这项工作得到了江苏省自然科学基金 (BK20140919)、中国国家自然科学基金 (31470375)、江苏高等教育机构重点学术项目开发和青兰项目的支持。

1. 植物的准备 (8 周)
<ol><li>将 50烟草烟种子放在一个1.5 毫升的离心管中, 其中含有1毫升5% 次氯酸和0.1% 海卫 X-100 (v/v) 溶液, 并留出5分钟来消毒种子。</li>
	<li>用无菌水冲洗种子5次, 在200µL 的无菌水中暂停种子。</li>
	<li>小心地将单个种子放到 MS 琼脂培养基上 (1 L: 1x 村 & Skoog 盐, 10 克蔗糖, pH 5.8 (KOH), 1% 琼脂), 并在4° c 中储存介质板, 3 天以同步发芽。 注意: 为了避免微生物污染, 在干净的?...

<ol>
	<li>Thines, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAZ+repressor+proteins+are+targets+of+the+SCF(COI1)+complex+during+jasmonate+signalling.">JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.</a> Nature. 448, 661-665 (2007).</li><li>Chini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAZ+family+of+repressors+is+the+missing+link+in+jasmonate+signalling.">The JAZ family of repressors is the missing link in jasmonate signalling.</a> Nature. 448, 666-671 (2007).</li><li>Jones, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border+control--a+membrane-linked+interactome+of+Arabidopsis.">Border control--a membrane-linked interactome of Arabidopsis.</a> Science. 344, 711-716 (2014).</li><li>Arabidopsis Interactome Mapping Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+network+evolution+in+an+Arabidopsis+interactome+map.">Evidence for network evolution in an Arabidopsis interactome map.</a> Science. 333, 601-607 (2011).</li><li>Yazaki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+transcription+factor+interactome+networks+using+HaloTag+protein+arrays.">Mapping transcription factor interactome networks using HaloTag protein arrays.</a> Proc Natl Acad Sci U S A. 113, 4238-4247 (2016).</li><li>Kenworthy, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein-protein+interactions+using+fluorescence+resonance+energy+transfer+microscopy.">Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.</a> Methods. 24, 289-296 (2001).</li><li>Walter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40, 428-438 (2004).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+complementation+imaging+assay+for+protein-protein+interactions+in+plants.">Firefly luciferase complementation imaging assay for protein-protein interactions in plants.</a> Plant Physiol. 146, 368-376 (2008).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein-protein+interactions+by+thermal+stability+shift+analysis.">Quantitation of protein-protein interactions by thermal stability shift analysis.</a> Protein Sci. 20, 1439-1450 (2011).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+determination+of+binding+affinity+and+kinetics+using+surface+plasmon+resonance+biosensors.">Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors.</a> Curr Opin Biotechnol. 8, 498-502 (1997).</li><li>Huang, X., Ouyang, X., Deng, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+repression+of+photomorphogenesis:+role+switching+of+COP/DET/FUS+in+light+signaling.">Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.</a> Current opinion in plant biology. 21, 96-103 (2014).</li><li>Liu, B., Zuo, Z., Liu, H., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+cryptochrome+1+interacts+with+SPA1+to+suppress+COP1+activity+in+response+to+blue+light.">Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.</a> Genes Dev. 25, 1029-1034 (2011).</li><li>Lian, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue-light-dependent+interaction+of+cryptochrome+1+with+SPA1+defines+a+dynamic+signaling+mechanism.">Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.</a> Genes Dev. 25, 1023-1028 (2011).</li><li>Zuo, Z., Liu, H., Liu, B., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light-dependent+interaction+of+CRY2+with+SPA1+regulates+COP1+activity+and+floral+initiation+in+Arabidopsis.">Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis.</a> Curr Biol. 21, 841-847 (2011).</li></ol>

这里描述的协议是简单和可再生的研究在体内蛋白质-蛋白质相互作用, 特别适合检测蛋白质相互作用动力学在外在治疗之下。本试验的关键步骤是烟浸润。为了确保渗透成功, 植物必须非常健康。另一个关键因素是何时检查渗透后的荧光活动。这个问题没有正确的答案。研究人员被鼓励监测荧光活动在不同的日子后, 渗透判断时, 荧光是表达的最高水平, 因为我们观察到的蛋白质表达?...

