我们感谢王实验室的成员提供了有益的讨论和意见。这项工作得到了中国国家自然科学基金 (自然科学基金, 31570001 号赠款) 和广东省和广州市自然科学基金 (2016A030313401 和 201707010024) 的支持。

1. 底漆设计策略和 DNA 放大
<ol><li>设计 DNA 片段分子克隆的引物。该引物包括20个 bp 基因特异结合序列和20到 25 bp 5 '-端悬架序列, 这是互补重叠序列的相邻 DNA 分子 (见表 1例)。 注: 每个 DNA 片段的后续组装, 不同蛋白表达盒的连接, 以及与最终表达载体的整合都取决于相邻重叠序列的识别。</li>
	<li>扩增 DNA 片段, 包括启动子、荧光记者、靶基因和终结器, 这些都是用标准 PCR 反应构建半独立蛋白表达盒的必要条件, 并与其相应的引物和高保真聚合酶。 注: 本研究中使用的用于 DNA 扩增的模板是从以前的研究15、22、23中得出的。PCR 反应的退火温度和延长时间为引物和基因依赖性。</li>
</ol>2. DNA 片段组装与蛋白表达盒的构建
<ol><li>通过 DNA 电泳和分光光度法对第一轮 PCR 产品的质量进行检验。检查1% 琼脂糖凝胶电泳是否发生 DNA 降解和污染。PCR 产品的 OD260/OD280 应介于1.6 和1.8 之间。</li>
	<li>将不同的 DNA 片段 (每个片段的 0.05-0.1 pmol) 混合成一个新的 PCR 管, 最终体积为5µL。 注: 混合 DNA 分子设计为同一蛋白表达盒一起在一个 PCR 管。避免将来自不同表达式卡带的 dna 混合在一起, 因为它将降低 dna 组装的效率, 因为 dna 分子的数量越来越多, 需要链接。 注意: 协议可以在这里暂停。</li>
	<li>准备 5x ISO 库存缓冲器: 500 毫米三盐酸, pH 值 7.5;50毫米氯化镁2;1毫米核苷酸 (dNTP);50毫米 dithiothreitol;25% 聚乙二醇 (PEG) 8000;5毫米烟酰胺腺嘌呤二核苷酸 (NAD)。</li>
	<li>使1毫升2x 主混合物从400µL 5x ISO 库存缓冲器, 7.5 单位的 T5 exonuclease, 62.5 单位的高保真 DNA 聚合酶, 5000 单位 Taq 聚合酶 (见材料表), 并灭菌双蒸馏 H2O。 注: 这是从以前的研究24,25,26适应;应优化这些卷。</li>
	<li>整除100µL 的2x 主混合物每管和存储在-20 摄氏度。 注意: 经常冷冻和解冻2x 主混合物会导致低 DNA 组装效率。</li>
	<li>添加15µL 2x 主混合物的5µL DNA 混合物和孵化50°c 60 分钟。</li>
</ol>3. 植物多嵌合体荧光融合蛋白共表达载体的构建
<ol><li>利用 0.5-1.0 µL 的第一轮等温反应作为模板和最外层的底漆 (例如, 1-FP35S 和 1-RNOS, 以第二轮 PCR 的方式放大整个半独立蛋白表达盒卡带 1)。<ol>
<li>使用1单位的高保真聚合酶在50µL 反应体积后30个周期 (94 °c 为三十年代, 55 °c 三十年代和68°c 为2分钟), 其次是最后的延长在68°c 为5分钟。</li>
	</ol>
</li>
	<li>进行线性化最后的蛋白表达主干载体 pUC18 和 pCAMBIA1300, 分别设计为蛋白质瞬时表达和遗传转化, 通过增加4个单位Sma I 到最后10µL 反应量和孵化 1-2小时25摄氏度。在65摄氏度处孵化20分钟, 禁用限制酶。</li>
	<li>将蛋白表达盒和线性化最终载体的摩尔 DNA 分子混合成5µL 的最终反应量。然后, 进行第二轮 DNA 重组, 混合15µL 2x 主缓冲和孵化在50摄氏度60分钟。</li>
