Name: peptide-derived method to transport genes and proteins across cellular and organellar barriers in plants
Uploaded: 2016-12-16T14:00:00.000+00:00
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作者要感谢来自日本科学技术振兴机构的探索性研究的先进技术（JST-ERATO）中，新能源产业技术综合开发机构（NEDO）和跨部级战略创新促进计划（SIP），日本的资金。

1.基于肽制剂的制备<ol><li>制备的每种肽的储备溶液如下:（KH）9 -BP100（1毫克/毫升或800纳米），Cytcox-（KH）9（1毫克/毫升），BP100（1毫克/毫升）和（BP100）2 K 8（70微米）。称重所需的每种肽的量在1.5ml微量离心管中，并添加蒸压超纯水以溶解肽。直到得到澄清的溶液反复吹打拌匀。 </li><li>扩增并纯化质粒DNA（质粒DNA），双链DNA（dsDNA）的和根据标准分子方法的双链RNA（dsRNA）的。在1.5ml微量离心管，使储备溶液具有1毫克/毫升（质粒DNA和双链DNA）或400nM的（dsRNA）的浓度。 </li><li>制备蛋白原液以7μm的浓度，以1 mg蛋白质（ 例如 ，醇脱氢酶，ADH）粉末溶解在1毫升碳酸钠溶液（0.1M，pH为9）。标签使用f蛋白质根据标准方法luorescent探针如若丹明B（罗丹明B），以使递送到细胞中的蛋白质的微观可视化。 </li><li>结合在1.5ml微量离心管中的各个部件。 <ol><li>对于肽的pDNA制剂靶向细胞质中，添加6.4微升（KH）9 -BP100（1毫克/毫升），以20微升的pDNA（1毫克/毫升），并通过移液混合均匀。使混合物在25℃下稳定15分钟。添加773.6微升灭菌超纯水稀释至800微升的最终体积。使用质粒DNA构建体设计用于核表达（P35S-与Rluc-TNOS， 表1）。 </li><li>对于肽的pDNA制剂靶向线粒体，添加6.6微升Cytcox-的（KH）9（1毫克/毫升），以20微升的pDNA（1毫克/毫升），并通过移液混合均匀。使混合物在25℃下稳定15分钟，然后添加BP100 2.4微升（1毫克/毫升）。使混合物在25稳定15分钟6，下再15分钟。添加771微升灭菌超纯水稀释至800微升的最终体积。使用质粒DNA构建体设计用于线粒体表达（PDONR-COX2:与Rluc， 表1）。 </li><li>对于肽双链DNA的配方，添加5.1微升（KH）9 -BP100（1毫克/毫升），以8微升双链DNA（1毫克/毫升），并通过移液拌匀。使混合物在25℃下稳定15分钟。添加786.9微升灭菌超纯水稀释至800微升的最终体积。 </li><li>对于肽dsRNA的配方，加入50微升（KH）9 -BP100（800纳米）至50μl双链RNA（400纳米）和吹打拌匀。使混合物在25℃下稳定15分钟。加入700微升的无RNase水稀释该溶液到800微升的最终体积。 </li><li>对于肽蛋白制剂，加入16微升（BP100）2 K 8（70微米）到ADH的16微升（7微米），混合深受移液。使混合物在25℃下稳定15分钟。添加768微升灭菌超纯水稀释至800微升的最终体积。 </li></ol></li><li>允许制剂在25℃下稳定15分钟。 </li></ol> 2.基于肽制剂的表征<ol><li>每个转移溶液（800微升）转化为动态光散射（DLS）分析比色皿。确定形成的复合物的流体动力学直径和多分散性指数用的ζNanosizer测，使用633纳米的氦氖激光器，在25℃下的173°的反向散射检测角度。 </li><li>以下大小的测量，将各溶液（800微升）在介电常数，折射率和水粘度的默认参数，在25℃转移到折叠毛细管单元用于zeta电位测量。 </li><li>观察小体积络合物溶液，用于DLS分析，通过原子力显微镜（AFM）。存10微升络合物溶液到云母片的新暴露的切割表面和离开该云母，以空气中的有盖的塑料培养皿干燥过夜。 </li><li>在轻敲模式27,28使用硅悬臂具有1.3牛顿/米的弹簧常数在25℃获得在空气中的复合物的图像。 </li></ol> 3.植物叶片渗透<ol><li>用3周龄的土壤中生长的植物（ 拟南芥 24， 本生烟 24或26杨树）。转染至少3片叶，作为一式三份的基因表达或蛋白质输送的量化。 </li><li>装载1毫升的无针注射器，塑料用100微升一个叶转复杂的解决方案。叶子的背面表面上的注射器的尖端的位置。 </li><li>按压叶注射器尖端稍慢慢压低注射器活塞，而施加反压的我胶乳手套的手的手指ndex从相对侧。成功的浸润，可以观察到在叶的水浸泡部的扩散。为便于识别标签的渗透叶子。 </li><li>孵育优化持续时间的转叶以下用肽的pDNA（12小时），肽的双链DNA（12小时），肽的dsRNA浸润 - 在下述条件下（9 48小时）和肽 - 蛋白（6小时）的配方:16小时光/在29℃，22℃， 拟南芥和杨树，或24小时恒亮的烟草本塞姆氏 8小时黑暗。 </li></ol> 4.评价转染效率<ol><li>切除小型工厂的整体转叶（ 例如 ， 拟南芥 ）或渗透区域的大型水泥厂1厘米2部分（ 例如 ， 烟草本塞姆氏 ）。 </li><li>对于使用Renilla荧光素酶（与Rluc）报告载体转染实验，确定transfectio氮效率定量使用它们间检测试剂盒。 <ol><li>放置在1.5ml微量离心管各切叶或叶节。加入100微升1×它们间测定裂解液每管。以相同的方式，制备非转染的对照叶（一式三份）的裂解物。 </li><li>磨用均质杵的叶和孵育在25℃下将所得溶胞产物为6 - 10小时。 </li><li>离心在12470×g下的裂解物中为1分钟的离心。转移20微升澄清的裂解液的一个井在96孔微量培养板，以及用于使用根据制造商的协议的BCA蛋白质测定试剂盒总蛋白质浓度的定量剩余体积。 </li><li>加入100微升1×它们间检测底物（使用它们间试验缓冲液）的入井，并缓慢吹打混合。放置在一个多模酶标仪微孔板并启动测量。 </li><li>减去背景发光（指的是没有正转从每个实验样品的发光式三份）。计算的光致发光，以蛋白（毫克）的量的比率（相对光单位，RLU）。 </li></ol></li><li>对于使用绿色荧光蛋白（GFP）的报告载体或荧光标记的蛋白质的转染实验（ 例如 ，ADH-罗丹明B），使用共聚焦激光扫描显微镜观察荧光。 <ol><li>切全叶（以有助于除去空气空间）的边缘，而可使用一个切片叶原样。从10毫升注射器卸下柱塞，并放置在注射器的每个切叶或叶节。 </li><li>更换柱塞，轻轻推到注射器的底部，但不会破碎的叶子。打水入注射器，直到它被大约一半填充。 </li><li>指向上的注射器和柱塞推到从通过尖端注射器除去空气。覆盖注射器的尖端并拔出柱塞向下慢慢从LEA排出空气F。重复此过程数次，直到叶出现半透明。 </li><li>覆盖胶带显微镜载玻片的表面上。切割胶带大到足以容纳使用刀片叶样品上的正方形区域，和剥离的正方形片胶带的关闭用镊子以创建一个样品室。磁带将作为幻灯片和盖玻片之间的间隔物。 </li><li>放置叶朝上的背面室和装满水的剩余室面积。密封用玻璃盖玻片的腔室中的叶片和用胶带固定盖玻片的边缘。 </li><li>检查使用下一个40倍物镜的共聚焦激光扫描显微镜或63X水浸物镜的叶样品。 GFP或罗丹明荧光可以在分别488纳米或555纳米，激发波长可视化。 </li></ol></li></ol>

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S., Serino, G., Chaudhuri, S., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+transformation.">Plastid transformation.</a> Arabidopsis thaliana Plant Cell Rep. 18, 20-24 (1998).</li><li>Svab, Z., Hajdukiewicz, P., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+plastids+in+higher+plants.">Stable transformation of plastids in higher plants.</a> Proc. Natl. Acad. Sci. U. S. A. 87, 8526-8530 (1990).</li><li>Liu, Y. G., 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=Complementation+of+plant+mutants+with+large+genomic+DNA+fragments+by+a+transformation-competent+artificial+chromosome+vector+accelerates+positional+cloning.">Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 6535-6540 (1999).</li><li>Park, H. S., Lee, B. M., Salas, G. M., Srivatanakul, M., Smith, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shorter+T-DNA+or+additional+virulence+genes+improve+Agrobactrium-mediated+transformation.">Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation.</a> Theor. Appl. Genet. 101, 1015-1020 (2000).</li><li>Frary, A., Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+and+stability+of+high+molecular+weight+DNA+transformation:+an+analysis+in+tomato.">Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.</a> Transgenic Res. 10, 121-132 (2001).</li><li>Que, Q., 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=Maize+transformation+technology+development+for+commercial+event+generation.">Maize transformation technology development for commercial event generation.</a> Front. Plant Sci. 5, 379 (2014).</li><li>Miranda, A., Janssen, G., Hodges, L., Peralta, E. G., Ream, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+transfers+extremely+long+T-DNAs+by+a+unidirectional+mechanism.">Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.</a> J. Bacteriol. 174, 2288-2297 (1992).</li><li>Loeb, T. A., Spring, L. M., Steck, T. R., Reynolds, T. L., Bajaj, Y. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+wheat+(Triticum+spp.).">Transgenic wheat (Triticum spp.).</a> Transgenic Crops I. , 14-36 (2000).</li></ol>

作者什么都没有透露。

已经凭经验确定的协议中的关键步骤进行了讨论。通过注射器渗入，基于肽的制剂引入到通过气孔植物叶内的空域。以确保溶液的最大吸收，渗透过程应进行时的植物有利于气孔开口即条件下，提供足够的水，并在光周期下。至于转染复合物的制备，对涉及两个肽（线粒体靶向DNA递送）的组合制剂，用于添加各成分的顺序是至关重要的，不应该被反转。 有许多可行的修改的过程。对于植物细胞的浸润另一种选择是使用真空浸润，这是能够在复杂的溶液引入整个植株和/或部分组织，包括顶端分生组织。对于此替代过程，苗浸没在转染溶液，并基于由真空产生的压力，所述肽 - 货物复合物通过气孔和进植物细胞（吉住，T。，未发表的数据）被迫。 瞬时基因表达的基础上，使用动物细胞的研究中，已被证明能增加与更高的DNA浓度29-31。要注意的是肽的DNA的比例会影响复合物的生物物理性质（大小，表面电荷）是非常重要的（ 图4），从而影响转染效率;因此，最佳的比率需要甚至具有增加的DNA浓度被维持。 之一的在使用肽作为基因/蛋白输送剂中的主要优点是，它的顺序是适合于调谐到满足所希望的功能。例如，所述载体肽的线粒体靶向域在该螺柱描述y可能与本地化这些细胞器叶绿体或过氧化物酶体靶向序列所取代。而叶绿体转化使用基因枪法32-34在几种植物是可能的，既不是农杆菌也不是生物射弹方法能够将基因导入线粒体或其它细胞器除了核（ 表2）。 有用于基于肽的转染没有刚性宿主范围限制，不像农杆菌基于方法（ 表2）。到目前为止，对于拟南芥 ， 烟草本塞姆氏和杨树的转染制剂已被优化（ 表1），但也可以适用于烟草的方法中， 番茄品种微汤姆，稻（基于预备实验），和其它的单 - 和双子叶植物。 迄今为止，有在转基因大小没有已知的限制时肽用于一小号转染载体。与此相反，已发现大量的DNA分子，以减少的土壤杆菌介导的方法35,36的转化效率，以及用于已报道37-39的约200kb的转基因的大小上限。使用基因枪，在另一方面，大的DNA片段可以制备或输送到植物中38中剪切。虽然没有上限被确定为生物射弹转化，到目前为止，已发现物理约束来限制可被转移到远小于150 kb的40 DNA的大小。使用相比于采用任何农杆菌或微粒轰击现有方法，以上所讨论的用于基因转移的基于肽的系统的主要优点，归纳在表2中 。 一些限制这种方法存在，因为它主张。首先，有针对性的pDNA的递送至特定的细胞器，如MITochondria，虽然效率较低已被证实可以通过DNA结合，细胞穿透和细胞器转运序列的简单组合。转基因表达可以被检测到，通过共聚焦显微术，仅在细胞内的线粒体的一个小口。因此，进一步的改进是必要的:（ⅰ）提高整个小区/细胞器膜更配合物的易位，以及（ii）提高的解离和从载体肽的pDNA转移到靶细胞器中表达。其次，使用该DNA递送系统中，外源报道基因的瞬时表达，成功在细胞的胞质和线粒体隔室来实现的。在植物核/细胞器基因组中导入的基因的稳定掺入和表达都尚未建立，但是，由于缺乏合适的选择策略。 虽然承认有改善或再经过D区才有发展，这里所描述的肽衍生战略仍然是一个简单而通用的技术已经铺平了交付各种货物到不同植物类型的方式。

Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant's own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.

<table><tbody><tr><td>(KH)&lt;sub&gt;9&lt;/sub&gt;-BP100 peptide</td><td>Custom-synthesized by RIKEN Brain Science Institute</td><td>N/A</td><td>Sequence: KHKHKHKHKHKHKHKH-&lt;br/&gt; KHKKLFKKILKYL</td></tr><tr><td>(BP100)&lt;sub&gt;2&lt;/sub&gt;-K&lt;sub&gt;8&lt;/sub&gt; peptide</td><td>Custom-synthesized by RIKEN Brain Science Institute</td><td>N/A</td><td>Sequence: KKLFKKILKYLKKLFKKIL-&lt;br/&gt; KYLKKKKKKKK</td></tr><tr><td>BP100 peptide</td><td>Custom-synthesized by RIKEN Brain Science Institute</td><td>N/A</td><td>Sequence: KKLFKKILKYL</td></tr><tr><td>Cytcox-(KH)&lt;sub&gt;9&lt;/sub&gt; peptide</td><td>Custom-synthesized by RIKEN Brain Science Institute</td><td>N/A</td><td>Sequence: MLSLRQSIRFFKKHKHKH-&lt;br/&gt; KHKHKHKHKHKH</td></tr><tr><td>P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS</td><td>N/A</td><td>N/A</td><td>Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan &lt;em&gt;et al.&lt;/em&gt; Biomacromolecules 2013, 14, 10)</td></tr><tr><td>pDONR-cox2:gfp and pDONR-cox2:rluc</td><td>N/A</td><td>N/A</td><td>Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah &lt;em&gt;et al.&lt;/em&gt; Sci Rep 2015, 5, 7751)</td></tr><tr><td>dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS)</td><td>N/A</td><td>N/A</td><td>Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan &lt;em&gt;et al.&lt;/em&gt; Plant Biotechnol 2015, 32, 39)</td></tr><tr><td>GFP5 siRNA</td><td>N/A</td><td>N/A</td><td>For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata &lt;em&gt;et al.&lt;/em&gt; Plant Biotechnol J 2014, 12, 1027)</td></tr><tr><td>CHS siRNA</td><td>N/A</td><td>N/A</td><td>For RNA interference-mediated silencing of chalcone synthase (Ref: Numata &lt;em&gt;et al&lt;/em&gt; Plant Biotechnol J 2014, 12, 1027)</td></tr><tr><td>1 ml and 10 ml Plastic Syringes</td><td>TERUMO Corporation</td><td>SS-01T, SS-10ESZ</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tube</td><td>AS ONE Corporation</td><td>151212</td><td></td></tr><tr><td>96-Well Flat-Bottom Plate</td><td>Asahi Glass Co., Ltd.</td><td>3860-096</td><td></td></tr><tr><td>Adhesive Tape</td><td>Sekisui Chemical Co., Ltd.</td><td>No. 835</td><td></td></tr><tr><td>Alcohol Dehydrogenase</td><td>Sigma-Aldrich Co., LLC.</td><td>A-7011</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>Seiko Instruments Inc.