Name: geomagnetic field (gmf) and plant evolution: investigating the effects of gmf reversal on arabidopsis thaliana development and gene expression
Uploaded: 2015-11-30T17:00:00.000+00:00
Duration: 11 min 4 s
Description: Watch this Scientific Journal Video about Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression at JoVE.com

This work was supported by a grant to MEM from the Dept. of Life Sciences and Systems Biology of the University of Turin, Italy. The authors thank G. Gnavi for technical assistance during some experiments. CTR is funded by the Wellcome Trust and the Royal Society (Grant Number 098436/Z/12/Z).

1.设置三轴线圈的 注 :图1示出了用来反转GMF三轴线圈。 <ol><li>打开三轴磁力计，其探头插入三轴线圈。 </li><li>打开电脑并启动磁力仪软件，它允许数据从三轴磁力计采集。 </li><li>使用所报告的磁力计来计算GMF的量值的分量值。例如，对于磁力计的值:Bx的= 6.39μT，通过= 36.08μT，BZ = 20.40μT计算的41.94μT的场强通过使用以下方程:B = GMF + B 附加 ，其中B 额外 =（Bx的2 +由2 + BZ 2）1/2（即，41.9μT中的例子中） </li><li>打开三个直流电源（双范围:0-8V / 5A和0-20V / 2.5A，50W），每一个连接到三个couplHelmholtz线圈ES和经由一个GPIB连接（图1B）连接到计算机。 </li><li>电源的组电压，以产生所需的磁场与反相磁场矢量。例如，对于乙GMF如在步骤1.3和此处所述的仪器的线圈尺寸，设定电压为V X = 0.00，V Y = 30.52，V Z = 0.00 V，以产生一个新的所得乙=乙GMF + B 三轴线圈 =（6.38，-36.08，20.39）μT。 即，一个新的领域的大小相同乙GMF而是指向不同的方向。 </li><li>通过使用1.3中描述的步骤，检查新的领域与磁力计的软件。 </li><li>暴露植物正常和反转基因食品的条件用培养皿中第2节。 </li><li>通过保持字段等于的幅度进行假暴露实验|乙GMF |守信字段等于基因改造食品，但垂直分量改变所述场的水平分量的方向（即"北，东或西"）与相对于磁场反向条件在三轴线圈的电流相等。通过改变线圈的电压如1.5中所述要这样做。 注意:此假曝光排除了潜在的微妙的加热或从任一线圈本身或从用于控制所述线圈的电子振动的影响。 </li><li>通过施加从人员盲现场条件进行实验的其余部分和/或解释的数据运行双盲实验。 </li></ol> 2.选煤厂材料和条件对植物生长的<ol><li>使用拟南芥的种子，生态型哥伦比亚0（西0），将接成1.5毫升管和表面进行处理消毒用5％（重量/体积）次氯酸钙溶液和0.02％（体积/体积）的Triton X-100在80％的乙醇（EtOH），10-12分钟，在25-28℃下，连续振荡。然后用80％的乙醇冲洗两次，洗涤用100％的乙醇，最后用无菌蒸馏水冲洗。 </li><li>制备1升Murashige和Skoog 22（MS）的改良培养基中加入:2.297克的MS（0.5×MS基础盐混合物），10克蔗糖，去离子水至1升，pH值5.8-6.0用KOH调节。将16克琼脂和高压釜20分钟，120℃。 </li><li>凝固前，倒80毫升培养基到每个（120×120 平方毫米）正方形培养皿。播种30无菌的种子上板，然后封板与蜡状膜。 </li><li>在黑暗水平Vernalize板在4℃下2天以增强和同步发芽，然后暴露培养皿为正常或颠倒GMF。 </li><li>在内外的三轴线圈和三轴线圈外侧平行实验的竖直位置暴露种子在气候受控环境在22℃下在8小时黑暗，16小时光照光周期的时间表，使用高压钠灯（220×10 -6ê米 -2 秒 -1）。