为了协调生长与环境, 植物已经进化了优雅的信号通路, 感知, 传感器, 并响应信号提示。就像接力赛中的赛跑者一样, 蛋白质是植物信号传导的必要参与者。人们普遍认识到蛋白质-蛋白质相互作用 (PPI) 在细胞通讯中起着重要作用。蛋白质的磷酸化、再乙酰化和降解都依赖于靶蛋白与其修饰酶之间的物理相互作用。例如, 茉莉以域蛋白 (JAZ) 家族蛋白与转录因子 MYC2 相互作用并抑制其转录活性1,2。鉴于 PPI 的重要性, 植物中的 large-scale 蛋白 interactomes 最近已被探索3,4,5。这些 interactome 的结果进一步揭示了复杂的调控网络, 协调不同的细胞功能。
有一些既定的方法来监测 PPI。酵母两种杂交 (Y2H) 法是 PPI 检测中应用最广泛的方法。Y2H 很容易执行, 适合快速检测蛋白质相互作用。Y2H 也广泛用于筛选未知的交互合作伙伴的特定 protein-of-interest。然而, 由于 Y2H 完全是在酵母中进行的, 它不能反映植物细胞的真实情况, 并带来高假阳性率。另一个类似的策略是下拉法。一般来说, 两个蛋白质的表达和纯化从大肠杆菌细胞, 然后混合和固定的检测蛋白相互作用。虽然此方法不费时, 但它也无法获取在体内交互结果。对于体内交互目的, co-immunoprecipitation (co) 是最常用的检测方法, 它要求高质量的抗体 immunoprecipitate protein-of-interest, 不能排除间接质子的可能性。此外, 由于联合 IP 中的复杂实验程序, 结果通常与个人专长不同。
基于记者的重组分析大大推进了对体内PPI 的检测。这些方法包括荧光共振能量传递 (烦恼)6, 双荧光互补 (BiFC)7, 和萤火虫荧光互补成像 (的) 试验8。虽然这三种方法比 co 更好地反映直接的在体内PPI, 烦恼, 和 BiFC 需要特定的显微镜来检测荧光信号, 不能轻易量化的互动强度。相比之下, 该公司利用萤火虫荧光, 这将发光后与它的基底素反应。更重要的是, PPI 强度可以从荧光活动的价值来确定。因此, 它不仅表明两种蛋白质是否相互作用, 而且还提供了治疗时相互作用动力学的信息。虽然有一些建立的方法来监测交互动力学 (基于表面等离子体共振或热稳定转移)9,10, 这些方法需要微妙的仪器或特定的标签。相比之下, 它更易于执行和检测。
该方法的原理是将氨基末端和羧基末端的荧光 (分别命名为 n-卢克或 C-卢克) 与蛋白 A 和 B 融合, 并在植物细胞中同时表达这两种融合蛋白。如果蛋白质 a 与蛋白质 B 相互作用, 那么荧光的两个半部分将被重组为活性荧光酶。荧光活动可以通过计或 CCD 摄像头检测到。没有必要获得稳定的转化。瞬态表达式足以获得高质量的交互结果。
环型 E3 泛素连接本构 photomorphogenic 1 (COP1) 与无数转录因子相互作用, 通过26S 蛋白酶体11促进它们的降解。其中一些 COP1-targeted 转录因子是光的阳性调节剂。在黑暗中, COP1 与光敏 A-105 1 (SPA1) 的抑制器交互, 这增强了 COP1 E3 活动8。在对光的知觉之后, 光将废除 COP1-SPA1 相互作用抑制 COP1 活动, 然后稳定 COP1 基板12,13,14。这个例子说明了研究 ppi 和确定 ppi 动力学的生物学意义。