</td><td>SPI3800, SPA 300HV</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>Hitachi High-Tech Science Corporation</td><td>AFM5300E</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific Inc.</td><td>23227</td><td></td></tr><tr><td>Cantilever</td><td>Hitachi High-Tech Science Corporation</td><td>K-A102001593</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Carl Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Cork Borer</td><td>Sigma-Aldrich Co., LLC.</td><td>Z165220</td><td>For excision of leaves into 1-cm diameter disks</td></tr><tr><td>Coverslip</td><td>Matsunami Glass Ind., Ltd.</td><td>C02261</td><td></td></tr><tr><td>Cuvette</td><td>Sarstedt</td><td>759116</td><td></td></tr><tr><td>Folded Capillary Cell</td><td>Malvern Instruments, Ltd.</td><td>DTS1070</td><td></td></tr><tr><td>Forceps</td><td>Shimizu Akira Inc.</td><td>Stainless pincet 150</td><td></td></tr><tr><td>Homogenization Pestle</td><td>Ieda Trading Corp.</td><td>9993</td><td></td></tr><tr><td>Mica</td><td>Nisshin EM Co., Ltd.</td><td>LC23Z</td><td></td></tr><tr><td>Microcentrifuge</td><td>Beckman Coulter</td><td>BKA46472</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular Devices Corporation</td><td>Spectra MAX M3</td><td></td></tr><tr><td>Microscope Slide</td><td>Matsunami Glass Ind., Ltd.</td><td>S011120</td><td></td></tr><tr><td>Pipette</td><td>Eppendorf Research&amp;reg; plus</td><td>3120000909</td><td></td></tr><tr><td>Pipette Tips</td><td>AS ONE Corporation</td><td>2-5138-01, 2-5138-02, 2-5138-03</td><td></td></tr><tr><td>Plastic Petri Dish</td><td>AS ONE Corporation</td><td>1-7484-01</td><td></td></tr><tr><td>Renilla Luciferase Assay Kit</td><td>Promega corporation</td><td>E2810</td><td></td></tr><tr><td>Rhodamine B Isothiocyanate</td><td>Sigma-Aldrich Co., LLC.</td><td>283924</td><td></td></tr><tr><td>RNase-Free Water</td><td>Qiagen</td><td>129112</td><td></td></tr><tr><td>Sodium Carbonate</td><td>Wako Pure Chemical Industries, Ltd.</td><td>199-01585</td><td></td></tr><tr><td>Surgical Blade and Handle</td><td>FEATHER Safety Razor Co., Ltd.</td><td>Stainless steel No. 14 (blade), No. 3L (handle)</td><td></td></tr><tr><td>Syringe Tip Cap</td><td>Musashi Engineering Inc.</td><td>NC-3E</td><td></td></tr><tr><td>Weighing Balance</td><td>Sartorius</td><td>CPA225D</td><td></td></tr><tr><td>Zeta Potentiometer</td><td>Malvern Instruments, Ltd.</td><td>Zetasizer Nano-ZS</td><td></td></tr></tbody></table>

peptide-derived method to transport genes and proteins across cellular and organellar barriers in plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

