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Genetics

机器人平台的高通量原生质体分离和转型

Published: September 27, 2016
doi:
10.3791/54300
, ,
1Department of Plant Sciences,University of Tennessee, Knoxville, 2Center for Renewable Carbon,University of Tennessee, Knoxville, 3Department of Mechanical, Aerospace, and Biomedical Engineering,University of Tennessee, Knoxville

Summary

高通量，自动化，烟草原生质体的生产和改造的方法进行描述。该机器人系统可以在模型BY-2系统应该是翻译到非模式作物大规模并行基因表达和发现。

Abstract

在过去的十年里，已经在使用植物原生质体，范围从模型物种作物物种，信号传导途径，转录调控网络，基因表达，基因组编辑，和基因沉默的分析的回潮。此外，显著进展已在植物的原生质体的再生，这已产生在使用这些系统用于植物基因组的甚至更多的利益作出。在这项工作中，一个协议已使用机器人平台已经开发了用于原生质体分离和转化的自动化从'亮黄色'2（BY-2）烟草悬浮培养。使用花椰菜花叶病毒35S启动子（35S）的控制下，为橙色荧光蛋白（OFP）报告基因（pporRFP）的转化方法进行了验证。原生质体OFP表达，荧光显微镜证实。分析还包括使用propidiu原生质体生产效率的方法米碘。最后，被用于原生质体分离过程的低成本食品级酶，绕过用于实验室级的酶，它们成本昂贵的自动化原生质体分离和分析的高通量的需求。根据此工作开发的协议，从原生质体分离到转化完成过程可以在4小时进行中，在不脱离操作者的任何输入。而在这一工作开发的协议是与BY-2细胞培养物中证实，程序和方法应该是可平移的任何植物悬浮培养/原生质体系统，该系统应使作物基因组学研究的加速度。

Introduction

近年来，一直放置在转基因作物的设计克服了各种疾病1，赋予抗除草剂2，赋予干旱3,4和耐盐5显著动力，防止虫食6，提高生物产量7，降低细胞壁顽抗8。这种趋势已经通过新的分子工具的发展，用于产生转基因植物，包括使用CRISPR和TALENS 9基因组编辑，基因通过的dsRNA 10，miRNA的11，和siRNA 12沉默辅助。虽然这些技术简化了转基因植物的一代，他们还创建了一个瓶颈，其中产生的转基因植物的数量之多，无法使用依赖于植株再生的传统系统进行筛选。与此相关的瓶颈，而沉默和基因组编辑构建体能够迅速地插进植物，许多目标性状不能产生所期望的效果，这通常没有发现直到植物在温室中进行分析。在这项工作中，我们开发了用于植物原生质体的快速，自动化，高通量筛选的方法，具体而言，以解决在大量的基因组编辑和基因沉默靶的早期筛查的当前瓶颈。

使用原生质体的，相对于完整的植物细胞，具有几个优点对自动化平台的开发。首先，原生质体的植物细胞壁的消化后分离，以及与此屏障不再存在，转化效率提高13。在完整的植物细胞，只有两种用于转化很好地建立的方法，基因枪法14和农杆菌介导的转化15。这些方法都不能够容易地转化为液体处理平台，生物射弹需要TRANSFO专门的设备细则第十五，而农杆菌介导转化需要共培养和随后的去除细菌。无论是适合于高通量方法。在原生质体的情况下，转换是经常使用聚乙二醇（PEG）介导的转染16，其仅需要几个溶液的交流，并非常适合用于液体处理平台进行。第二，原生质体，顾名思义，是单细胞培养物，从而与在植物细胞培养物结块和链的形成相关的问题，没有在原生质体观察。在使用基于平板分光光度计，细胞凝集快速筛选而言，或细胞在多个平面将导致难以取得一致的测量。因为原生质体也比其培养基稠密，它们沉淀到孔的底部，形成一单层，这有利于对基于板状光度法。最后，尽管植物细胞悬浮培养物是PRIMAR随手从愈伤组织17衍生的原生质体可以从多种植物组织的收获，从而导致识别组织特异性表达的能力。例如，能够分析根级或一个基因的叶特异性表达可以是表型的预测很重要的。由于这些原因，在该工作开发的协议使用从广泛使用的烟草（ 烟草属）“亮黄色'2（BY-2）悬浮培养物分离的原生质体进行了验证。

在BY-2悬浮培养已被描述为高等植物的“的HeLa”细胞，因为在植物细胞中18的分子分析其无处不用途。最近，BY-2细胞已被用于研究植物的影响压力19-22，细胞内蛋白质定位23,24，以及基本的细胞生物学25-27表明在植物生物学这些培养物的广泛的实用性。 BY-2培养物的一个附加的优点是到培养物与阿非迪霉素同步，这可导致增强的基因表达的重现能力研究28。此外，已开发了用于使用低成本酶29,30 BY-2原生质体的提取，如传统上用于产生原生质体的酶是成本过高的高通量系统。因此，下面描述的协议已被使用BY-2悬浮培养验证，但它应该是易于进行的任何植物细胞悬浮培养。证明的概念实验使用CaMV 35S启动子的控制下，从硬盘珊瑚滨橙色荧光蛋白（OFP）报告基因（pporRFP） 滨 31进行。

