Efficient isolation of protoplasts from freesia callus and its application in transient expression assays
Efficient isolation of protoplasts from freesia callus and its application in transient expression assays
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小苍兰愈伤组织原生质体的高效分离及其在瞬时表达测定中的应用
DOI：
10.1007/s11240-019-01649-9
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发表时间：
2019-09-01
影响因子：
3
通讯作者：
Wang, Li
中科院分区：
文献类型：
--
作者：
Shan, Xiaotong;Li, Yueqing;Wang, Li
小苍兰是由野生种驯化而成的著名切花品种之一，以花色艳丽、香气芬芳著称。它有可能成为一种模式植物，以揭示蝴蝶科植物特化代谢产物生物合成的分子基础，这些代谢产物的生物合成对花色和花香等观赏性状的遗传修饰至关重要。然而，从组织培养中繁殖出来的漫长的生命周期和极低的转化率严重减慢了通过转基因方法进行基因功能鉴定的速度。以广受欢迎的小苍兰品种‘红河’的幼花芽为材料，建立并优化了一种高效的原生质体分离方法，并将其应用于基因瞬时表达系统。结果表明，该系统具有较强的通用性，可广泛应用于蛋白质免疫印迹、酶活性、蛋白质亚细胞定位、蛋白质-蛋白质相互作用、蛋白质-DNA相互作用以及基因转录调控特性分析等方面。此外，从小苍兰的另一个品种‘Ambiance’诱导的愈伤组织也可以用于分离原生质体进行基因瞬时转基因分析，表明该系统具有广泛的适用性。本研究结果为在体内阐明基因功能提供了新的方法，对其他生活史较长或难以转化的物种，如蝴蝶科植物也具有一定的借鉴意义。
Freesia hybridais one of the most famous cut flower variety domesticated from wild species inFreesiagenus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar ofFreesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar ofFreesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function inFreesiain vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae.