Name: agrobacterium-mediated genetic transformation, transgenic production, and its application for the study of male reproductive development in rice
Uploaded: 2020-10-06T14:00:00
Description: Watch this Scientific Journal Video about Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice at JoVE.com

作者感谢陈小飞为幼米提供花序，协助制作水稻组织培养媒介。这项工作得到了国家自然科学基金（31900611）的支持。

1. sgRNA-CAS9植物表达载体 构造和农业细菌-中栽转化
<ol><li>根据28日出版的文献，在水稻中瞄准雄性无菌基因OsABCG15。</li>
	<li>在 OsABCGG15 的第二个 exon 中，为位于 106-125 bp 之间的目标站点设计 sgRNA（图 1）。</li>
	<li>使用 T4 多核苷酸激酶合成 sgRNA 寡糖 （sgR-osABCG15-F： 5'TGGCAAGCACATCCTCAAGAT3' 和 5'sgR-OsABCG15-R：ACATCCCCTGTGTGTT'）。</li>
	<li>使用内核...

<ol>
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人工原生雄性无菌突变体传统上由随机的物理、化学或生物突变产生。虽然这些都是强大的技术，他们的随机性质未能利用大量的现代基因组知识，有可能提供定制的改进分子育种32。CRISPR-Cas9系统由于其操作和编辑DNA29，33的简单而经济实惠的手段，在植物中得到了广泛的应用。与传统的诱变方法相比，它具有节省时间的潜力。
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稻米是最重要的粮食作物，特别是在发展中国家，是世界人口一半以上的主食。总体而言，对稻谷的需求正在增长，预计到2030年将增长50%，到2050年将增长100%。,2未来水稻产量的提高需要利用多样化的分子和遗传资源，使水稻成为单体植物研究的优秀模型。其中包括一个有效的转换系统，先进的分子图，和公开访问的数据库的表达序列标签，这是多年来产生的,3，4。提高作物产量的一个策略是杂交种子生产5，其中一个核心要素是操纵男性生育能力。了解谷物作物中男性肥力的分子控制有助于将关键知识转化为实用技术，以提高杂交种子产量，提高作物,产量。
基因转化是基础研究和商业农业的关键工具，因为它能够引进外来基因或操纵作物中的内源基因，并产生转基因线。适当的转化方案可以帮助加速遗传和分子生物学研究，以基本理解基因调控8。在细菌中，遗传转化是自然发生的;然而，在植物中，它是使用分子生物学技术9，10,人为执行的。农业细菌Tumefaciens是一种土壤传播的、格拉姆阴性细菌，通过T-DNA（Ti质粒的一个区域）通过IV型分泌系统11，12将T-DNA转移到植物细胞中，导致植物冠胆病。在植物中，A.tumefaciens-中照转化被认为是一种广泛的基因修饰方法，因为它导致T-DNA稳定且低拷贝数集成到宿主基因组13中。转基因水稻是1990Agrobacterium年代中期在雅波尼卡品种14中首次通过农业细菌培养基因转化产生的。利用该协议，在4个月内获得多条转基因线路，转化效率为10%~30%。研究表明，成功转化有两个关键步骤：一是胚胎基因从成熟种子中诱导，另一个是将乙酰胺酮（一种酚类化合物）加入到共同培养的细菌培养中，从而在14、15,植物中提高转化效率。该协议已广泛使用与小改动在japonica16，,17，,18， 19,19以及其他品种，如稻谷20， 21，,21,22，,23和热带贾波尼卡24，,25.事实上，超过80%的关于水稻转化的文章使用农业细菌中位基因转化作为工具13。迄今为止，已经开发出几种基因转化方案，利用水稻种子作为卡卢斯诱导16、17、18、19,17,18,的起始材料。然而，对于年轻的花序作为卡卢斯生产的外植，却很少为人所知。总体而言，为功能基因组学和作物改良研究建立快速、可重复、高效的基因转化和再生协议非常重要。
近年来，CRISPR-Cas9技术的发展产生了精确的基因组编辑机制，以了解基因功能，为植物育种提供农,27艺上的重要改进。CRISPR也为男性生殖发育和杂交生产提供了相当大的希望。在这项研究中，我们利用CRISPR-Cas9技术利用基因敲除系统，并结合使用幼年花序作为外植的高效水稻基因转化方案，从而为研究生殖发育创造稳定的雄性无菌线。

