Name: lignin down-regulation of zea mays via dsrnai and klason lignin analysis
Uploaded: 2014-07-23T13:00:00
Description: Watch this Scientific Journal Video about Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis at JoVE.com

显微成像是通过美国密歇根州立大学中心高级显微镜的服务进行。玉米愈伤组织爱荷华州立大学的玉米转化中心购买。笔者想感谢密歇根州立大学植物研究实验室的杰夫·韦瑟他对碳水化合物分析技术援助。这项研究是由慷慨密歇根州的玉米营销计划（CMPM）及财团的植物生物技术研究（CPBR）资助。

1。dsRNAi构造用于下调ZmCCR1的制备<ol><li>设计基因特异性引物，包括必要的限制性酶切位点用于制作dsRNAi构建以敲除的ZmCCR1基因。两个引物组被设计以扩增ZmCCR1 cDNA的片段2段：一个523 bp的片段从核苷酸748到1271，和一个285 bp的片段从核苷酸986至1271的ZmCCR1的cDNA是从亚利桑那州基因组研究所（AGI）提供。更多细节在图1中描述。 </li><li>通过从ZmCCR1 cDNA模板?...

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微生物纤维素酶对植物细胞壁多糖的可访问性在很大程度上取决于其所用酚醛聚合物23相关联的程度。从木质纤维素生物质的转化率为可发酵的糖是负沉积在植物secondadry细胞壁木质素含量相关。这种相关性是归因于木质素的物理性质如疏水性24，化学不均一性，以及没有定期水解intermonomeric连杆25。 在这项研究中，一个dsRNAi技术诱发不同程度的遗传靶?...