使用蓝色明胶薄膜为聚光灯以减少灯的红色分量。 </li><li> RNA提取到正常（对照）和反转（治疗）GMF条件之前暴露10天的植物。 </li><li>曝光后，利用培养皿图片。 </li><li>使用ImageJ的软件计算根长和叶面积。 <ol><li>简言之，测量培养皿的一侧，然后打开培养皿，并通过使用"直"线选项画一条线，恰好穿过板侧的图像。 </li><li>在"分析"菜单中选择"设置规模"，并插入实际距离的"已知距离"框（ 如 120毫米），然后插入的长度（mm）的单位;最后单击"全局"选项，以使可用于所有的测量设置。 </li><li>对于袋鼠长度T，使用手绘工具认真遵循根的形状。通过使用"分析"菜单中的"测量"选项测量长度。继续测量画面中的所有根和保存用于进一步的统计学分析该文件。 </li><li>对于叶面积，从"图像"菜单中的调节选项，然后在"颜色阈值"。选择单个叶片，并在"分析"菜单中选择"分析粒子"。保存用于统计分析的单独的测量。 </li></ol></li></ol> 3. 拟南芥总RNA提取，实时定量PCR（定量PCR）反应条件和引物对拟南芥 <ol><li>分别收集30芽和根30，并立即在液氮中冷冻。然后研磨在液氮与研钵和杵。 </li><li>用纯化试剂盒和RNA酶的DNA酶处理试剂盒通过厂家专业提取总RNA呃的说明。 </li><li>检查样品的质量和数量通过根据制造商的说明使用RNA纳米试剂盒和毛细管凝胶电泳。确认RNA的定量分光光度。 </li><li>用2微克总RNA，并用cDNA的逆转录试剂盒根据制造商的建议，以获得第一链cDNA随机引物的。 </li><li>使用SYBR Green I的使用ROX作为内部装载标准进行实时系统完成所有实验。 </li><li>执行与25μl的混合物组成的12.5微升2×SYBR绿定量PCR主混合物，0.5微升的cDNA和100​​nM的引物的反应。使用表 1中所列的引物。包含在控制非实时控制（使用总RNA未经逆转录以监视基因组DNA污染）和非模板对照（水代替模板）。 </li><li>计算底漆效率使用标准的所有引物对曲线法23。 </li><li>使用以下PCR条件:CRU3，COTP1，RRTF1。在95℃下10分钟，在95℃，30秒58℃，和30秒72℃40个循环的15秒; UBP6，eEF1Balpha2，ACT1，GAPC2，CAT3，TAPX，APX1，RbohD，FeSOD1 10分钟，在95℃，在95℃，20秒在57℃，和30秒72℃40个循环的15秒。 </li><li>阅读的荧光在每次退火和延伸阶段。对于所有的运行，通过在热曲线的解离段执行从55到95℃的解链曲线分析。使用解离曲线屏幕，通过成果标签访问以查看的解离曲线（荧光作为温度的函数的曲线图）。确保该实验的离解段期间收集数据集被选择用于分析使用Analysis /设定屏幕。 </li><li>确定第l通过利用基因由三个独立的RNA提取三个技术复制24,25分析了十倍系列稀释液（1至10 3倍 ）inear范围模板浓度阈值周期（Ct值）的。 </li><li>分析所有扩增曲线的实时PCR仪软件获得Ct值。校准和规范相对RNA水平与最佳的看家基因如下级别:3.11.1）通过结果选项卡进入扩增曲线屏幕上，选择斜坡或平台的数据应该使用Analysis Selection / Setup界面进行分析，然后从命令面板上的荧光菜单选择DRN（基线校正标准化荧光）。进入板样品值筛选通过的结果选项卡，显示Ct值的采样井。 </li><li>使用四个不同的参考基因[例如，细胞质甘油醛-3-磷酸脱氢酶，（GAPC2），泛素specif集成电路蛋白酶6（UBP6），Actin1（ACT1）和延伸因子1B的α-亚基2（eEF1Balpha2）]正常化的实时PCR的结果。选择顶部使用分析软件26排基因;并使用最稳定的基因进行标准化。 <ol><li>简言之，将组织在Excel表单与含有含样本名基因名与第一行第一列中的输入数据。然后从菜单栏上的分析软件。使用对话框中选择输入数据。 </li><li>接下来，检查领域的样品名称，基因名称和简单的输出而已。点击开始按钮来进行分析。选择排名最高的基因（具有最小稳定性的值），为候选基因最稳定表达。 </li></ol></li><li>绘图数据通过显示在这两个芽和根的差异倍数变化的表达。 </li></ol> 4.统计分析<ol><li> Express数据为平均值±标准差。比较合作ntrol和治疗组通过进行方差分析和Tukey检验和Bonferroni和邓恩-Sidak调整的概率试验（0.95％置信度）的分析。 </li></ol>