	<li>使用第二轮等温重组反应的最终产物, 根据标准程序27转换合格的大肠杆菌细胞 (如DH5α)。通过菌落 PCR 和 DNA 测序对阳性菌落进行双重检查。用微型质粒提取试剂盒从大肠杆菌中提取阳性粒, 并贮存在-80 摄氏度。 注意: 对于长期贮存, 在 TE 缓冲器或双蒸馏 H2O 中溶解的质粒比保持大肠杆菌菌株更稳定。</li>
</ol>4. 枪法-轰击介导的多嵌合体荧光融合蛋白在植物中的瞬态联合表达
<ol><li>准备烟草 BY-2 悬浮细胞和拟南芥幼植物进行轰炸。<ol>
<li>培养 BY-2 细胞在 Murashige 和 Skoog (MS) 培养基由传代每星期两次在25°c 在 130 rpm 设置的振动筛。过滤器收集30毫升 3 d 培养 BY-2 细胞到一块70毫米蒸压过滤纸通过真空泵通过设置真空压力到40毫巴。</li>
		<li>为了防止细胞在以下步骤中干燥, 在放置过滤纸之前, 在培养皿中加入几滴 BY-2 细胞液体培养基 (下一步)。</li>
		<li>将过滤纸与 BY-2 细胞转移到一个新的培养皿 (85 毫米 x 15 毫米)。</li>
		<li>表面消毒拟南芥(Col-0) 种子的涡流混合物与 70% (v/v) 乙醇含有0.05% 吐温20为10分钟。</li>
		<li>用台式离心机以最大速度旋转2秒的种子, 取出上清液, 并在三十年代一次性用100% 乙醇清洗种子. 将种子移出新的无菌滤光纸中。然后, 风干的种子, 并传播到½ MS 琼脂板块。</li>
		<li>将板材在4摄氏度前贮存48小时, 然后将其转移到植物生长室, 其设置如下:16 h 光/8 h 暗与 120-150 µm m-2 s-1强度, 22 °c.
</li>
		<li>将7天的老样本植物转移到一个直径为30毫米的新½ MS 中板中心, 以提高轰击效率。 注意事项: 在转移和放置到新的½ MS 板上时避免重叠植物。</li>
		<li>在植物或组织的表面添加几滴½ MS 液体培养基, 以保持水分, 并防止在以下步骤中干燥植物。</li>
	</ol>
</li>
	<li>用质粒 DNA 涂层金颗粒。<ol>
<li>涡金载体溶液彻底, 3 分钟, 准备一个新的1.5 毫升管。</li>
		<li>按顺序将以下溶液加入管和涡:25 µL (1.5 毫克) 金粒子, 涡旋十年代;10µL 25.46 毫克/升胺, 漩涡十年代;5µL 1µg/µL 质粒 DNA, 涡旋3分钟;25µL 277.5 毫克/升 CaCl2溶液, 涡旋1分钟。 注意: 在此步骤中保持涡流。</li>
		<li>在5秒的最大速度下, 用一台台式离心机将金载体, 仔细地取出上清液, 而不干扰颗粒。用200µL 的绝对乙醇洗净, 并在 5-十年代的漩涡中重新悬浮颗粒. 以最大速度旋转5秒, 然后取出乙醇。</li>
		<li>在18µL 的绝对乙醇和整除6µL 颗粒悬浮在三 macrocarriers 中间, 重新悬浮金粒子。让他们风干。</li>
	</ol>
</li>
	<li>通过粒子轰击将 DNA 转移到植物中。<ol>
<li>将粒子输送系统设置为: 1100 psi、28毫米汞真空、1厘米间隙距离和9厘米粒子飞行距离。</li>
		<li>在三不同位置上轰击琼脂培养基板上的细胞/植物三次, 然后在轰炸后立即将细胞/植物置于黑暗中。 注意事项: 在操作颗粒输送系统时佩戴安全眼镜, 因为高压气体和高速粒子与系统的关联。</li>
	</ol>
</li>
