Arabidopsis Cell-Free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures
Arabidopsis Cell-Free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures
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DOI：
10.1093/pcp/pcr080
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发表时间：
2011-08-01
影响因子：
4.9
通讯作者：
Naito, Satoshi
中科院分区：
文献类型：
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作者：
Murota, Katsunori;Hagiwara-Komoda, Yuka;Naito, Satoshi
无细胞提取物系统的使用极大地促进了植物转录后调控机制的分析。拟南芥作为模式系统提供了广泛的遗传资源，然而，迄今为止，还没有合适的拟南芥无细胞翻译系统。在这项研究中，我们设计了一个拟南芥无细胞提取物（ACE）用于体外翻译研究。从拟南芥幼苗的愈伤组织培养物制备原生质体，通过Percoll梯度离心将原生质体抽空后制备无细胞提取物。新的ACE系统具有与小麦胚芽提取物系统相当的翻译活性。我们证明了从拟南芥的5 '-3'核糖核酸外切酶缺陷突变体xrn 4 -5制备的ACE，与野生型拟南芥相比，表现出增加的脱帽mRNA的稳定性。我们应用ACE系统研究AtCGS 1的转录后调控。AtCGS 1编码胱硫醚γ-合酶（CGS），其催化植物中甲硫氨酸和S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine，S-Met）生物合成的第一个关键步骤，并且通过mRNA降解与翻译延伸停滞耦合来反馈调节。ACE系统能够复制翻译延长停滞和随后的AtCGS 1 mRNA降解，这是由ATPMet诱导的。这里描述的ACE系统可以在种子播种后一个月内制备，并且将使研究植物基因的转录后调节成为可能，同时利用拟南芥的遗传学。
The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine gamma-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.