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Manage. 13 (1), (2013).</li><li>Khandelwal, A., Elvitigala, T., Ghosh, B., Quatrano, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+transcriptome+reveals+control+circuits+regulating+redox+homeostasis+and+the+role+of+an+AP2+transcription+factor.">Arabidopsis transcriptome reveals control circuits regulating redox homeostasis and the role of an AP2 transcription factor.</a> Plant Physiol. 148 (4), 2050-2058 (2008).</li><li>Haddad, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-sensing+mechanisms+and+the+regulation+of+redox-responsive+transcription+factors+in+development+and+pathophysiology.">Oxygen-sensing mechanisms and the regulation of redox-responsive transcription factors in development and pathophysiology.</a> Respirat. Res. 3 (1), (2002).</li></ol>

The authors have nothing to disclose.

我们最近发现，转基因食品的逆转，当大多数家族被子植物谱系转移发生2时之间存在惊人的相关性。然而，尽管刺激假设和研究上的变化的基因改造食品强度效果的过多，假设GMF逆转自身可诱导在植物基因表达和形态学显著变化从未被证实。这里，我们表明，第一次，使用三轴八角亥姆霍兹线圈扭转GMF在我们的实验室，并且环境磁场的反转会导致表型变化和基因表达的植物中调制的方法。 为了获得GMF反转（或修改）在足够体积的植物生长试验（2×2×2米3），我们建立了一个八角亥姆霍兹线圈系统。该系统是不可商购（通常亥姆霍兹线圈环形和更小）和建设的费用是相当大的。重要的是，该系统提供了强大的现场修改，具有优异的时间稳定性和均匀性在修改后的磁场。 该系统的设计和建造，以减少GMF的值的正常条件千分之一或扭转任何磁场的三个方面的。然而，线圈的设计不允许以产生高磁场强度。因此，该仪器在本形式不适合用来评估的高磁场强度对植物或其它生物的影响的实验。 在实验室中，改变的GMF类似于在此方法中所描述的已用不同的方法获得，包括屏蔽由铁磁金属板具有高导磁率，其中偏离磁场和金属本身内集中他们周围的实验区。钍Ë优点使用亥姆霍兹线圈的是，该系统允许植物暴露于更多的自然条件下（光，空气流通等 ），从而使其不仅适合在体外研究 （与使用的培养皿），但也用于体内植物生长和发育的实验。我们的系统的尺寸创造一个空间，它允许抑制到天然基因食品&lt;1 / 1,000整个25×25×25厘米3的球形体积（ 见图 1A），从而使托管多个佩特里细菌培养皿或一些小盆植物生长。 这里介绍的方法已被应用到植物生物学研究;然而，该系统允许大范围的实验，包括病毒学，微生物学，以及对线虫（例如， 秀丽隐杆线虫 ），节肢动物和小动物（包括小鼠和大鼠）的研究。因此，在假设的测试，反转基因食品能以诱导形态学和转录变化也可以扩大到许多其他生命系统，可能最终甚至到人细胞。 该转基因食品是不断变化和波动。因此，在我们的实验中的一个主要挑战是提供基因改造食品的一个常数补偿，以获得所需的新基因改造食品的值。通过磁场值的连续控制，通过读取磁强计值和电压补偿时才可以实现。因此，该系统可以补偿基因改造食品的缓慢变化的一部分，但它不执行任何对于较高频率的波动。 总之，利用三轴线圈扭转GMF矢量是有助的，以证明这种逆转的GMF的载体是能够诱导植物的形态变化和差别基因表达。用该方法获得的结果提供了令人信服的证据支持的假设，即转基因食品重新versals可能是植物进化的驱动力之一在地质时间尺度2。