	<li>在观察荧光信号之前, 在植物生长室中保持6到72小时在黑暗中被轰击的细胞/植物。将植物生长室设置为 24 h 暗和22°c。 注意: 表达效率和荧光信号强度均为启动子、基因和植物/组织依赖性。</li>
</ol>5. 通过农杆菌介导的转化, 生成稳定的转基因拟南芥共同表达多嵌合荧光蛋白。
<ol><li>在冰上解冻农杆菌主管细胞 (PMP90), 等待30分钟, 然后将2µL 二进制向量 (100-200 ng) 添加到主管细胞中。把混合物放在冰上坐10分钟。</li>
	<li>将混合物转化为预冷的0.1 厘米电穿孔试管。将试管插入电穿孔系统并进行电穿孔, 其设置如下: 1.6 伏, 600 欧姆, 25 µF。</li>
	<li>在电穿孔后立即在试管中加入1毫升的 SOC 液体培养基, 将细胞注入新的1.5 毫升管, 并在28摄氏度的水平轨道振动筛中孵育200转120分钟。</li>
	<li>离心细胞在 2348 x g 室温5分钟, 丢弃大部分上清, 轻轻地重新悬浮颗粒细胞与吸管尖端, 传播他们在一个含有50毫克/升潮霉素 B 的 LB 板, 并孵化28摄氏度 2-3 天。</li>
	<li>将拟南芥Col-0 植物与农杆菌相结合, 其中含有与多蛋白表达盒集成的二进制向量 pCAMBIA1300, 由花浸28法, 如前所述生成稳定的转基因植物。</li>
	<li>将转基因拟南芥种子的表面与含有0.05% 吐温20的 70% (v/v) 乙醇混合, 杀菌。旋涡为10分钟. 用台式最高离心机在2秒的最大速度下旋下种子, 取出上清, 并在三十年代用100% 乙醇清洗种子。</li>
	<li>将种子移到无菌的滤光片中。此后, 空气干燥并将其传播到含有潮霉素 B 的½ MS 琼脂板上, 以筛选阳性后代。</li>
	<li>在4摄氏度孵化出2天的盘子。然后, 将它们转移到植物生长室和培养7天。</li>
	<li>通过在荧光显微镜下检查荧光信号, 选择7天的老存活幼株, 然后将植物转化为土壤, 进一步筛选纯合植物。</li>
</ol>6. 药物治疗
<ol><li>对于药物治疗, 在细胞或植物孵化前, 将液体 MS 培养基中的每种药物稀释到适当的工作浓度。<ol>
<li>Wortmannin 处理: 通过将 Wortmannin 粉溶于亚砜, 并贮存在-20 摄氏度的库存, 制备出1毫米的 Wortmannin 溶液。将植物细胞或幼株转移到含有16.5 µM wortmammin 的液体 MS 培养基中, 在成像前孵化 30-45 分钟。</li>
		<li>Brefeldin (博鳌): 通过在亚砜中溶出博鳌粉末, 并储存在-20 摄氏度的股票, 准备1毫米的亚洲论坛的库存解决方案。在成像前, 将植物细胞或幼植物转移到含有10µg/mL 论坛的液体 MS 培养基中, 30-45 分钟。</li>
	</ol>
</li>
</ol>7. 共焦显微镜成像和蛋白质亚细胞共定位分析
<ol><li>将幼株或悬浮细胞转移到传统的玻璃滑梯上, 用标准共焦激光扫描显微镜轻轻地将盖子滑动在顶部进行成像。使用以下设置: 63X 水目标 (1.4), 0 背景, 700 增益, 0.168 毫米像素尺寸, 光电倍增管探测器。激发 GFP 标记的蛋白质在485毫微米和检测荧光在 525 nm。对于 RFP 标记的蛋白质, 激发在514毫微米和检测在 575 nm。</li>
	<li>用图像 J 软件 (https://imagej.nih.gov/ij/) 与皮尔逊-长矛相关 (PSC) 的联合本地化插件计算荧光信号的共定位比, 如前所述8。皮尔逊相关系数或长矛的秩相关系数显示在结果中 (图 4)。所生成的 r 值将从-1 到1。0显示两个信号没有可检测的相关性, 而 +1 和-1 分别显示两个信号的完全正和负相关。</li>
</ol>

