在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析
Summary
生物钟的调节有关拟南芥转录组的三分之一，但基因的反馈到报时的比例尚不清楚。在这里，我们可视化的方法来使用一昼夜记者在原生质体瞬时表达的荧光成像拟南芥突变体的任何行快速评估昼夜的表型。
Abstract
The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.
Introduction
大多数生物体具有内源性计时员，以帮助每日变动对环境的有效协商。此计时，生物钟，调节生物体的代谢和生理学的许多方面以预料的外部环境可预见的变化。在例如植物，在时间上可预见的病原体或草食动物攻击的预期强烈降低了整体的易感性1 – 4。淀粉代谢紧紧生物钟调节到最后保证淀粉的储备，直到天亮在或长或短日照条件下5是否增长。事实上，与之搭配的24小时环境显示出增加的增长率生物钟植物固碳率和存活率相比，运行时间不匹配6的时钟植物。昼夜节律在所有这些过程中广泛的影响产生在很大程度上从多达三分之一有节奏的调控在拟南芥7的总转录。这些节律的基因产物参与广泛的细胞过程，包括代谢途径，激素信号，和应激反应7。最重要的是，蛋白质功能的直接昼夜调节是通过磷酸化8昼夜调控最有可能其他翻译后后修饰成立。
在驱动这每天转录重新编程生物钟的中心位于转录网络/翻译反馈回路（TTFLs），包括早晨表达基因生物钟ASSOCIATED 1（CCA1）和LATE芒胚轴（LHY），该抑制的表达CAB的1（TOC1），GIGANTEA（GI）和晚上复杂（EC）的成员日晚基因时序 ; LUX ARRHYTHMO（LUX），早开花3（ELF3）和ELF4 9 – 11。一起机智小时假响应调节 -genes（PRR3，5，7和9），TOC1压制CCA1 / LHY表达而EC充当阳性调节器12 – 14。所述TTFL网络组件的附加反馈机制，转录后和翻译后调节起着调谐昼夜节律的重要作用。到目前为止，上确定生物钟蛋白质最丰富的PTM是磷酸化15。 CCA1由酪蛋白激酶2（CK2）的磷酸化会影响其结合靶子16和为生物钟17的温度补偿重要。该PRR蛋白磷酸化差异在昼夜周期，TOC1的磷酸化影响其负调节因子的相互作用，晚上分阶段时钟组件ZEITLUPE（ZTL）18。这些实施例说明如何翻译后修饰和蛋白质 – 蛋白质相互作用调TTFL网络到24小时的转录振荡器。对于更详细的介绍了转录时钟网络及其调控，近期出色的评语是可用的（ 如参考15）。
然而，仍不太清楚的是，以有节奏调节过程和细胞代谢的其它方面反馈到计时机构本身的程度。时钟缺陷拟南芥突变体的筛选粗放可能获得进一步的了解它的机制或信号通路可能在24小时节律的产生涉及从TTFL网络。为此，金和索先前发表的时钟功能的任何拟南芥线19中的高效率和快速分析的突破方法。基于原生质体采用瞬时表达，这种方法超越了稳定的转基因株系的需要或超过到发光的时钟标记线。在这里，我们可视化高产原生质体隔离和与优化的协议合并正方法20筛选通过的瞬时表达的时钟标记发光成像昼夜缺陷在96孔板阅读器。
我们将野生型协作0拟南芥比较良好的特点时钟突变体，确认这里描述成功检测改变昼夜节律的方法。从这些行衍生的原生质体转染表达与昼夜CCA1子萤火虫荧光素酶报道构建; CCA1pro：LUC 19。萤光素酶催化的多步反应，其中萤光素转化成电子激发氧化萤光素发射光21，其被成像在一段时间，以评估在这些原生质体的期间，振幅和转录节律的相位。
该协议包括三个主要部分组成;分离的原生质体，用报道质粒，和发光即时转染的原生质体老化。这三个部分应该总是在一天用新鲜制备的缓冲液和试剂进行。植物生长，并有足够量的报道质粒的纯化应提前进行，并且在该协议中不可见。
Protocol
Representative Results
Discussion
在这里，我们提出了一个快速检测筛选拟南芥线昼夜周期缺陷，包括原生质体分离，转染，并luminecent成像。在野生型的昼夜周期拟南芥和时钟突变CCA1 / LHY，ZTL 和 CCA1 -OX被从一个CCA1pro发光的测量计算：LUC报告并发现与使用更多时间从整个植物产生的公布的数据一致耗时转基因方法（ 表2）。使用该测定以筛选拟南芥属突变体避免了初始要求产生转基因发光线，这是耗时，并且可能仅揭示的特定突变显示出野生型的节奏。该协议的一个明显优势是任何拟南芥行可以在很短的时间进行筛选，改变昼夜节律的转录，这将有助于影响植物报时更多基因的鉴定。
<p class="“jove_content”">原生质体的分离可以是费力的，特别是如果该突变行显示一个矮化表型。产生原生质体先前报道的方法涉及切割叶或苗成细条，真空渗透与酶液条，使酶达到细胞壁26,27。随后的消化需要至少3小时温育在黑暗中的原生质体释放到酶溶液中。当不采用真空浸渗，孵化时间长达18小时建议。在这里介绍的协议，因为难以渗透到酶表皮细胞层通过胶带除去真空渗入是不必要的。当通过切割植物材料产生的原生质体，有必要从消化后的细胞壁碎片分离原生质体;这通常是通过过滤所述溶液或纯化它的糖梯度27,26完成。这里，任何未消化的组织将保持高压釜后胶带上消化，所以可以省略原生质体的过滤和糖梯度纯化。还有的是，从长期的原生质体程序产生的应力影响细胞活力和生物钟的机会。这里可视化的基于磁带的原生质体法20产生了大量的原生质体;和给定细胞的存活率超过昼夜时间序列和原生质体和整个幼苗（ 表2）的匹配期间的长度，在这里所描述的方法优于其他方法来产生原生质体。该协议的转染步骤是关键步骤，关于原生质体的生存力的影响。所述PEG溶液使在该溶液中被传递到细胞中的DNA，并且两个孵育时间（步骤3.4），并应根据经验确定的PEG的百分比。标准浓度为20％，用较低的浓度降低了转化效率和高浓度降低了生存能力28。孵育时间短五分钟可产生原生质体27的有效转染。