地球的磁场（或等价地磁场，GMF）是所有生活在地球上的生物，包括植物一个无法回避的环境因素。在转基因食品一直是地球的自然功能，因此在进化过程中，所有生物经历了行动。越来越多的证据体表明GMF是能够影响许多生物过程1。该基因改造食品是不均匀的并且有在地球的表面上在它的大小和方向显著地方的差异。在转基因食品在地球表面显示了广泛的幅度，从不到30μT几乎70μT。该转基因食品通过磁层2偏转大部分带电粒子保护地球及其生物圈来自太阳风的致命影响。 植物对环境刺激作出反应;和古典响应非生物因素如光和重力已经吨通过定义所谓的向光性和向地响应horoughly说明。很少或没有，被称为悟性和植物磁场反应的机制，尽管发表了关于这个话题，最近回顾1篇论文多如牛毛。不同的是引力场，在转基因食品植物进化过程中始终如一地改变，从而表示最近已被认为是潜在的动力，最终促进植物多样性和物种2重要的非生物胁迫因素。地磁反转（或偏移）的变化，转基因食品的极性。在地球的生命史，转基因食品的逆转发生几次。这些每一个极性转换过程中暴露出来的星球降低GMF实力周期。一些作者已经推测的低强度GMF这些过渡期间可能已经允许电离辐射从太阳风到达地球表面，从而诱导一一贯强调要活的生物体，这本来是强大到足以引起基因的改变最终导致植物进化2。 实验说明磁场对植物的影响进行详细的分析表明，大量的矛盾的报告，其特点是似是而非的生物物理相互作用机制的缺乏。许多实验都根本不现实，而另一些人缺乏检验的假设，最终都难以令人信服3。在过去几年中，在植物磁场的影响的进展和研究现状进行了审查2,4-11。近日，低和高磁场的影响进行了深入的讨论1，特别侧重于基因食品逆转事件的植物进化2的参与。 最直接的手段来证实这一假设转基因食品的逆转影响植物的进化是合成一种转基因食品逆转在实验室中通过检测植物对正常和反向的磁场条件下的反应。为了检验这一假设，我们因此建立了一个三轴亥姆霍兹八角线圈对磁场的补偿系统（三轴线圈），其能够准确地扭转正常GMF条件。 我们用拟南芥为模式植物，我们测试了逆转基因食品对一些重要基因的基因表达的影响: 十字花科3（CRU3），其编码12S种子贮藏蛋白，这种蛋白酪氨酸磷酸化和磷酸化状态被调制响应对ABA在拟南芥种子12,13; 铜运输蛋白1（COTP1），编码重金属运输/解毒家族蛋白在土壤中铜的采集和花粉发育14的主要功能;和氧化还原响应转录因子1（RRTF1）编码该ERF（乙烯反应因子）亚家族B-3的ERF / AP2转录因子家族，其中包含一个AP2结构域促进协同共激活的基因表达的途径和赋予交叉耐受性的非生物和生物胁迫15中的一员。 此外，我们还分析了五个基因参与氧化应激反应: 铁超Dismutase1，（FSD1），编码胞质酶酶促并迅速转换超氧阴离子（O 2 - ）和水 （H 2 O）与过氧化氢 ​​（H 2 O 2）和分子氧（O 2），16; Catalase3（CAT3），即编码和酶，其催化的H击穿2 O 2成水和氧17,18; Thylakoidal抗坏血酸过氧化物酶（TAPX），其编码叶绿体类囊体过氧化物酶能清除H 2 O的2 19; 抗坏血酸Peroxidase1（APX1），其编码胞质过氧化物能清除H 2 O的2和表示由NO-衍生分子20介导的翻译后修饰的潜在目标之一;和NADPH- 呼吸爆发氧化酶蛋白D（RbohD）编码生成O 2的酶-和调节生长，发育和应激反应在拟南芥21起着举足轻重的作用。 我们的现场逆转方法提供了第一手的证据表明转基因食品反转可诱导A的形态学和基因表达显著的变化拟南芥根与芽。这个协议提供了一种创新的方法来评估基因食品逆转对植物形态和基因表达的效果，并且可以用来评估GMF逆转对植物的行为的其他方面的潜在影响，并从而引导ロ讨论乐反转基因食品对植物进化的。