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作者没有什么可透露的。

在此, 我们展示了一种新的方法, 以有力地共同表达嵌合体荧光融合蛋白的植物。它既可用于瞬态表达和遗传转化, 又能与当前基于荧光蛋白的生物成像、分子、生物化学应用和技术相兼容9、10、13.此外, 它克服了传统方法的困难, 使用几个单独的表达质粒的蛋白质共同表达。相比之下, 它使用一个单一的表达载体, 其中包含多个蛋白表达盒与他们自己的个人启动器, 荧光标签, 靶蛋白, 和终结者。此外, 蛋白表达盒可以独立管理, 以满足不同的表达要求, 如使用特定的启动子和 n-或 C 终端嵌合体融合的荧光蛋白与靶蛋白。因此, 蛋白表达盒的功能就像一个基本的 "乐高" 元素, 在质粒中半独立工作。此外, 它也是一个高度多才多艺的系统, 其中基因编辑, 更换和组装都可以很容易地实现一步等温重组反应, 没有额外的酶消化和结扎过程。通过测试不同浓度的 T5 exonuclease、Phusion DNA 聚合酶和 Taq 聚合酶, 我们优化了先前研究的等温重组反应的效率, 如步骤2.4 所述。此外, 建议每一步等温重组反应的 DNA 片段浓度为0.05 至 0.1 pmol, 以达到最大结扎效率。
转基因的过度表达通过取代其内源启动子与强大和连续的启动子, 如 ubiquitin-10 启动子 (UBQ10), 并引入更多的基因副本是一个广泛使用的方法来研究其细胞功能和单元格15、32中的底层工作机制。然而, 意外下降调节和强烈抑制的基因表达有时被发现, 以及33,34。在这些情况下, 不可预知的基因沉默率为2到 100%, 在这些情况下33,35。同时, 基因沉默在多个基因的表达上有较高的发生几率, DNA 高拷贝的转化, 基因转录水平的显著增加9,33,24 ,35。为了最大限度地减少基因沉默在鲁棒多融合蛋白共表达系统中的发生, 我们选择了不同的主动启动子来驱动不同蛋白表达盒, 同时表达多种融合蛋白。此外, 我们避免了不断使用相同的启动器不同的表达式磁带。此外, 这个协议的另一个潜在的限制是降低效率的一步 DNA 等温重组造成的数量越来越多的蛋白表达盒的共同表达。此外, 在最终表达质粒中可以承载的表达盒的数量主要依赖于主干质粒24、25、35的传代。
结合起来, 我们开发了一个强大的系统, 以共同表达多嵌合体荧光融合蛋白方便地在植物36。它克服了经典方法的局限性, 并以一种悠久的方式对 dna 互连和质粒结构进行了优化的一步 dna 组装反应。通过 AtVSR2 和 AtSCAMP4 在植物细胞中的表达,通过瞬时表达和遗传转化, 验证了该技术的进步。因此, 通过对植物中嵌合体荧光融合蛋白的联合表达, 证明了科学发现的不同方面具有令人信服和新颖的方法。此外, 通过单表达载体对多嵌合体荧光融合蛋白共表达的概念和原理, 完全符合 CRISPR-Cas9、rna 干扰和蛋白质超表达技术, 研究其功能和多基因在植物中的相互作用37,38,39。