生物燃料由木质纤维素生物质生产是由于其大量存在于美国1非常可取的，并在农业和林业废弃物可持续收获的情况下，不直接用于食品和动物饲料的生产耕地竞争的能力。然而，与玉米粒，这是目前在美国产生生物燃料的主要来源，木质纤维素材料是显著更加复杂和难以打破。除了长链碳水化合物，纤维素和半纤维素，其是糖的主要来源的木质纤维素原料的发酵过程中，多种类型的植物细胞壁也含有木质素，苯丙聚合物，可提供强度，抵御病原体的攻击，和疏水性细胞壁。而必需的植物的生长和存活，木质素也提出了显著障碍的纤维素和hemicellu的成功的酶转化失去可溶性糖。具有高木质素含量的材料一般为生物燃料（通过生物转化途径），且由于对加工性能和产品质量的负面影响的纸浆和造纸工业不太理想的材料。因此，植物材料的木质素减少在不与作物的结构强度和防御系统干扰电平的遗传操作可能是对双方的木质纤维素生物燃料和纸浆和造纸工业的生产成本的降低非常重要的。 在玉米（ 玉蜀黍 ），木质素共价交联通过阿魏酸和diferulate桥2的初级细胞壁半纤维素。木质素半纤维素复合物通过氢键结合到纤维素微纤维，形成了复杂的矩阵赋予完整性和强度的次生细胞壁。植物细胞壁的机械强度在很大程度上是由利的类型确定gnin亚基3-5。在以前的研究中，改变木质素亚基的比例显示在酶解6-11没有明显的趋势。然而，减少的木质素含量通常显示在转换12,13的改进，并且可以是键，由水解酶包括endocellulases，纤维二糖水解，和β-葡糖苷酶14增加植物材料的可消化性。 基因工程来调节转录物的表达水平已被广泛实行，以提高作物的性状。先进的技术，包括反义15和共抑制16的技术，能够有效地下调靶基因。完整的基因敲除也被用用发夹结构17编码内含子剪接的RNA基因结构来实现。此外，双链RNA干扰（dsRNAi）技术， 即一个强大和有效的基因表达媒体器的工作原理是针对两种转录本的降解或翻译抑制，提供了一种有效的手段，引导广泛的对靶mRNA 18抑制效果。基因沉默技术显示一些局限性。这些技术不能精确地调节转录水平，并可能导致对其他同源基因沉默意想不到的效果。 在该方法中，我们采用的粒子轰击进行的dsRNAi构造成玉米基因组中。迄今为止，植物种类繁多已成功利用粒子轰击， 农杆菌介导的转化，电穿孔，显微注射和方法转化。在玉米中的遗传转化，粒子轰击方法是有利的，对所有的其它方法，因为它是最有效的。粒子轰击是不依赖于细菌，所以该方法是游离的生物因素如基因，物种基因的大小或igin，或植物的基因型。物理转基因传送系统，使高分子量DNA和多个基因在高转化效率19被引入到植物基因组中，并在某些情况下进入叶绿体。在叶中肋的血管系统中的木质素还原可以通过扫描电子显微镜（SEM）进行可视化是有益的用于检查的样品的形貌和组成。 在玉米植株，两个肉桂酰辅酶A还原酶（ZmCCR1：X98083和ZmCCR2：Y15069）基因被发现在玉米基因组中20。肉桂酰基-CoA还原酶可催化羟基肉桂酰-CoA酯转化成肉桂醛。我们选择了ZmCCR1基因下调这种酶是因为基因的表达在所有的组织木质化。在523个核苷酸的ZmCCR1基因的3&#39;末端被选为一个dsRNAi构造，因为序列似乎是而相对于ZmCCR2的更加多样化。因此，dsRNAi结构会正是只绑定到ZmCCR1，避免脱靶沉默21。甲ZmCCR1_RNAi构建体设计到细胞质表达系统ImpactVector1.1-标签（Ⅳ1.1）包含绿色组织特异性启动子，核酮糖1,5 -二磷酸羧化酶加氧酶（Rubisco）。 研究的dsRNAi构造上的转基因植物的影响，木质素含量进行定量。的Klason（酸不溶性）木质素测量已知相比，其溶解一些木质素22的酸性洗涤木质素的定量方法，以更准确。因此，Klason木质素测定转基因玉米秸秆。这个过程包括两个步骤的酸水解，转换聚合碳水化合物转化为可溶性单糖23。水解的生物量，然后分馏成酸可溶性和不溶性MATERIALS和酸不溶木质素是根据以前的研究23,24测量。理想情况下，木质素分析应包括在水解之前步骤中，为了除去可影响结果可溶性材料，木质素残渣占存在于所述残基的任何灰分的后燃烧水解萃取，用水和乙醇。没有这些步骤，样品的木质素含量可以人为地抬高。完整的方法，这里介绍的，但是我们的实验中，我们无法因体积小的材料可用于测试执行这两个步骤其他两个细胞壁成分，纤维素和半纤维素中的木质素下调的转基因玉米系进行了分析。它已经报道，已下调或者他们的苯丙氨酸解氨酶（PAL）25，4 -香豆酸的转基因植物：辅酶A连接酶（4CL）26，或肉桂一lcohol脱氢酶（CAD）27显示增加在其它细胞壁结构组分。如在我们的研究中的第一步骤，结晶纤维素是使用Updegraff方法28测定。最初被设计用于测定纤维素的大量纤维素分解细菌和真菌的这种方法。简言之，将磨碎的玉米库存均与Updegraff试剂（：硝酸：乙酸的水）处理，以除去半纤维素，木质素和xylosans。结晶纤维素，通过加入H 2 SO 4完全水解成经由Saeman水解葡萄糖。结晶纤维素，然后用比色蒽酮法测定29。核实，如果半纤维素含量发生了变化，从磨碎的茎的提取物的单糖用三氟乙酸水解，用醛醇乙酸酯法衍生化，然后用气相色谱（GC）30进行分析。的详细程序结晶CELlulose含量和基质多糖成分分析是在福斯特等人（2010）31。 在这里，我们描述了用于木质素的程序下调通过RNAi技术，粒子轰击转化和木质素分析玉米木质纤维素生物质解构加速成可发酵糖生物燃料的玉米。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Chemical compositions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6OSM</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Osmotic medium)</td>
			<td>1 ml/l N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.69 g/L proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/l sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l sorbitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l mannitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6E</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Callus induction)</td>
			<td>1 ml/l (1000X) N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.76 g/l proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/l sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6S media</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Selection media)</td>
			<td>1 ml/l N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.69 g/L proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l sorbitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l mannitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Regeneration medium</td>
			<td>4.3 g/L MS salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 ml/L (1000X) MS vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>60 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3 g/L gelrite, pH 5.8 (100x25 mm petri-plates)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized bialaphos (3 mg/L) added after autoclaving.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rooting medium</td>
			<td>4.3 g/L MS salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 ml/L MS vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3g/L gelrite, pH 5.8 (100x25 mm petri-plates).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Specific materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-top high pressure tubes</td>
			<td>Pressure tube (#8648-27); Ace Glass, Vineland, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Plug (#5845-47); Ace Glass, Vineland, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral buffered formalin&#160;</td>
			<td>100ml of formalin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(1 liter)</td>
			<td>900ml of ddH2O</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>4.0 g of Sodium dihydrogen phosphate, monohydrate&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>(NaH2PO4.H2O)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad PSD-1000/He Particle Delivery device (Hercules, CA, United States)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss PASCAL confocal laser scanning microscope (Carl Zeiss, Jena, Germany)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excelsior ES Tissue Processor (Thermo Scientific, Pittsburgh, PA, United States).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCentre III Embedding Station (Thermo Scientific, Pittsburgh, PA, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Microtome Model Reichert 2030 (Reichert, Depew, NY, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emscope Sputter Coater model SC 500 (Ashford, Kent, England)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JEOL JSM-6400V Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fitzpatrick JT-6 Homoloid mill; Continental Process Systems, Inc., Westmont, IL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MA35 Moisture Analyzer; Sartorius</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Critical point dryer, Balzers CPD (Leica Microsysstems Inc, Buffalo Grove, IL, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lignin down-regulation of zea mays via dsrnai and klason lignin analysis

