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?...

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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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 &amp;micro;m</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;amp;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)。?...

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 ...

co-expression-multiple-chimeric-fluorescent-fusion-proteins-an

Research

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

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

遗传学

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

免疫与感染

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

生物学

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...