嵌合荧光融合蛋白被认为是研究细胞内动力学和亚型定位的有用工具, 进一步了解其生理功能和工作机制1,2,3,4. 与所讨论的蛋白质共同表达知名的细胞器报告蛋白, 以更好地说明其在单元格中膜系统中的时空原理、分布和功能, 尤其有益.4,5,6,7,8。
通过瞬时表达和稳定的遗传转化, 可以在植物中表达嵌合体荧光融合蛋白, 其各自的优点和局限性分别为9、10、11。蛋白质的瞬时表达是一种方便的方法, 包括枪法轰击-, 聚乙二醇 (PEG)-, 或电穿孔介导的 DNA 瞬时表达在原生质体和农杆菌介导的叶浸润完整的植物细胞, 如图 1A所示,B12,13,14,15,16。然而, 多嵌合体荧光融合蛋白在单个植物细胞中的联合表达需要多种独立表达质粒的混合物。因此, 在植物中使用多种质粒进行蛋白质共表达的缺点是, 由于几种粒度的几率大大降低, 同时进入同一细胞, 而与单粒,每种质粒的无控制随机量导致的蛋白质表达水平的变化转移到细胞17,18。此外, 在技术上有挑战性的是引入几个独立的表达质粒成一个单一的农杆菌蛋白共表达9,10,11。因此,农杆菌介导的烟叶浸润的蛋白质瞬时表达只能一次表达一种质粒, 如图 1B所示。相反, 产生荧光融合蛋白的转基因植物通常是由带有二进制转化载体的农杆菌实现的。然而, 将基因转移和插入到植物基因组中的二进制载体只能表达单一的荧光融合蛋白 (图 1B)9、10、12。生成一种能同时表达几种嵌合荧光蛋白的转基因植物需要多轮的基因交叉和筛查, 这可能需要数月到数年, 这取决于要共同表达的基因的数量。
就业一个单一表达载体的多种蛋白质的共同表达在植物已经报告了几个以前的研究19,20,21。然而, 在蛋白质共表达或过度表达的最终质粒的生成中, 通常需要对 dna 分子和主干载体进行多轮酶消化和 dna 结扎。在这里, 我们开发了一种新的和稳健的方法来共同表达多嵌合体荧光蛋白在植物中。它是一种高效、方便的方法, 在植物中实现了多种蛋白质的共同表达, 既具有瞬态表达, 又能稳定地转化为一种时间的方式。它使用一个单一的载体, 包含多个功能独立的蛋白表达盒, 为蛋白质的共同表达, 从而克服了传统方法的弊端。此外, 它是一个高度多才多艺的系统, 其中 dna 操作和组装是通过简单的一步优化反应, 没有额外的步骤 dna 消化和结扎。工作原理如图 2所示。此外, 它完全兼容目前的细胞, 分子和生物化学的方法, 基于嵌合体荧光融合蛋白。

<table>
	<tbody>
		<tr>
			<td>KOD-FX Polymerase</td>
			<td>TOYOBO</td>
			<td>KFX-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sma I</td>
			<td>NEB</td>
			<td>R0141L/S/V</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>BBI</td>
			<td>A600194-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>BBI</td>
			<td>A601336-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>NEB</td>
			<td>#N0447V</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>BBI</td>
			<td>C4H10O2S2</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>BBI</td>
			<td>A100159-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>BBI</td>
			<td>A600641-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>T5 exonuclease</td>
			<td>Epicentre</td>
			<td>T5E4111K</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>NEB</td>
			<td>B9022S</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture(MS)</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>BBI</td>
			<td>A500737-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI</td>
			<td>A600560-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BBI</td>
			<td>A505255-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>BBI</td>
			<td>A614270-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold microcarrier particles</td>
			<td>Bio-Rad</td>
			<td>165-2263</td>
			<td>1.0 &#181;m</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>BBI</td>
			<td>CD0050-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrocarriers</td>
			<td>Bio-Rad</td>
			<td>165-2335</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture disk</td>
			<td>Bio-Rad</td>
			<td>165-2329</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopping screen</td>
			<td>Bio-Rad</td>
			<td>165-2336</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BBI</td>
			<td>A610476-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>BBI</td>
			<td>A610440-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>BBI</td>
			<td>A600219-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Genview</td>
			<td>AH169-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wortmannin</td>
			<td>Sigma</td>
			<td>F9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>SML0975-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulphoxide (DMSO)</td>
			<td>BBI</td>
			<td>A600163-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Panasonic</td>
			<td>MLR-352H-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>PSD-1000/He particle delivery system</td>
			<td>Bio-Rad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Bio-Rad</td>
			<td>1652083</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5427000097</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 7 DUO (780&#38;7Live)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>EPS-300 Power Supply</td>
			<td>Tanon</td>
			<td>EPS 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Mshot</td>
			<td>MF30</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrose</td>
			<td>BBI</td>
			<td>A600234</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>BBI</td>
			<td>A100339</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetie Acid</td>
			<td>BBI</td>
			<td>B300599</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants

关于蛋白质的时空亚细胞定位的信息对于了解细胞的生理功能至关重要。荧光蛋白和荧光融合蛋白的生成已被广泛地应用于直接可视化细胞内蛋白质定位和动力学的有效工具。这是特别有用的比较, 他们与知名的细胞器标记后, 共同表达与蛋白质的兴趣。然而, 植物中蛋白质共表达的经典方法通常涉及多个独立的表达质粒, 因此有缺点, 包括低的协同表达效率, 表达水平的变化, 和高的时间基因交叉和筛查的支出。在本研究中, 我们描述了一种健壮和新颖的方法, 共同表达的多嵌合体荧光蛋白在植物。它克服了传统方法的局限性, 采用了由多个半独立表达式盒组成的单表达载体。每个蛋白表达盒都含有其自身的功能蛋白表达元素, 因此可以灵活调整以满足不同的表达需求。同时, 通过优化一步反应, 无需额外的消化和结扎步骤, 在表达质粒中进行 DNA 片段的组装和操作是很容易和方便的。此外, 它完全兼容目前的荧光蛋白衍生生物成像技术和应用, 如烦恼和 BiFC。作为对该方法的验证, 我们采用了这种新的系统来共同表达荧光融合泡排序受体和分泌载体膜蛋白。结果表明, 它们的透视亚细胞定位与以往的研究一样, 都是由植物的瞬时表达和遗传转化引起的。