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		<col style="width:140pt" width="186" />
		<col style="width:140pt" width="187" />
		<col style="width:114pt" width="152" />
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	<tbody>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Transformation solution</td>
			<td class="xl16" style="width:140pt" width="186"></td>
			<td class="xl16" style="width:140pt" width="187"></td>
			<td style="width:114pt" width="152"></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">10 mM Morpholineethanesulfonic acid</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900336&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">27.8 mM Glucose</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900392</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl18" height="32" style="width:293pt;height:24.0pt" width="390">10 mM MgCl2&#215;6H2O</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900020&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">150 &#956;M Acetosyringone</td>
			<td class="xl17" style="width:140pt" width="186">ALDRICH</td>
			<td class="xl17" style="width:140pt" width="187">D134406&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">pH 5.7</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Luciferin working buffer</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="56" style="height:42.0pt">
			<td class="xl17" height="56" style="width:293pt;height:42.0pt" width="390">5 mM Luciferin potassium salt</td>
			<td class="xl17" style="width:140pt" width="186">GOLD BIOTECHNOLOGY</td>
			<td class="xl17" style="width:140pt" width="187">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">0.025% Triton X-100</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900502&#160;</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl17" height="32" style="width:293pt;height:24.0pt" width="390">H2O to 10 ml</td>
			<td class="xl21"></td>
			<td class="xl21"></td>
			<td></td>
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luciferase complementation imaging assay in nicotiana benthamiana leaves for transiently determining protein-protein interaction dynamics

蛋白质-蛋白质相互作用是在大多数细胞过程中中继信号传导的基本机制;因此, 鉴定新的蛋白质-蛋白质相互作用对和监测蛋白质相互作用动力学是特别感兴趣的揭示植物如何响应环境因素和/或发展信号。已经开发出过多的方法来检测蛋白质-蛋白质相互作用, 无论是体外还是体内。其中, 最近建立的荧光互补成像技术是最简单和最快的方法来演示体内蛋白-蛋白质相互作用。在这种检测中, 蛋白质 a 或蛋白 B 与氨基末端或羧基末端荧光的一半分别融合。当蛋白质 a 与蛋白质 B 相互作用时, 荧光的两个半部分将被重组, 形成功能性和活性的荧光酶。荧光活动可以用计或 CCD 摄像机记录。与其他方法相比, 基因组检测显示蛋白质-蛋白质相互作用的定性和定量。农杆菌在烟草烟叶片中的浸润是一种广泛应用的瞬态蛋白表达系统。通过 COP1 和瞬态表达式的结合, 表明茉莉处理后, SPA1 的物理相互作用逐渐降低。

在这个荧光的互补协议中, 有三主要的步骤可用于研究蛋白质-蛋白质相互作用的体内, 包括植物生长、烟草浸润和荧光测定。该协议中最关键的步骤是将液体A. 农杆菌渗透到N. 烟叶 (图 1)。
下面是这个技术的一个例子, 它证实了茉莉减少 COP1-SPA1 的相互作用。荧光的氨基末端和羧基端部...

Watch this Scientific Journal Video about Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics at JoVE.com