<table>
	<tbody>
		<tr>
			<td>1-Naphthaleneacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0640</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#65292;4-Dichlorophenoxyacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7299</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Benzylaminopurine (6-BA)</td>
			<td>Sigma-Aldrich</td>
			<td>B3408</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10000561</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10002918</td>
			<td></td>
		</tr>
		<tr>
			<td>Aneurine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Sangon Biotech</td>
			<td>A100636</td>
			<td></td>
		</tr>
		<tr>
			<td>Beef extract</td>
			<td>Sangon Biotech</td>
			<td>A600114</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10004808</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>20011160</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein acid hydrolysate</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>C184</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(&#8545;) chloride hexahydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10007216</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(&#8545;) sulfate pentahydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10008218</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>63005518</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>63011037</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, Disodium Salt, Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>EOS Digital SLR and Compact System Cameras</td>
			<td>Canon</td>
			<td>EOS 700D</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully Automated Rotary Microtome</td>
			<td>Leica Biosystems</td>
			<td>Leica RM 2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10000208</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>62011516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>H370</td>
			<td></td>
		</tr>
		<tr>
			<td>Inositol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>63007738</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10011517</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(&#8545;) sulfate heptahydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10012116</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycine</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>K378</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinetin</td>
			<td>Sigma-Aldrich</td>
			<td>K0753</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>62004034</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>62004736</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>G229</td>
			<td></td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>P698</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10013018</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese sulfate monohydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10013418</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td>NIKON</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>MS</td>
			<td>Phytotech</td>
			<td>M519</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0765</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10016308</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10017608</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10017160</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium nitrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>1001721933</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine Hydrochloride (B6)</td>
			<td>Sigma-Aldrich</td>
			<td>47862</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>R501</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019718</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium molybdate dihydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019816</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscopes</td>
			<td>Leica Microsystems</td>
			<td>Leica M205 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10021418</td>
			<td></td>
		</tr>
		<tr>
			<td>Technovit embedding Kits 7100</td>
			<td>Heraeus Teknovi, Germany</td>
			<td>14653</td>
			<td></td>
		</tr>
		<tr>
			<td>Timentin</td>
			<td>Beijing XMJ Scientific Co., Ltd</td>
			<td>T869</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>T3260</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for paraffin sections</td>
			<td>Leica Biosystems</td>
			<td>Leica HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sangon Biotech</td>
			<td>A515245</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10024018</td>
			<td></td>
		</tr>
	</tbody>
</table>

agrobacterium-mediated genetic transformation, transgenic production, and its application for the study of male reproductive development in rice

雄性不育是杂交种子生产的重要农艺特征，通常以雄性生殖器官/游戏体的功能缺陷为特征。CRISPR-Cas9基因组编辑技术的最新进展使特定位点内源候选基因的编辑效率高，而且可以节省时间。此外， 水稻的农业细菌中位基因转化也是基因改造的关键方法，已被许多公立和私营实验室广泛采用。在这项研究中，我们应用CRISPR-Cas9基因组编辑工具，通过目标基因组编辑 OsABCG15在japonica 培养中成功生成三 条雄性无菌 突变线。我们采用一 种改良的农业细菌调剂水稻转化方法，为水稻杂交种子生产提供了优良的遗传消瘦手段。转基因植物可以在2~3个月内获得，通过PCR扩增和桑格测序通过基因分型筛选同源转化剂。通过对水稻雄性生殖器官的微观观察，通过碘碘化钾（I2-KI）染色半薄横截面进行花粉生存能力分析，对雄性无菌同源线进行基本表型表征。

这里演示的是利用基因编辑技术，为水稻中农业细菌的遗传转化的未来研究创建一条雄性无菌线。为了创建雄性无菌线osabcg15，CRISPR-CAS9中位突变用于二元向量构造。sgRNA由OsU3启动子驱动，而hSpCas9的表达盒是由双35S启动子驱动的，中间载体组装在一个二进制载体pCAMBIA1300中，专为农业细菌中调解的水稻稳定转化而设计。图1A提供了用于CRISPR/Cas9中切水稻突变的构造的<strong ...

Watch this Scientific Journal Video about Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice at JoVE.com