原生质体可以在任何发光成像平台进行成像。在此视频，我们已经使用配备有外部光的发光板读数器（红色和蓝色发光二极管，630和470处，分别在〜20μE的组合强度），其允许高通量筛选和频繁的测量。其他成像设备，其检测发光，例如基于一个电荷耦合器件（CCD）照相机替代板读取器或设置，可以应用同样只要照明可用于光合作用。使用安装在一可控制柜CCD照相机的优点在于，光线和温度可被编程。一个缺点是较长的捕获时间，引入黑暗的长时间可导致应力到原生质体中除扰乱ING内源性报时。无论选择哪个实验平台，设置调整可能相比，苗或成熟植物设置是必要的。根据我们的经验，在成像缓冲器转染和/或萤光素过程中增加的报告质粒的浓度可能会解决可能发生在最初的实验中的任何不稳定或快速衰减的节奏。
在以后的实验中，这里描述的方法可以用于研究任何突变拟南芥线的昼夜表型。稍作修改，EG的各种药物对计时的影响可能在此设置进行研究。此外，转染可被修改以包括用于基因沉默19人工微RNA，进一步扩大该协议的通用性。协议生成大米原生质体成熟29和这里介绍的成像协议可能同样适用于我们进一步ü在经济上重要的单子叶作物植物时钟nderstanding。
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Prof. David Somers and Dr. Jeongsik Kim for the luciferase reporter plasmid and initial method development. Prof. Karen Halliday kindly provided all the circadian mutant lines used in this study. This research was supported by Royal Society research grants RS120372 and RS140275. GvO is a Royal Society University Research Fellow (UF110173).
Materials
| CELLULYSIN Cellulase, Trichoderma viride | Merck Millipore | 219466 | |
| Pectinase R10 | Sigma Aldrich | P2401 | |
| D-mannitol | Sigma Aldrich | M4125 | |
| CaCl2 | Sigma Aldrich | C4901 | |
| KCl | Sigma Aldrich | P3911 | |
| Bovine Serum Albumin | Sigma Aldrich | A4503 | |
| MES | Acros Organics | 327765000 | |
| NaCl | Acros Organics | 327300010 | |
| Glucose | Fischer Scientific | G/0500/60 | |
| MgCl2 | Sigma Aldrich | M2670 | |
| Polyethylene glycol (PEG) 4000 | Acros Organics | 434630010 | |
| Fetal Bovine Serum | Sigma Aldrich | F4135 | |
| D-Luciferin, Firefly | Biosynth AG | L-8200 | Dissolve luciferin powder in 0.1M trisphosphate buffer pH 8.0 to a concentration of 50mM |
| Ampicillin | Sigma Aldrich | A9618 | |
| Comply Steam Indicator Tape | 3M | 1222 | |
| Magic Tape | 3M | 819-1460 | |
| Petri Dishes | Scientific Laboratory Supplies | SLS2002 | |
| Microplate, 96 well, flat bottom, white | Greiner Bio-One | 655075 | |
| TopSeal-A, adhesive lids for 96-well plates | Perkin Elmer | 6050195 | |
| Syringe, 10 ml, w/o needle | BD Plastipak | 302188 | |
| Syringe filter 0.22 µm | Merck Millipore | SLGP033RS | |
| Biological Rhythms Analysis Software System (BRASS) | Free download at amillar.org | ||
| QIAfilter Maxiprep Kit | Qiagen | 12262 | |
| Modified Fuch-Rosenthal counting chamber, double cell | Hawksley | AC6000 | |
| Bright field microscope | Optika Microscopes | B-150POL-B | |
| LB942 TriStar2 multimode reader, with luminescence module | Berthold Technologies Ltd | 57947/57948 |
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