为了便于使用木质纤维素生物质作为替代生物能源资源，在生物转化过程中，前处理工序是必要的，开放的植物细胞壁的结构，增加了细胞壁碳水化合物的可访问性。木质素，多酚材料存在于许多细胞壁类型，被称为是一个障碍显著酶连接。减少木质素的含量为不与植物的结构完整性和防御系统干扰电平可能是一个有价值的步骤，以减少生物乙醇生产的成本。在这项研究中，我们已经通过基因的双链RNA干扰技术下调1的木质素生物合成相关的基因，肉桂酰基-CoA还原酶（ZmCCR1）。使用粒子轰击法ZmCCR1_RNAi构建体已整合到玉米基因组中。转基因玉米植物正常生长相比于野生型对照植物在无与生物生长或防御机制terfering，除在转基因植物叶片中肋，壳显示棕颜色的，和茎。微观分析，与组织学检测相结合，发现叶厚壁纤维变薄，但其他主要血管系统部件的结构和尺寸没有改变。在转基因玉米中的木素含量降低了7-8.7％，结晶纤维素含量响应于木质素还原性增大，半纤维素保持不变。的分析可以表明碳流可能已被移离木质素生物合成的纤维素生物合成。本文界定了用于下调通过RNAi技术在玉米中的木素含量的方法，并在细胞壁组成分析用于验证的修改对细胞壁结构的影响。

我们已经证明通过RNAi技术在玉米植株的木质素含量降低。粒子轰击转化方法取得了30％左右trnasformation效率。 ZmCCR1的基因沉默在T0-T2世代中观察到的一致。木质素降低转基因同样成长为野生型的玉米植株，除了在叶片中肋，壳，茎和显示棕色着色。组织学分析表明，该突变体品系表现出显著减少在玉米叶中肋18的厚壁组织纤维细胞壁厚度。尽管细胞壁厚度的减小，其它主要血管系统?...

Watch this Scientific Journal Video about Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis at JoVE.com

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

双链RNA干扰（dsRNAi）技术以下调玉米肉桂酰辅酶A还原酶（ZmCCR1）基因为低植物木质素含量。木质素下调从细胞壁是由微观分析和可视化的Klason法进行定量。在半纤维素和结晶纤维素组成的变化进行了分析。

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Research

JoVE Journal

Bioengineering

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Polymer Microarrays for High Throughput Discovery of Biomaterials

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

子结构分析仪：用于荧光显微镜图像中细胞体快速探索和精确分析的用户友好型工作流程

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Engineering Antiviral Agents via Surface Plasmon Resonance

通过表面等离子体共振 设计 抗病毒药物

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...