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Abstract
Introduction
Protocol
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Materials
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Biology

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Published: January 25, 2018
doi:
10.3791/57258
,
1Department of Biology,California State University, Northridge

Summary

我们开发了一个简单和有效的协议, 以制备大量的大豆原生质体研究复杂的调控和信号机制的活细胞。

Abstract

大豆 (甘氨酸最大值(L.)含笑) 是一种重要的农作物品种, 已成为研究遗传和生物化学途径的豆科动物模型。因此, 建立高效的大豆瞬时基因表达系统是十分重要的。在这里, 我们报告了一个简单的协议, 大豆原生质体的制备及其应用的瞬态功能分析。我们发现, 大豆幼苗的幼单叶叶片产生了大量高质量的原生质体。通过优化 PEG-钙介导的转化方法, 利用大豆单叶原生质体实现了高转化率。该系统为检测活大豆细胞中复杂的调控和信号机制提供了一个高效、通用的模型, 有助于更好地了解豆类的不同细胞、发育和生理过程。

Introduction

原生质体是细胞壁被移除的植物细胞。由于它们保持植物细胞的大部分功能和活动, 原生质体是一个良好的模型系统, 观察和评价各种细胞事件, 是有价值的工具, 研究体细胞杂交1和植物再生2。原生质体也被广泛用于植物转化3,4,5, 因为细胞壁会阻止 DNA 进入细胞。原生质体具有完整植物的一些生理反应和细胞过程, 因此为研究亚细胞蛋白定位的基础研究提供了基本的价值6,7,8,蛋白质-蛋白质相互作用9,10和启动子活动11,12,13在活细胞中。

首次报道了植物原生质体的分离, 在 1960年14 , 对原生质体分离和转化的协议进行了开发和优化。原生质体分离的标准程序包括叶片的切割和细胞壁的酶消化, 然后分离出未被消化的组织碎片释放出的原生质体。转换策略包括电穿孔15,16, 微注射17,18, 和聚乙二醇基 (PEG)4,5,19方法。广泛的物种已报告原生质体分离成功, 包括柑桔20, 芸薹21, 茄22和其他观赏植物系列23,24。虽然不同的组织类型用于各种物种, 一个系统的瞬态表达在拟南芥叶肉原生质体 (TEAMP) 分离从模型植物的叶子拟南芥已经建立良好的25并广泛应用于不同的应用程序。

大豆 (甘氨酸最大值(L.)含笑.) 是最重要的蛋白质和油料作物之一26。与拟南芥和大米不同, 获得转基因大豆植株是相当困难和低效率的。农杆菌介导的浸润已普遍用于烟草表皮细胞的瞬态基因表达研究27和幼苗在拟南芥28,29, 而农杆菌杆菌已用于大豆毛状根的转化,30。病毒诱导的基因沉默方法已被用于下调的目标基因31,32和瞬态表达式33系统的方式。原生质体为这些方法提供了一个有价值的和多才多艺的替代品。原生质体可以从大豆的地上材料中获得, 并允许快速、同步的转基因表达。然而, 自从大豆原生质体在 198334初期成功分离后, 大豆原生质体的应用有了有限的报告35、36、37、38, 主要是由于大豆原生质体的产量相对较低。

本文介绍了一种简单有效的大豆原生质体分离方法及其在瞬态基因表达研究中的应用。利用大豆幼苗的幼单叶叶, 我们能够在几个小时内获得大量的重要原生质体。此外, 我们还优化了 PEG-钙介导的转化法, 使 DNA 转化为高效的大豆原生质体是简单、低成本的。

Protocol

1. 植物的生长 母猪 5-10 大豆种子 (威廉斯 82) 在一个 13 cm 罐在温室在长的天情况下 (16 h 光在1500µmol m-2 s-1) 在25° c 在风俗土壤混合为大豆 (1:1: 1 土、珍珠岩和鱼雷砂的比值。 2. 质粒 DNA 的制备 使用无菌吸管尖端或牙签, 选择一个单一的殖民地或冷冻甘油库存的大肠杆菌携带的质粒含有的利益基因和接种它在20毫升 Luria Bertani (LB) 液体?…