核酸和蛋白质的货物的阵列被成功地引入到使用设计的肽作为递送载体的各种植物。阳离子肽和带负电荷的货物之间的静电相互作用导致转染复合物的形成，可直接渗入植物使用无针注射器（ 图1）叶。优化制剂的植物细胞的转染（在这些研究中21,23-26经验确定）列在表1中 ，其中的大范围递送货物（质粒DNA，双链DNA，双链RNA和蛋白质）的各类型的代表。所有基于肽的制剂的平均直径为在150的大致范围内 - 300纳米。根据DLS分析，所有制剂显示相对低的大小多分散性，这表明形成肽 - 货物聚集体具有均匀的尺寸分布。形貌肽和质粒DNA（ 图2A）或蛋白质（ 图2B）之间的复合物，在云母，由原子力显微镜成像。观察均匀的球状物为肽的pDNA和肽 - 蛋白质组合，与从DLS测量数据的协议。在配合物（ 表1）的ζ电位而言，pDNA-和双链DNA衍生的复合物具有净负表面电荷，而基于双链RNA复合物具有一个接近中性表面电荷。肽 - 蛋白质复合物，在另一方面，被带正电。 肽质粒DNA，并在调解拟南芥或烟草本塞姆氏作为模式植物系统的转肽dsDNA抗体制剂的效率进行了定量评价以及定性。在与Rluc基因表达测定法用于基因表达水平的定量（ 表1），因此，对于这个实验的pDNA或双链DNA编码与Rluc基因必须用于络合与各自的载体肽。使用（KH）9 -BP100 /质粒DNA制剂，核靶向递送和质粒DNA的表达，可以实现以下12小时的潜伏期，以大约1×10 5的估计RLU /毫克值。对于质粒DNA，肽的组合的线粒体靶向递送和表达，Cytcox-（KH）9和BP100，需要为复合物形成。用12小时的相同的优化潜伏期，然而，获得转染（约1×10 3 RLU /毫克）的低得多的水平。同时，需要类似潜伏期（12小时）和基因表达水平（约1×10 3 RLU /毫克）/记录基于双链DNA复合物，使用（KH）9 -BP100肽还配制。基因表达的定性评估，通过复合prepar处理的叶片直接显微镜观察下进行ED使用质粒DNA的双链DNA或编码GFP报告基因。与非靶向肽pDNA复合转染的细胞，弥散性对应与GFP表达清楚地观察，发现在胞质溶胶（ 图3A）来本地化绿色荧光。在绿色荧光的本地化模式截然不同的差异与线粒体靶向肽pDNA复合浸润细胞明显。这里，点状的绿色荧光与线粒体染色共定位是可见的，证实基因表达的特异性只在细胞的线粒体隔室（ 图3B）。 在肽 - 蛋白制剂的情况下，蛋白质的货物（ADH）以荧光团（罗丹明B）的共轭将使在胞内区室的递送蛋白可视化。在6小时的潜伏期短，ADH-蛋白罗丹明B（蓝色）被发现在整个分发细胞质和浸润细胞的液泡（ 图3C）。与此同时，基因表达的快速和有效的下调可以使用肽的dsRNA制剂各种植物来实现。在第一个实验中， 拟南芥叶用肽的dsRNA渗透络合物沉默的查耳酮合成酶基因（CHS）负责在干旱条件下花青素（红色颜料）的生物合成。在拟南芥的外观差异叶在正常（ 图3D，a）和干旱条件（ 图3D，B）提供了一种简单的方法来评价社区卫生服务利用优化肽双链RNA沉默配方（箭头1指示渗透区域）。在第二个实验中，肽的dsRNA复合物渗入转基因拟南芥的表达黄色荧光蛋白（YFP）的叶子。在YFP表达明显减少可能会在表皮细胞9小时P为可见OST转染（ 图3E，F）。在下调的基因表达的制剂在不同的植物系统（杨树，12小时后转染）的效力也进行了验证（ 图3G中的H）。 <img alt="图1" src="/files/ftp_upload/54972/54972fig1.jpg" /> 图 1: 核酸和蛋白质的货物交付到活体植物肽基配方。 所设计的载体肽包括结合到细胞穿透或细胞器转运序列聚阳离子。聚阳离子启用从内涵体隔室下面的内化到细胞结合和/或带负电荷的货物的缩合以及逃逸。货物递送到细胞中，随后对特定的细胞器是通过细胞穿透序列和细胞器转运序列分别介导。各种货物，可能是事业有成LLY输送到植物包括核酸如质粒DNA，双链DNA和双链RNA，以及诸如牛血清白蛋白（BSA），醇脱氢酶（ADH）和黄晶（黄色荧光蛋白的变体）的模式蛋白。生物活性分子能够形成与通过静电相互作用，其被引入到植物的叶通过注射器浸润肽缀合物转染复合物。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54972/54972fig2.jpg" /> 图2: 基于肽制剂的形态。 （A）AFM幅度（KH）在N / P 0.5 9 -BP100 /质粒DNA配方的形象。 （B）（BP100）在摩尔慧慧2 K 8 / ADH配方AFM高度图像Ø10.转载许可已公布的资料23,24。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54972/54972fig3.jpg" /> 图3: 使用优化的基于肽制剂转染效率的微观评价。 （A）的细胞溶质的GFP表达（绿色），来自叶绿体自发荧光（红色）明确区分，在拟南芥的海绵组织细胞中观察到叶渗入带（KH）9 -BP100 /质粒DNA制剂（N / P 0.5; 12小时）。 （B）GFP表达（绿色）与Cytcox-（KH）渗透拟南芥叶9 / BP100 /质粒DNA制剂（N / P 0.5的每个肽类表皮细胞线粒体（红色）检测Ë; 12小时）。与GFP表达线粒体的放大图像显示在最右侧的面板中。 