<table><tbody><tr><td>Three-axis magnetometer</td><td>Bartington&amp;nbsp;</td><td>Mag-03MC</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>Magnetometer power supply</td><td>Bartington&amp;nbsp;</td><td>Mag-03PSU</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>Magnetometer software</td><td>Bartington&amp;nbsp;</td><td>Mag03DAM</td><td>triaxial fluxgate magnetometer</td></tr><tr><td>DC power supply&amp;nbsp;</td><td>Agilent Technologies</td><td>E3642A</td><td></td></tr><tr><td>Calcium hypochlorite&amp;nbsp;</td><td>Sigma</td><td>211389</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma</td><td>X100&amp;nbsp;</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>2860</td><td></td></tr><tr><td>GroLux Sodium vapor lamps&amp;nbsp;</td><td>OSRAM Sylvania</td><td>600W</td><td></td></tr><tr><td>RNeasy Plant RNA kit&amp;nbsp;</td><td>Qiagen</td><td>74903</td><td></td></tr><tr><td>RNase-Free DNase&amp;nbsp;</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2938B</td><td></td></tr><tr><td>NanoDrop ND-1000&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>not available</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit&amp;nbsp;</td><td>Applied Biosystems</td><td>4368813</td><td></td></tr><tr><td>Mx3000P</td><td>Agilent Technologies</td><td>401512</td><td></td></tr><tr><td>2x MaximaTM SYBR Green qPCR Master Mix&amp;nbsp;</td><td>Fermentas International, Inc</td><td>K0221</td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519&amp;nbsp;</td><td></td></tr><tr><td>Petri dish square (120 x120 mm&lt;sup&gt;2&lt;/sup&gt;)</td><td>Sigma</td><td>Z692344&amp;nbsp;</td><td></td></tr></tbody></table>

geomagnetic field (gmf) and plant evolution: investigating the effects of gmf reversal on arabidopsis thaliana development and gene expression

其中最刺激的观察在植物进化是地磁场的发生（GMF）逆转（或偏移）和被子植物的辐射的瞬时之间的相关性。这导致了在基因改造食品极性的改变可能在植物进化中起作用的假说。在这里，我们描述了一个方法，通过使拟南芥人为地逆转GMF条件下检验这一假设。我们使用了三轴磁力计和收集到的数据被用于计算该转基因食品的大小。三个DC电源被连接到三个亥姆霍兹线圈对以及由计算机得到控制，以改变GMF条件。在培养皿中生长的植物被暴露在正常和反向的基因改造食品的条件。还进行深水暴露实验。在实验过程中暴露的植物进行拍照和图像进行分析，以计算​​根长和叶面积。拟南芥提取总RNA和定量分析在十字花科3（CRU3），铜转运蛋白1（COTP1），氧化还原响应转录因子1（RRTF1），铁超氧化物歧化酶1，（FSD1），Catalase3（CAT3），Thylakoidal的基因表达进行实时PCR（qPCR的） 抗坏血酸过氧化物酶（TAPX），胞质抗坏血酸Peroxidase1（APX1），和 NADPH /呼吸爆发氧化酶蛋白D（RbohD）。四个不同的参考基因进行分析，以归一化的qPCR结果。的四个基因最好选择和使用了最稳定的基因进行标准化。我们的数据显示，在第一次，使用三轴线圈扭转GMF极性具有对植物生长和基因表达显著影响。这支持了GMF反转有助于诱导的变化在植物发育的假设，可能证明较高的选择压力，最终导致为p兰特演变。