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

这篇手稿描述了一个简单和快速的实验程序, 以确定蛋白质-蛋白质相互作用的基础上荧光活动的测量。

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

The overall goal of this procedure is to quantitatively detect protein-protein interactions in a transient expression system. This measure can help answer key questions in quan-signal transaction fields such as how to quantitatively monitor our protein protein directions after a chemical or environmental treatment. The main advantage of this technic is that it uses a luminometer or a CCD camera to detect the luciferous activity, which represents the protein protein direction intensity so the results are more accurate.To begin this procedure, add one milliliter of a solution containing five percent sodium hypochlorite and zero point one percent triton x one hundred to a one point five milliliter microcentrifuge tube containing seeds. Let the solution rest for five minutes to sterilize the seeds. Then, wash the seeds with sterile water five times.Suspend the washed seeds in two hundred microliters of steril water. Using fine tips, carefully place individual seeds onto the surface of plated MS agar medium. Store the plates at four degrees Celsius for three days to synchronize germination.After this, keep medium plates under normal growth conditions for ten days. Transfer the germinating seedlings into soil, making sure not to damage the roots. Cover the tray with a transparent plastic cover overnight to maintain humidity.Grow plants in a controlled growth room for seven weeks, when they'll be ready for agrobacterium tumefaciens infiltration. To begin, deliver one hundred and fifty nanograms of plasmids into the a-tumefaciens competent cell strain GV3101. Place the tubes containing the a-tumefaciens culture in a shaker, and incubate for four hours.Using a glass rod, transfer the culture from the tubes to LB mediums. Culture a-tumefaciens and LB agar medium at 28 degrees celsius for four days. Next, prepare to inoculate a single a-tumefaciens colony from each plate into its own tube containing three milliliters of LB liquid medium.Shake at 280 RBM at 28 degrees Celsius for 24 hours to propagate the culture. Then, transfer 75 microliters of the bacterial culture into 15 milliliters of fresh LB liquid medium containing 15 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin. Culture the bacteria at 220 RPM at 30 degrees Celsius for approximately 8 hours until OD600 is between 0.5 to 1.After this, transfer the culture to 15 milliliters centrifuge tubes. Centrifuge at 4000 times gravity and four degrees Celsius for 15 minutes. Discard the supernatant and resuspend the pallet in 15 milliliters of transformation solution.Centrifuge at 4000 times gravity and 4 degrees Celsius for 15 minutes. Then, repeat the suspension and centrifugation step once more. Discard the supernatant and resuspend the pallet in 5 milliliters of transformation solution.Let the resuspended pallet rest for two hours at room temperature in dark conditions. Then, either add transformation solution into the OD600 is 0.5 or combine two samples with equal volume for testing paired protein interactions. Using a one milliliter needle of syringe, infiltrate suspended cells into the fourth to seventh leaves.After this, immediately cover the plants with a black plastic cover for 12 hours. Firstly, plants should be very healthy to ensure success. Secondly, make sure not to damage any leaves during the infiltration process.Keep the tray in darkness for 12 hours. Then grow the plants under normal conditions for 2 to 4 days before detecting the luciferase activity. To begin the imaging method, detach an infiltrated leaf.Place the entire leaflet on a plate of MS agar medium. Using a 50 milliliter spray bottle, spray luciferon working buffer onto the leafs adaxial side. Then, keep in the dark for 5 minutes to quench the chlorophyll autofluorescence.Use a lower light cooled CCD imagine apparatus cooled to negative 30 degrees Celsius to capture images with a light filled exposure time of 50 milliseconds, and luminescence detection time of 1 minute. After this, cut out leaf fragments from the infiltration area of the N-benthamiana leaves. Immerse the fragments in 100 microliters of deionized water in the wells of 96-well white plate.Add ten microliters of luciferon working buffer. Let the plate rest 5 minutes. Then, perform any desired specific treatment to study protein interaction dynamics, and measure the luminescence directly with a commercial luminometer.Shown here is the demonstration of luciferous activity to represent of COP 1 SPA 1 interactions. The luciferous activity, which is detected by luminescence, is imaged by CCD camera. COP 1 SPA 1 interactions are seen to result in copper mutation of functional luciferase.The luciferase activity under Jasmine 8 treatment over time is then determined. COP 1 SPA 1 interactions represented by luciferase activity are seen to gradually decline in darkness. Treatment with Jasmine 8 further reduces these interactions.Once mastered, this technic can be done in 1 week, after the plants are ready if it's performed properly. While attempting this procedure, it is important to remember to maintain healthy plants. Infiltrate leaves carefully and include controls properly.After it's development, this technic paves the way for researchers in the field of signal transactions to explore protein protein interaction dynamics in a rapid way. After watching this video, you should have a good understanding of how to grow tobacco plants, infiltrate agrobacterias into tobacco leaves, and to detect luciferous activities.

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Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

13.6K Views.  Nanjing Normal University. The overall goal of this procedure is to quantitatively detect protein-protein interactions in a transient expression system. This measure can help answer key questions in quan-signal transaction fields such as how to quantitatively monitor our protein protein directions after a chemical or environmental treatment. The main advantage of this technic is that it uses a luminometer or a CCD camera to detect the luciferous activity, which represents the protein protein direction intensity so the ...