建立了一种健壮高效的多嵌合体荧光融合蛋白在植物中的联合表达方法。它突破了传统方法的障碍使用多种分离质粒进行蛋白质共表达, 如图 1AB所示,通过瞬时表达或稳定的遗传转化。在这一新的方法中, 我们生成一个单一的表达载体, 由多个蛋白表达盒组成, 以实现蛋白质共表达一次 (图 1C, D)。蛋白质表达盒功能半独立与它自己必要的脱氧核糖核酸元素为蛋白质表达。因此, 每个蛋白表达盒可以根据不同的蛋白质表达要求独立定制。对于最终的蛋白质共表达载体, 它的蛋白质表达盒作为基本的 "乐高" 元素, 可以修改, 重新构建。和重新放置方便。此外, 还简单地实现了 dna 分子组装、几种蛋白表达盒的连接以及 dna 片段与最终目标向量的结合, 以实现嵌合体荧光融合蛋白的联合表达的方法。通过优化的等温体外重组反应没有进一步的额外步骤 DNA 消化和互连。图 2说明了等温体外重组反应的工作原理。多 dna 分子的联系 (例如, 图 2所示的三个代表性 DNA 片段 1-3) 和它们与最终表达载体的融合是通过一步反应简单有效地实现的 (见图2).它是根据先前的研究, 通过重叠重组 dna 分子介导的重叠短序列, 以实现融合的 dna 片段和建设的质粒25,26。
作为试验方法, 我们选择了泡排序受体 (VSR) 和分泌载体膜蛋白 (流氓), 这是两个记者蛋白参与蛋白分泌和吞途径, 分别为6,22 ,23,29,30。VSRs 是 I 型整体膜蛋白, 介导分泌通路的生物合成蛋白流量到液泡, 主要本地化 prevacuolar 室 (PVCs) 在植物6,22,23。与此相反, 分泌载体膜蛋白 (流氓) 是参与植物吞通路的 IV. 型膜蛋白。它本地化的等离子膜 (PM) 和反式高尔基网络 (TGNs), 作为早期内涵体22,29,30。我们构建了两个蛋白表达盒, 宿主拟南芥VSR2 (AtVSR2) 嵌合体融合的 RFP 和拟南芥SCAMP4 (AtSCAMP4) 与 GFP, 如图 3所示。为了确保 RFP-AtVSR2 可以转化为 ER, 信号肽 (SP) 添加到 RFP 之前, 如前所报告的6,31。两个单独的蛋白表达盒是进一步相互联系和结扎的最终蛋白表达载体 pUC18 或 pCAMBIA1300 蛋白质的共同表达, 无论是通过蛋白质瞬态或植物稳定转化, 如所示图 3。AtVSR2 和 AtSCAMP4通过粒子轰击成功地在烟草 BY-2 细胞中共同表达, 并显示出正确的定位 (图 4A)。RFP-AtVSR2 显示了一个点状模式, 这与 AtSCAMP4-GFP 的等离子膜定位和一些胞浆点状斑点不同。此外,通过农杆菌介导的转化, AtSCAMP4-GFP 和 RFP-AtVSR2 共同表达的拟南芥转基因植株。图 4B 和 4E显示了 RFP-AtVSR2 和 AtSCAMP4-GFP 在根和根毛细胞中表达的亚细胞定位。转基因拟南芥RFP-AtVSR2 和 AtSCAMP4-GFP 的共表达结果与 BY-2 细胞一致。此外, 对转基因拟南芥进行了 wortmannin 和博鳌30分钟的治疗, Wortmmanin 导致 RFP-AtVSR2 标记 PVCs 形成一个小的环状结构, 和博鳌诱导 AtSCAMP-GFP 标记 TGN 聚合, 如图4C 所示. , D, F, G。此外, 在烟草 BY-2 细胞和拟南芥根和根毛细胞中可以检测到小 autofluorescent 信号, 方法是应用与图 4相同的图像收集设置 (补充图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57354/57354fig1.jpg"> 图 1: 一种强健的系统, 用于植物中多种嵌合荧光融合蛋白的联合表达。(A) 通过电穿孔、粒子轰击和 PEG 介导的转化, 通过混合几种独立的表达来实现植物中多个荧光报告蛋白瞬态共表达的常规方法向量。(B) 农杆菌利用单一荧光融合蛋白表达载体进行植物遗传转化的常规方法。为了在转基因植物中共同表达多种嵌合荧光融合蛋白, 需要进一步的遗传杂交和多轮筛查才能获得纯合后代。(C)和(D)替代性新的蛋白质联合表达方法。它利用单一表达载体, 它由多种蛋白表达盒组成, 能够通过瞬时表达和遗传转化, 在植物中共同表达几种嵌合荧光报告蛋白。这一数字的细节首先发表在中等。201736 (转载许可; 植物科学中的版权前沿)。<a href="/files/ftp_upload/57354/57354fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57354/57354fig2.jpg"> 图 2: 用等温复合反应法演示一步 DNA 组装方法的工作原理.在 5 '-悬重叠区之间, 以 dna 聚合酶为载体, 通过连接酶, 将 dna 片段和线性化质粒与重叠序列 (OSs) 连接在一起。<a href="/files/ftp_upload/57354/57354fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57354/57354fig3.jpg"> 图 3: 构建用于共表达多嵌合体荧光融合蛋白的单一质粒的策略.该图展示了构建单个表达质粒的策略, 用于在植物中进行瞬时表达或稳定转化的多嵌合体荧光融合蛋白的共表达。该表达载体由两个蛋白表达盒组成, 每一个都含有自己的蛋白质表达所必需的元素, 并在表达其单独嵌合荧光融合蛋白半独立的功能。通过与重叠 dna 片段进行介导的优化等温体外重组方法, 可方便地实现所有 dna 分子的组装和互连。这一数字的细节首先发表在中等。201736 (转载许可; 植物科学中的版权前沿)。<a href="/files/ftp_upload/57354/57354fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57354/57354fig4.jpg"> 图 4: 植物细胞中 VSR 和流氓嵌合体荧光融合的代表性图像。 (A)在烟草 BY-2 悬浮细胞中通过微粒轰击 RFP-AtVSR2 和 AtSCAMP4-GFP 的共同表达。(B) RFP-AtVSR2 和 AtSCAMP4-GFP 的转基因拟南芥根细胞的代表性图像。( C)和(D)转基因拟南芥根共表达 RFP-AtVSR2 和 AtSCAMP4-GFP 分别以 wortmannin 和博鳌为30分钟。(E)转基因拟南芥根发 RFP-AtVSR2 和 AtSCAMP4-GFP 的代表性图像。( F)和(g ) 转基因拟南芥根毛共表达 RFP-AtVSR2 和 AtSCAMP4-GFP, 以 wortmannin 和博鳌为30分钟. (D)和(G)箭头表示的箭聚集。rp = 皮尔逊相关系数;rs = 长矛的秩相关。刻度条在(A)-(D)是50µm, (E)-(G)是30µm. 这一数字的详细信息首次发表在中等。201736 (转载许可; 植物科学中的版权前沿)。<a href="/files/ftp_upload/57354/57354fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="3">表达式卡带1</td>