该协议的目的是提供一种方法，以评估逆转地磁场（GMF）是否可能影响植物的发育和拟南芥生态型胶原0三轴线圈的基因表达，如图1A被用来反转GMF时集与适当的驱动电压（图1B），获得如在步骤1.5在协议中所述。的三轴线圈的尺寸为〜2×2×2米3，允许与反转GMF条件托管多个培养皿足够的空间。对照培养在相同的环境条件，并在正常的基因改造食品的值。经过10天的接触正常或反转基因食品的条件下，植物的表型表现出明显的形态学改变。 如图2，对照植物（即，在正常GMF条件下生长）表明根长度与显著（唐恩-Sidak和邦费罗尼调整机会率&lt;0.001;学生的t = 10.68，自由度= 31）更高的值（29.41毫米; SEM = 1.04; N = 32），相对于暴露在逆转GMF植物（17.53毫米; SEM = 0.58; N = 36）。在GMF反转植物，芽的形态，也改变了显示单张扩张减少了开发。暴露在正常条件下植株4.95 平方毫米（SEM 0.025，N = 54），而暴露在扭转GMF条件植株显著（邓恩-Sidak和邦费罗尼调整机会率= &lt;0.001的平均叶面积;学生t = 31.32，DF = 53）较低的叶面积值（3.71毫米2; SEM = 0.032; N = 54）。因此，拟南芥暴露于反相GMF条件诱导两根长，叶面积的减少。 展叶和根系的生长依赖这两个分裂和细胞27的延伸上。因此，植物发育，生产力和综合健身依赖于一个最佳的拍摄开启和根系统架构&lt;SUP&gt; 28。暴露于扭转GMF条件的植物的减少的根长，叶的大小表示的感测系统的存在不仅能够感知变化的磁场强度，也能以变化的磁场"方向"相比，重力作出响应。基因改造食品的逆转可能影响植物生长的假说认为在我们的实验中，这表明转基因食品反转条件显著影响植物生长发育有说服力的证据。 还伴随着改变基因表达的形态学变化。中持家基因，最稳定的基因是延伸因子1B的α-亚基2。基因（CRU3，COTP1，RRTF1）的第一组出现了大幅度改变的基因中的表达（图3）。拍摄所有三种基因在植物中表达了显著升高（P &lt;0.05）约2.5倍，暴露在储备ð转基因食品的条件。 CRU3根表达上调在暴露在正常条件下基因食品植物根部，但显著（P &lt;0.05），下调逆转转基因食品的条件。相反，发现对COTP1和RRTF1，这是下调在正常条件下和上调GMF反转的存在（图3）。 十字花科（一个12秒球蛋白）是A的种子最丰富的储藏蛋白拟南芥和其他十字花科植物和被合成为在粗面内质网的前体。它被运送到蛋白质贮藏液泡13。幼苗发芽要求十字花科，它被用来作为氮气的初始来源的击穿。下调十字花科降解受损害的细胞结构或细胞成分的发展29,30降低胚胎发育。我们的研究结果表明，CRU3的上调与相关低级展叶和减小的根长，从而表明该基因在基因改造食品逆转并且其过度表达敏感可能有助于植物发展的减少。此外，转基因食品逆转诱导CRU3在根部显著下调，这与减少的根长。铜是用于在植物关键过程的重要辅助因子，但它施加时在过量的有害影响;因此，过表达铜运输损害植物的生长。 GMF逆转的效果COTP1在这两个芽和根一显著过表达，从而解释了降低植物生长。离子应力损害叶绿体代谢，这是紧密相连的细胞的氧化还原状态。在拟南芥转录因子RRTF1为相关联的能力的基因的表达很重要的调节氧化还原变化31。因此，当植物暴露于能够改变他们的生理和发育的外部刺激人的程序这一重要的转录因子的表达期望。对转基因食品的逆转诱导RRTF1在这两个芽和根一显著的过度表达，从而表明植株较高的氧化应激反应，逆转基因改造食品的条件。 有趣的结果是通过分析参与氧化应激五个基因获得。在一般情况下，提取和分析在枝条的所有基因没有显示当植物生长在正常或反向GMF条件（图4和图5）显著差异（P&gt; 0.05）。然而，显著下调总是暴露在相反的转基因食品的条件植物的根部观察。特别是，CAT3表现出最高的下调（图5），接着在由APX1，FSD1，RBOHD和TAPX（图4）顺序的下调。 交叉耐受性到生物和生物胁迫是由参与几个生化途径不同的基因的激活提供。RRTF1转录因子促进协同共激活这些途径15,31的基因表达，并且可以潜在地参与氧化应激32。因此，预计RRTF1的上调时除氧降低。根清除酶的下调相关与RRTF1的上调，其作用在响应于氧化应激增加。 CAT3，APX1和TAPX的戏剧性根下调表示根细胞以清除H 2 O的2，这是伴随着由FSD1下调到dismutate超氧阴离子的能力降低的能力降低。氧化应激反应是根，这似乎是相反的转基因食品认知的主要部位高。 图1.地磁场补偿系统。 （A）的三轴线圈（包括一对线圈八角每个三个方向轴的）用于反向地磁场矢量。 （B）的一种计算机控制的电源被连接到各对亥姆霍兹线圈。 （图中电压是任意） <a href="https://www.jove.com/files/ftp_upload/53286/53286fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53286/53286fig2.jpg" /> 图2.