Effect of Leaf Incubation Temperature Profiles on Agrobacterium tumefaciens-Mediated Transient Expression
Effect of Leaf Incubation Temperature Profiles on Agrobacterium tumefaciens-Mediated Transient Expression
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DOI：
10.1002/btpr.2077
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发表时间：
2015-05-01
影响因子：
2.9
通讯作者：
Dandekar, Abhaya M.
中科院分区：
文献类型：
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作者：
Jung, Sang-Kyu;McDonald, Karen A.;Dandekar, Abhaya M.
众所周知，农杆菌介导的瞬时表达高度依赖于培养温度。与早期在恒温下进行的研究相比，我们考察了不同的叶片孵化温度对瞬时表达的影响。作为一个模型系统，利用真空渗透技术，合成内切葡聚糖酶(E1)和内切木聚糖酶(Xyn10A)基因在向日葵叶片中瞬时表达，用于生物燃料的应用。我们发现，瞬时表达的动力学强烈依赖于温度变化的时间和叶片孵化温度。令人惊讶的是，我们发现较高的培养温度(27-30℃)不适合T-DNA转移，如果在叶片培养的后期(第3-6天)施加高温，则显著增强瞬时表达，而在早期(0-2天)培养温度在20-25℃的范围内会导致较高的产量。在这些结果的基础上，我们认为在具有不同温度依赖动力学的植物细胞中，瞬时表达受T-DNA转移和蛋白质合成的共同控制。由于这两个阶段在时间上是分开的，并且具有不同的最适温度，因此我们能够开发出一种新的叶片孵化温度的两阶段优化策略。应用随时间变化的温度分布，我们能够使蛋白质积累比在20摄氏度恒定温度下的对照增加五倍。据我们所知，这是第一份说明变温分布对改善瞬时表达的影响的报告。(C)2015年美国化学工程师学会
Agrobacterium tumefaciens-mediated transient expression is known to be highly dependent on incubation temperature. Compared with early studies that were conducted at constant temperature, we examined the effect of variable leaf incubation temperature on transient expression. As a model system, synthetic endoglucanase (E1) and endoxylanase (Xyn10A) genes were transiently expressed in detached whole sunflower leaves via vacuum infiltration for biofuel applications. We found that the kinetics of transient expression strongly depended on timing of the temperature change as well as leaf incubation temperature. Surprisingly, we found that high incubation temperature (27-30 degrees C) which is suboptimal for T-DNA transfer, significantly enhanced transient expression if the high temperature was applied during the late phase (Day 3-6) of leaf incubation whereas incubation temperature in a range of 20-25 degrees C for an early phase (Day 0-2) resulted in higher production. On the basis of these results, we propose that transient expression is governed by both T-DNA transfer and protein synthesis in plant cells that have different temperature dependent kinetics. Because the phases were separated in time and had different optimal temperatures, we were then able to develop a novel two phase optimization strategy for leaf incubation temperature. Applying the time-varying temperature profile, we were able to increase the protein accumulation by fivefold compared with the control at a constant temperature of 20 degrees C. From our knowledge, this is the first report illustrating the effect of variable temperature profiling for improved transient expression. (C) 2015 American Institute of Chemical Engineers