Protocol

1.悬浮培养的建立 BY-2介质通过加入4.43克Linsmaier＆Skoog基础介质30克蔗糖，200毫克KH 2 PO 4制备的液体，和200微克至900的2,4-二氯苯氧乙酸（2,4-D）毫升蒸馏水水和pH至5.8，用0.1M的KOH。调节pH值之后，调节最终体积至1000毫升蒸馏水和高压釜中。介质可以在4℃保存最多2周。 接种用100ml液体BY-2的媒体和单片BY-2愈伤组织（> 1厘米直径）上生长固BY-2介质的250ml Erlenmeyer烧瓶（?…

Representative Results

在目前的研究中，BY-2的倍增速率从14-18小时取决于在该培养物温育的温度，用15小时的平均细胞周期长度的先前的报告一致变化。与此倍增速率，以1：100起始接种物用于启动培养，导致培养物在5-7天的50％的收集细胞体积（PCV）。在当前的协议，其中培养物在200ml培养基中生长，在7天，这提供了足够的细胞以填充33 6孔板生成100ml的PCV。在原生质体产率而言，产生在协议中?…

Discussion

上述的协议已被成功验证为原生质体分离，计数，以及使用该BY-2烟草悬浮细胞培养物转化;然而，该协议可以很容易地扩展到任何植物悬浮培养。目前，原生质体分离和改造已经在众多的植物，包括玉米（ 玉米 ）10，红萝卜（ 胡萝卜 ）32，杨树（ 胡杨 ）33，葡萄（ 葡萄 ）34，油棕（ 油棕 ）35实现，生菜（ 莴苣 <su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by Advanced Research Projects Agency – Energy (ARPA-E) Award No. DE-AR0000313.

Materials

Orbitor RS Microplate mover Thermo Scientific
Bravo Liquid Handler Agilent
Synergy H1 Multi-mode Reader BioTek
MultiFlo FX Multi-mode Dispenser BioTek
Teleshake Inheco 3800048
CPAC Ultraflat Heater/cooler Inheco 7000190
Vworks Automation Software Agilent Software used to control and write protocols for Agilent Bravo
Momentum Software Thermo Scientific Task scheduling software for controlling Orbiter RS
Liquid Handling Control 2.17 Software Biotek Software used to control and write protocols for MultiFlo FX
IX81 Inverted Microscope Olympus
Zyla 3-Tap microscope camera Andor
ET-CY3/TRITC Filter Set Chroma Technology Corp 49004
Rohament CL AB Enzymes sample bottle low-cost cellulase
Rohapect UF AB Enzymes sample bottle low-cost pectinase
Rohapect 10L AB Enzymes sample bottle low-cost pectinase/arabinase
Linsmaier & Skoog Basal Medium Phytotechnology Laboratories L689
2,4 dichlorophenoxyacetic acid Phytotechnology Laboratories D295
propidium iodide Sigma Aldrich P4170
Poly (ethylene glycol) 4000 Sigma Aldrich 95904-250G-F Formerly Fluka PEG
Propidium Iodide Fisher Scientific 25535-16-4 Acros Organics
CaCl2 Sigma Aldrich C7902-1KG
Sodium Acetate Fisher Scientific BP333-500
Mannitol Sigma Aldrich M1902-1KG
Sucrose Fisher Scientific S5-3
KH2PO4 Fisher Scientific AC424205000
KOH Sigma Aldrich P1767
Gelzan CM Sigma Aldrich G1910-250G
6-well plate Thermo Scientific 103184
96-well 1.2 ml deep well plate Thermo Scientific AB-0564
96 well optical bottom plate Thermo Scientific 165305
Finntip 1000 Wide bore Pipet tips Thermo Scientific 9405 163
NaCl Fisher Scientific BP358-10
KCl Sigma Aldrich P4504-1KG
MES Fisher Scientific AC17259-5000
MgCl2 Fisher Scientific M33-500
DOWNLOAD MATERIALS LIST

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Protoplast IsolationHigh-throughput ScreeningPlant BiotechnologyAutomated Protoplast ProductionProtoplast TransformationBY-2 Suspension CultureMicroplate MoverLiquid HandlerMulti-mode Plate ReaderMulti-mode DispenserPlate HeaterPlate ChillerRobotics SystemPlate Mover SoftwareLabwareContainer TypesStart And End Positions
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Dlugosz, E. M., Lenaghan, S. C., Stewart, Jr., C. N. A Robotic Platform for High-throughput Protoplast Isolation and Transformation. J. Vis. Exp. (115), e54300, doi:10.3791/54300 (2016).
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