影响地磁逆转对拟南芥的形态 。曝光后，控制植物十天（ 即那些暴露在正常条件下GMF）显示显著更大的根长，更EXPANDED传单相比，暴露于扭转GMF条件的植物。公制吧= 18毫米。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig2large.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/53286/53286fig3.jpg" /> 图对拟南芥基因表达3.影响地球磁场逆转。曝光后十天，控制和处理的植物的总RNA提取和实时​​PCR表达分析来分析。反转的转基因食品的效果，诱导该测试CRU3，十字花科3所有基因的基因表达的急剧变化; COTP1，铜传输蛋白1; RRTF1，氧化还原响应转录因子1。线表示标准误差;星号表示显著（P &lt;0.05）复之间的差异暴露在扭转，转基因食品的正常条件下的蚂蚁。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig3large.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/53286/53286fig4.jpg" /> 图对拟南芥的抗氧化剂相关基因的表达4.影响地球磁场逆转。曝光后十天，控制和处理的植物的总RNA分离，并使用实时PCR用于基因表达分析。反转的转基因食品的作用是诱导芽基因表达无显著的变化;然而，大幅下调，观察植物的根系基因表达逆转基因食品的条件下生长TAPX，Thylakoidal抗坏血酸过氧化物 ; APX1，抗坏血酸Peroxidase1; FSD1，铁超Dismutase1; RbohD，NA。DPH /呼吸爆发氧化酶的活性蛋白D线表示标准误差;星号表示受到扭转，转基因食品的正常条件下植物之间显著（P &lt;0.05）的区别。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53286/53286fig5.jpg" /> 图5.影响地球磁场逆转对拟南芥过氧化氢 ​​酶3（CAT3）基因的表达 。曝光后十天，控制和处理的植物的总RNA分离，并使用实时PCR用于基因表达分析。反转的转基因食品的作用是诱导芽基因表达无显著的变化;然而，急剧下调中观察到颠倒GMF条件下生长的植物的根的基因表达。线表示标准埃罗- [R;星号表示受到扭转，转基因食品的正常条件下植物之间显著（P &lt;0.05）的区别。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression at JoVE.com

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

地磁场（GMF）和植物进化:调查逆转基因食品对影响<em&gt;拟南芥</em&gt;开发与基因表达

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

geomagnetic-field-gmf-plant-evolution-investigating-effects-gmf

Research

JoVE Journal

Developmental Biology

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

13.2K 次观看.  University of Turin. This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

视频: Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

科研

遗传学

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

发育生物学

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

地磁场（GMF）和植物进化:调查逆转基因食品对影响

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提高吞吐量的轰击大麦糊粉层层中基因表达的测定

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

表征在拟南芥胚方法

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

高效，快速的早期胚胎分离

种皮床品检测到基因探索

测量时空的Ca

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定

成像使用烟草瞬时表达系统MAPK信号通路的空间重组