		</tr>
<tr>
<td>1-FP35S</td>
			<td colspan="2">
GAATTCGAGCTCGGTACCCACATGGTGGAGCACGACACA</td>
		</tr>
<tr>
<td>1-RP35S</td>
			<td colspan="2">
AGGACGCGGGCGTGGGCCATTATCACATCAATCCACTTGC</td>
		</tr>
<tr>
<td>1-FRFP</td>
			<td colspan="2">
GCAAGTGGATTGATGTGATAATGGCCCACGCCCGCGTCCT</td>
		</tr>
<tr>
<td>1-RRFP</td>
			<td colspan="2">
ATTATCCATATCACTCCCCAGGCGCCGGTGGAGTGGCGGC</td>
		</tr>
<tr>
<td>1-FAtVSR2</td>
			<td colspan="2">
GCCGCCACTCCACCGGCGCCTGGGGAGTGATATGGATAAT</td>
		</tr>
<tr>
<td>1-RAtVSR2</td>
			<td colspan="2">
AAATGTTTGAACGATCGGGATTACAACTCTAGTTGAGAAG</td>
		</tr>
<tr>
<td>1-FNOS</td>
			<td colspan="2">
CTTCTCAACTAGAGTTGTAATCCCGATCGTTCAAACATTT</td>
		</tr>
<tr>
<td>1-RNOS</td>
			<td colspan="2">
GAGAATGGATGCGAGTAATGTCTAGTAACATAGATGACAC</td>
		</tr>
<tr>
<td colspan="3">表达式卡带2</td>
		</tr>
<tr>
<td>2-FP35S</td>
			<td colspan="2">
CATTACTCGCATCCATTCTCACATGGTGGAGCACGACACA</td>
		</tr>
<tr>
<td>2-RP35S</td>
			<td colspan="2">
TTAGGATCGTGTCGTGCCATTATCACATCAATCCACTTGC</td>
		</tr>
<tr>
<td>2-FAtSCAMP4</td>
			<td colspan="2">
GCAAGTGGATTGATGTGATAATGGCACGACACGATCCTAA</td>
		</tr>
<tr>
<td>2-RAtSCAMP4</td>
			<td colspan="2">
TCCTCGCCCTTGCTCACCATTAGTGCACGCATCAAGGTCG</td>
		</tr>
<tr>
<td>2-FGFP</td>
			<td colspan="2">
CGACCTTGATGCGTGCACTAATGGTGAGCAAGGGCGAGGA</td>
		</tr>
<tr>
<td>2-RGFP</td>
			<td colspan="2">
TAAAACCAAAATCCAGTGACTTACTTGTACAGCTCGTCCA</td>
		</tr>
<tr>
<td>2-FT35S</td>
			<td colspan="2">
TGGACGAGCTGTACAAGTAAGTCACTGGATTTTGGTTTTA</td>
		</tr>
<tr>
<td>2-RT35S</td>
			<td colspan="2">
GTCGACTCTAGAGGATCCCCGTCCGCAAAAATCACCAGTC</td>
		</tr>
</tbody></table>表 1: 本研究中使用的底漆设计策略和序列.每个底漆的名称都在左面板上给出。引物的互补重叠序列下划线。本表的详细资料首次发表在中等。201736 (转载许可; 植物科学中的版权前沿)。
补充图 1: BY-2 细胞和拟南芥根和根毛中自发荧光的代表性图像。(A)在与转化细胞相同的成像条件下检测出野生型 BY-2 细胞的自体荧光。( B)和(C)在相同的成像条件下, 用转化的拟南芥细胞检测拟南芥根毛和根细胞。刻度条在(A)-(C)是20µm, 差异干扰对比度。<a href="/files/ftp_upload/57354/11.Supplemental_Figure_1.tif">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

我们开发了一种新的方法来共同表达多嵌合体荧光融合蛋白在植物中克服传统方法的困难。它利用单一表达质粒, 包含多功能独立蛋白表达盒, 以实现蛋白质的共同表达。

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

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Nanyang Technological University

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JoVE Journal

Chemistry

9.6K 次观看.  South China Agricultural University. 我们开发了一种新的方法来共同表达多嵌合体荧光融合蛋白在植物中克服传统方法的困难。它利用单一表达质粒, 包含多功能独立蛋白表达盒, 以实现蛋白质的共同表达。

视频: Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

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Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

通过RAFT控制聚合轻便和高效制备三组分荧光Glycopolymers

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

点击化学可视化植物木动力学: 双标记是幸福!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...