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

这项工作描述了使用CRISPR-Cas9基因组编辑技术敲除内源 基因OsABCG15， 然后修改的农业细菌 中培养转化方案，在水稻中产生稳定的雄性-无菌线。

农业细菌-中生菌遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

年轻的花序作为卡卢斯生产的基因外显，对于建立快速有效的基因转化和再生方法揭示基因功能以及作物改良非常重要。年轻的花序作为卡卢斯生产的基因外显，对于建立快速有效的基因转化和揭示基因功能的再生方法至关重要。这种技术的视频演示将帮助观看者了解维护材料的过程，以产生所需的转基因对齐，而不会浪费时间和精力。首先从稻田或温室中采集幼米花序，确保它们被叶套覆盖。用 70%酒精拭子擦拭每个花序，在切割前晾干。将花序带到无菌实验室的长凳上，用消毒剪刀将其切成小块，然后将切口转移到含有 NBD2 介质的培养板中。在黑暗中26摄氏度的温度下孵育板10至14天，以诱导卡卢斯。要进行转化，在50毫升锥形无菌试管中，将一个带选择性抗生素的白色EB板转移到含有相同抗生素的5毫升液体YEB介质中。在轨道摇床上以250倍g和25至28摄氏度摇动管子，直到细菌长到0.5的OD 600。在250毫升锥形烧瓶中加入100毫升的YEB介质中1毫升的细菌悬浮液，并在250倍g和28摄氏度的轨道摇床上摇动烧瓶，4小时。在室温下将培养在4000克的培养器离心10分钟，以收集细菌。丢弃上一杯，用 AAM AS 介质重新悬浮颗粒，然后将悬浮液稀释至 0.4 的 OD 600。孵育后，将大约150个健康的浅黄色胚胎卡收集到150毫升的无菌烧瓶中。将50至75毫升的细菌细胞悬浮液加入烧瓶中，然后加入10至25毫升的新鲜AM AS介质，浸入10至20分钟，偶尔摇晃。小心地将细菌悬浮液从烧瓶中倒出，然后用无菌滤纸干燥卡利，然后将它们放在带 NBD AS 介质的培养皿上，然后用滤纸盖住它们。在黑暗中在25至28摄氏度的温度下孵育卡利三天，检查它们是否细菌过度生长。经过三天的共同栽培，使用滤纸将卡利转移到无菌培养皿中，然后在干净的长凳上晾干两个小时。确保卡利未粘附在滤纸上，并将它们传输到主选择介质 NBD2。两周后，均匀地将卡利转移到含有新鲜选择介质的新板中，然后将卡利移动到新鲜的MS介质进行分化，将芽移动到MS介质，每升10毫克的海格霉素可增殖。将新芽转移到半MS介质中进行根诱导，并在25至28摄氏度的光线下培养它们。转化的植物应在两周内产生根。要进行生殖表型观察，请从花光中去除古色，并在立体显微镜下成像整个表型。为了观察花粉的可行性，在花前挑选成熟的稻穗，取出古色和白草，释放花粉。拿六个安特，把它们放在玻璃滑梯上。加入一滴蒸馏水，用钳子碾碎，然后释放花粉颗粒，然后加入两到三滴碘溶液，用盖玻片盖住。在低放大显微镜下观察幻灯片。花粉粒染成黑色显示更旺盛的生存能力，而没有颜色的颗粒或那些染色的黄棕色的颗粒发育迟缓或退化。要进行横向分切显微镜，请使用 0.5% 的二丁蓝色溶液将幻灯片染色 30 分钟，然后用水冲洗并在烟罩中干燥。干燥后，用中性树胶密封幻灯片，轻轻将盖玻片放在幻灯片上，将其干燥在烟罩中，然后在显微镜下对样品进行成像。该协议用于通过农业细菌培养的水稻遗传转化创建男性无菌线。胚胎致病直接用于转化或亚培养增殖，以获得更多的卡皮进一步感染。经过48小时的共同培养，该卡利被转移到第二轮选择媒介。10到14天后，转换的卡利显示新形成的微卡利，而未转换的卡利变成棕色和死亡。后来，健康和奶油色的卡利被转移到再生介质。改造后的卡利逐渐显示出绿点。这些绿点在再生培养中培养，允许拍摄生长，然后转移到半MS培养中诱导根。后来，健康而充满活力的生长芽与发达的根收获。共获得21株再生幼苗。Hygromycin 区域的 PCR 扩增表明，转化频率约为 85.71% 转化剂被基因型，以识别 sgRNA 靶点处的突变。在18种转基因植物中，有8种异质线和3条同源线为CRISPR CAS9阳性。通过基本男性生殖器官观察验证了同源敲除突变体。osabcg15的外型比野生植物更小、更苍白，缺乏成熟的花粉颗粒。对osabcg15中的外向形态缺陷进行了横向剖面显微镜研究。在第10阶段，野生型微孢子变成圆形，并用深蓝色染色的外显子进行排泄。相比之下，osabcg15微孔折叠和降解。在尝试此协议时，必须确保收集的材料处于 meiosis 阶段，并控制 calli 的风干时间。

Research

JoVE Journal

Biology

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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

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

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 ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

转基因检测鼠类量化男性生殖细胞突变频率

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

使用形态基因的农业细菌 -顽固玉米近亲繁殖系的介导未成熟胚胎转化

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

用化学发光法实时检测水稻免疫反应中活性氧的产生

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...