ADH-罗丹明B（蓝色）的（C）的传递到拟南芥的海绵叶肉细胞叶由（BP100）2 K 8以10的肽/蛋白质的摩尔比介导的，可视化6小时后的渗透。 （D）的前拟南芥叶（a）和之后（b）与（KH）9 -BP100 /双链RNA制剂（摩尔比2; 48小时）处理，这导致了花青素生物合成途径的抑制。含有双链RNA GFP5了类似表述渗入到叶为阴性对照组（C）。箭头1和3表示的熔渗区域，而箭头2和4表示的叶内非渗入区。 （E） 拟南芥叶表达黄色荧光蛋白（YFP）和（F）在与浸润减少YFP荧光<html> （KH）9 -BP100 / dsRNA的配方（摩尔比2; 9小时）。 （G）的转基因杨树的叶子表达黄色荧光蛋白（YFP）和（H）的减弱YFP荧光下浸润（KH）9 -BP100 / dsRNA的配方（摩尔比2; 12小时）。从公布的资料来源21,23,24,26转载许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54972/54972fig4.jpg" /> 图4: 变异肽对DNA（N / P）比率及配合生物物理特性的影响。 随着肽DNA比，肽基配方的尺寸减小，而他们的ζ电位值过渡由负转正。/files/ftp_upload/54972/54972fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54972/54972table1.jpg" /> 表1: 表征及各种基于肽配方评价。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972table1large.jpg" target="_blank">请点击此处查看该表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54972/54972table2.jpg" /> 表2:完整植物为基础的肽与其他现有的DNA传递技术的比较。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972table2large.jpg" target="_blank">请点击此处查看该表的放大版本。</a>

Watch this Scientific Journal Video about Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants at JoVE.com

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

peptide-derived-method-to-transport-genes-proteins-across-cellular

Research

JoVE Journal

Genetics

10.5K 次观看.  RIKEN Center for Sustainable Resource Science. Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

视频: Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers 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

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

生物化学

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

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

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 ...

化学

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

细胞的细胞大分子运输检测在植物利用基因枪法

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

生物学

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

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 ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

使用直接输注装置在植物中递送分子

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

作者聚焦：用于植物分子递送的直接输注装置  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

用于植物分子输送的直接输液装置

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

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