cellulose and macerozyme were used to isolate protoplasts from true leaves of Gossypium hirsutum L. acc. TM-1. The effects of osmotic pressure, components of digestion buffer and digestion time on protoplast yield were studied and the validity of protoplasts were compared under different mannitol concentration and digestion time. To improve the transformation efficiency of cotton protoplast, the effects of mannitol and PEG concentration and buffers for protoplast culture were subsequently studied. In order to verify the optimized transient expression system, the vector 35S:LTP-GFP was constructed and transformed into protoplasts of Arabidopsis and cotton and tobacco epidermal cells followed by observation of fusion protein localization.【Result】High concentration of CaCl₂ in the digestion buffer significantly inhibited the isolation of protoplast from cotton true leaves, which was opposite of that using cotyledon. 10 mmol·L ^-1 CaCl₂ was employable for digestion buffer to isolate cotton protoplasts from true leaves. Mannitol concentration significantly affected protoplast yield that peaked under mannitol concentration of 0.5 mol·L^-1, and protoplast validity decreased moiety under 0.4 mol·L^-1 mannitol, suggesting that 0.5 mol·L^-1 mannitol was most suitable to maintain the osmotic pressure of cotton protoplasts. Cotton protoplasts displayed suitable size when isolated from newly flattened true leaves, while protoplast enlarged and yield decreased when produced from young leaves flattened 5 days. The protoplasts dissociate slowly until being digested 9 h when the yield reached the peak. The transformation efficiency was greatly improved under isotonic condition of 40% PEG buffer. While hypotonic condition that is commonly applied to facilitate transformation was against the entrance of exogenous DNA into cotton protoplasts. After transformation, the protoplast ruptured abundantly in WI buffer,whereas the shape maintained well in W5 buffer adding 0.5 mol·L^-1 mannitol. The transformation efficiency was improved to 90% using the optimized transient expression system. The subcellular location analysis results showed consistent GFP signal in protoplasts of cotton and Arabidopsis true leaf and epidermal cells of tobacco leaf.【Conclusion】Our study has optimized the cotton mesophyll protoplast transient expression system, which could produce 8.10×10 ⁶·mL^-1 fine protoplasts with validity above 95% and transformation efficiency reached to 90%. This system is applicable for analysis of subcellular location, protein interaction and research on metabolism and regulation network.

Key words: cotton, TM-1, isolation of protoplasts, transient expression

李青,鱼海鹏,张子豪,孙正文,张艳,张冬梅,王省芬,马峙英,阎媛媛. 棉花真叶原生质体分离及瞬时表达体系的优化[J]. 中国农业科学, 2021, 54(21): 4514-4524.

LI Qing,YU HaiPeng,ZHANG ZiHao,SUN ZhengWen,ZHANG Yan,ZHANG DongMei,WANG XingFen,MA ZhiYing,YAN YuanYuan. Optimization of Cotton Mesophyll Protoplast Transient Expression System[J]. Scientia Agricultura Sinica, 2021, 54(21): 4514-4524.

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https://www.chinaagrisci.com/CN/Y2021/V54/I21/4514

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