Representative Results

对10天的老大豆不同器官进行了原生质体准备 (图 1), 并在显微镜下观察了产量 (图 2)。从下胚轴和上的细胞壁几乎没有被消化, 有些细胞相互连接 (图 2B, 2C)。在子叶 (图 2D) 和根 (图 2A) 中, 单元格壁仅在单元格的一小部分中被移除。相比之下, 在使用…

Discussion

本方案对大豆原生质体的分离及在瞬态表达研究中的应用进行了全面的测试, 并在实验室中得到了很好的效果。程序简单易得, 需要普通设备和最低成本。我们的协议产生了大量的均匀, 高质量的原生质相比, 以前报告的方法34,35,36,37,38。然而, 由于有许多因素影响原生质体的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了来自国家科学基金会 (NSF-PGRP-IOS-1339388) 的植物基因组研究项目的支持。

Materials

MES Sigma Aldrich  M8250-100G
Cellulase CELF Worthington Biological Corporation LS002611
Pectolyase Y-23 BioWorld 9033-35-6
CELLULASE "ONOZUKA" R-10 yakult 10g
MACEROZYME R-10 yakult 10g
Mannitol ICN Biomedicals  152540
CaCl2 Fisher  C79-500g 
BSA NEB R3535S
DTT Sigma Aldrich  D5545-5G
NaCl Sigma Aldrich  S7653-1kg
KCl Fisher  P217-500g 
MgCl2 Sigma Aldrich  M8266-100g
PEG4000 Fluka 81240
nylon mesh carolina 652222N
Tissue Culture Plates  USA Scientific CC7682-7506
Razor Blades Fisher 12-640
hemacytometer hausserscientific 1483
QIAprep Spin Miniprep Kit Qiagen 27104
EZNA plasmid miniprep kit Omega D6942-01
GeneJET Plasmid Miniprep Kit Thermo Scientific K0502
Centrifuge 5810 eppendorf 5811000827
Centrifuge 5424 eppendorf 22620401
Jencons Powerpette Plus Pipet Controller Jencons 14526-202
Zeiss 710 Confocal Microscope Zeiss N/A
Nonstick, RNase-free Microfuge Tubes, 1.5 mL Ambion AM12450
15 mL Centrifuge Tubes Denville C1018-P
50 mL Centrifuge Tubes Denville C1060-P
Newborn Calf Serum Thermo Scientific 16010159
Soil Ingram's Nursery
perlite Vigoro 100521091
Torpedo Sand JKS Ventures
LB Broth, Lennox (Powder) Fisher BP1427-500
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References

