西瓜与瓶装葫芦之间的同源和异种移植在冷反应微 rna 研究中的应用
1Institute of Vegetables,Zhejiang Academy of Agricultural Sciences, 2State Key Lab Breeding Base for Sustainable Control of Plant Pest and Disease,Zhejiang Academy of Agricultural Sciences, 3Institute of Horticulture,Zhejiang Academy of Agricultural Sciences, 4Shanghai Biozeron Biotechnology Co., Ltd
Summary
本文提出了一个详细的方案, 有效地制作西瓜和葫芦之间的同质和异种移植, 除了组织取样、数据生成和数据分析的方法, 用于研究冷反应微 rna。
Abstract
微 rna (mirna) 是大约20-24 特的内生小非编码 rna, 已知在植物发育和适应中发挥着重要作用。有越来越多的证据表明, 某些 mirna 的表达在嫁接时发生了变化, 这是农民通常用来提高作物对生物和非生物胁迫能力的农业做法。与包括西瓜在内的许多其他主要黄瓜相比, 瓶葫芦是一种具有固有气候抵御能力的作物, 使其成为西瓜中使用最广泛的根茎之一。最近高通量测序技术的发展为研究冷反应 mirna 及其对异种移植优势的贡献提供了巨大的机会;然而, 适当的实验程序是实现这一目标的先决条件。在这里, 我们提出了一个详细的协议, 有效地生成同异种之间的冷易感西瓜和耐寒瓶葫芦, 除了组织取样, 数据生成和数据分析的方法。所提出的方法也适用于其他植物移植系统, 用于检测各种环境压力下的 mirna 法规, 如热、干旱和盐度。
Introduction
长期以来, 嫁接一直被用作提高作物产量和耐受生物和非生物胁迫 1,2,3的农业技术。在异质嫁接系统中, 精英根系可以提高植物的水分和养分吸收, 增强对土壤病原体的抵抗力, 并限制金属毒性 4,5的负面影响, 这可能会增加嫁接增长活力和提高对环境压力的耐受性。在许多情况下, 异种嫁接也会影响园艺植物中的果实品质, 从而改善水果风味, 增加与健康相关的化合物6,7的含量。研究发现, 植物激素、rna、肽和蛋白质在根茎和接穗之间的远距离转移是调节接穗植物生长发育的一个基本机制 8,9 ,10。接枝在与环境适应有关的远距离信号和运输研究中得到了广泛的应用。接枝实验对于在接收组织或血管液中明确检测传输分子以及由于信号传输而激活或抑制分子目标的作用特别大。
据报道, 非编码 rna 是一种在细胞中发挥重要调控功能的大一类 rna, 它在促进植物适应非生物胁迫方面发挥了作用.mirna 是大约 20-24 nt 的内生小非编码 rna. 研究揭示了 mirna 在植物活动各个方面的调控作用, 如幼苗生长、侧根形成14、15、16、营养吸收, 硫酸盐代谢和稳态 17,以及对生物和非生物胁迫的反应18。近年来, mirna 及其靶向基因的表达与异种嫁接黄瓜幼苗的耐盐胁迫性有关.在葡萄的种间移植中, mirna 表达对干旱胁迫的反应被发现是遗传依赖性的 20。
高通量测序技术的快速发展和成本的降低, 为研究农艺植物中的 mirna 规律提供了良好的契机。西瓜 (瓜兰特 [thunb.]曼斯夫) 是全世界种植的一种重要的葫芦作物, 容易受到低温的影响。瓶装葫芦是一种更具气候弹性的黄瓜, 农民通常用西瓜移植。本研究的主要目的是建立一种标准、高效、简便的西瓜之间异种移植方法.曼斯夫)和瓶装葫芦 (紫菜 [molina] standl).该协议还提供了一个详细的实验方案和分析程序, 用于研究接枝后 mirna 表达的调节, 这对于揭示异质接枝优势背后的机制是有用的。
本研究使用的植物材料包括西瓜品种和葫芦林。西瓜品种是一种产量高、但容易受到低温影响的商业品种。瓶装葫芦是一种很受欢迎的西瓜、黄瓜和葫芦嫁接的根茎, 因为它具有良好的耐低温能力.
Protocol
1. 种子灭菌和萌发 对于表面灭菌, 将瓶装葫芦种子浸泡在500毫升的烧杯中, 在58°c 下充满水, 偶尔搅拌, 直到水温下降到40°c。 同时, 将3公斤泥炭土放入尼龙袋中, 对其进行灭菌, 以 120°cse0.5 mpa 高压灭菌20分钟。 在不搅拌的情况下, 将葫芦种子再浸泡4-5小时。 一旦水达到室温, 用蒸馏水冲洗种子 2x-3倍。 将多余的水排干, 让种子在 2 8°c 的纱布袋中发芽, 在黑暗中?…
Representative Results
图 2: 在室温和冷应力条件下各种移植物的表型.(a) 该面板显示在室温下的同型和异种嫁接幼苗作为对照。(b) 该面板显示经过48小时的冷处理后的同型和异种嫁接幼苗。请点击这里查看此图的较大版本.</p…
Discussion
在该方案中, 我们详细介绍了一种高效、可重复的方法, 使西瓜与葫芦之间的同质和异种移植物。这种方法不需要特定的设备, 操作起来非常容易, 通常嫁接的存活率非常高。该方法还可用于制作其他黄瓜的嫁接物, 如西瓜、黄瓜和南瓜之间的嫁接物。
值得注意的是, 根茎和接穗的相对大小 (年龄) 对于成功嫁接至关重要 ( 《议定书》第2.2 步)。我们观察到, 如果所使?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金 (31772191)、浙江省公益研究项目 (2017c32027)、浙江省植物育种重点科学项目 (2017C32027) 和国家项目计划的支持。支持一流的青年专业人员 (到 p. x.)。
Materials
| TRIzol Reagent | Invitrogen | 15596026 | |
| RNA-free DNase I | Takara | D2270A | |
| Truseq Small RNA sample prep Kit | Illumina | RS-200-0012 | |
| 2100 Bionalyser | Agilent | 5067 | |
| DNA Polymerase | Thermo Fisher Scientific | F530S | |
| UEA sRNA workbench 2.4-plant version (software) | NA | NA | http://srna-workbench.cmp.uea.ac.uk/ |
| Rfam 11.0 database (website) | NA | NA | http://rfam.janelia.org |
| miRBase 22.0 (website) | NA | NA | http://www.mirbase.org/ |
| MIREAP(software) | NA | NA | https://sourceforge.net/projects/mireap/ |
| TargetFinder (software) | NA | NA | http://targetfinder.org/ |
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Wang, L., Wu, X., Li, G., Wu, X., Qin, D., Tao, Y., Xu, P. Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs. J. Vis. Exp. (141), e58242, doi:10.3791/58242 (2018).