  1. Melchers, G., Labib, G. Somatic hybridisation of plants by fusion of protoplasts. Mol. Gen. Genet. 135 (4), 277-294 (1974).
  2. Gresshoff, P. M. In vitro culture of white clover: callus, suspension, protoplast culture, and plant regeneration. Bot. Gaz. 141 (2), 157-164 (1980).
  3. Lörz, H., Baker, B., Schell, J. Gene transfer to cereal cells mediated by protoplast transformation. Mol. Gen. Genet. 199 (2), 178-182 (1985).
  4. Hayashimoto, A., Li, Z., Murai, N. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol. 93 (3), 857-863 (1990).
  5. Koop, H. -. U., et al. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta. 199 (2), 193-201 (1996).
  6. Hrazdina, G., Wagner, G. J., Siegelman, H. W. Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts. Phytochemistry. 17 (1), 53-56 (1978).
  7. Lin, W., Wittenbach, V. A. Subcellular localization of proteases in wheat and corn mesophyll protoplasts. Plant Physiol. 67 (5), 969-972 (1981).
  8. Vögeli-Lange, R., Wagner, G. J. Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves. Plant Physiol. 92 (4), 1086-1093 (1990).
  9. Chen, S., et al. A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol. Plant Pathol. 7 (5), 417-427 (2006).
  10. Wu, F. -. H., et al. Tape-Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method. Plant methods. 5 (1), 16 (2009).
  11. Christensen, A. H., Sharrock, R. A., Quail, P. H. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18 (4), 675-689 (1992).
  12. Marcotte, W. R., Bayley, C. C., Quatrano, R. S. Regulation of a wheat promoter by abscisic acid in rice protoplasts. Nature. 335 (6189), 454-457 (1988).
  13. Dron, M., Clouse, S. D., Dixon, R. A., Lawton, M. A., Lamb, C. J. Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts. P. Natl. A. Sci. USA. 85 (18), 6738-6742 (1988).
  14. Cocking, E. A method for the isolation of plant protoplasts and vacuoles. Nature. 187 (4741), 962-963 (1960).
  15. Zhang, H., et al. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep. 7 (6), 379-384 (1988).
  16. Fromm, M., Taylor, L. P., Walbot, V. Expression of genes transferred into monocot and dicot plant cells by electroporation. P. Natl. A. Sci. USA. 82 (17), 5824-5828 (1985).
  17. Crossway, A., et al. Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol. Gen. Genet. 202 (2), 179-185 (1986).
  18. Holm, P. B., Olsen, O., Schnorf, M., Brinch-Pedersen, H., Knudsen, S. Transformation of barley by microinjection into isolated zygote protoplasts. Transgenic Res. 9 (1), 21-32 (2000).
  19. Schapire, A. L., Lois, L. M. A simplified and rapid method for the isolation and transfection of Arabidopsis leaf mesophyll protoplasts for large-scale applications. Plant Signal Transduction: Methods and Protocols. 1363, 79-88 (2016).
  20. Niedz, R. P. Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes. Plant Cell Tiss. Org. Cult. 84 (3), 353-357 (2006).
  21. Sheng, X., et al. Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis). Plant Cell Tiss. Org. 107 (3), 513-520 (2011).
  22. Grun, P., Chu, L. -. J. Development of plants from protoplasts of Solanum (Solanaceae). Am. J. Bot. 65 (5), 538-543 (1978).
  23. Davey, M. R., Anthony, P., Power, J. B., Lowe, K. C. Plant protoplasts: status and biotechnological perspectives. Biotechnol. Adv. 23 (2), 131-171 (2005).
  24. Pitzschke, A., Persak, H. Poinsettia protoplasts-a simple, robust and efficient system for transient gene expression studies. Plant methods. 8 (1), 14 (2012).
  25. Yoo, S. -. D., Cho, Y. -. H., Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2 (7), 1565 (2007).
  26. Sedivy, E. J., Wu, F., Hanzawa, Y. Soybean domestication: the origin, genetic architecture and molecular bases. New Phytol. 214 (2), 539-553 (2017).
  27. Yang, Y., Li, R., Qi, M. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J. 22 (6), 543-551 (2000).
  28. Marion, J., et al. Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J. 56 (1), 169-179 (2008).
  29. Wu, H. -. Y., et al. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant methods. 10 (1), 19 (2014).
  30. Govindarajulu, M., Elmore, J. M., Fester, T., Taylor, C. G. Evaluation of constitutive viral promoters in transgenic soybean roots and nodules. Mol Plant Pathol. 21 (8), 1027-1035 (2008).
  31. Nagamatsu, A., et al. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotechnol. J. 5 (6), 778-790 (2007).
  32. Juvale, P. S., et al. Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean. Mol. Plant Pathol. 13 (9), 1140-1148 (2012).
  33. Zhang, C., Bradshaw, J. D., Whitham, S. A., Hill, J. H. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol. 153 (1), 52-65 (2010).
  34. Lin, W. Isolation of mesophyll protoplasts from mature leaves of soybeans. Plant Physiol. 73 (4), 1067-1069 (1983).
  35. Yi, J., et al. A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean. Plant J. 62 (6), 1019-1034 (2010).
  36. Faria, J. A., et al. The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway. BMC Plant Biol. 11 (1), 129 (2011).
  37. Kidokoro, S., et al. Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression. Plant J. 81 (3), 505-518 (2015).
  38. Sun, X., et al. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep-UK. 5, 10342 (2015).
  39. Karimi, M., Inzé, D., Depicker, A. GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7 (5), 193-195 (2002).
  40. Xia, Z., et al. Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering. P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).

Tags

Keywords: Soybean ProtoplastsTransient Gene ExpressionRegulatory NetworksProtein InteractionsUnifoliate LeavesEnzyme SolutionVacuum InfiltrationProtoplast DigestionW5 SolutionNylon MeshCentrifugation
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Wu, F., Hanzawa, Y. A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses. J. Vis. Exp. (131), e57258, doi:10.3791/57258 (2018).
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