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Trichome Isolation and Integrity Test from Brassica villosa and Other Species
芸薹属红丁香和其它物种的表皮毛分离和完整性检测   

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本实验方案简略版
Plant Molecular Biology
Jul 2014

Abstract

The outward growths from one or more epidermal cells are well known as trichomes (plant hair cells) (Levin, 1973; Mathur, 2006). Preparation of pure intact non-glandular trichomes from trichome-rich Brassica villosa depended on rendering the trichomes sufficiently stiff to dislodge them from the leaves in an undamaged state, which can be further used for structural, transcriptome, genome and biochemical or chemical analysis. Dislodging the flexible trichomes from Brassica villosa (B. villosa) with a paintbrush [as in Zhang and Oppenheimer (2004)] proved too gentle, and scraping trichomes off the leaves with a razor blade [as in Zhang and Oppenheimer (2004)] resulted in trichome cell disruption. Aziz et al. (2005) reported isolating alfalfa glandular trichomes by shearing in liquid nitrogen (N2). In the present study, a similar method was used. Non-glandular leaf trichomes were isolated by treating tissue with liquid N2 to stiffen the flexible trichomes, followed by 1 min of shearing force with a common vortex mixer to dislodge the trichomes from their leaf bed (Nayidu et al., 2014). Up-to ~20 % of the trichomes were removed from the leaf surface and the majority were unbroken (confirmed by no staining by trypan blue; Figure 1). Trichomes were then purified by straining a tissue/trichome/water mixture through a sieve. However, leaf tissues also became very brittle after dipping in liquid N2 and broke up into very small pieces if the leaf tissue was agitated for a longer time period. Hence, N2-treated leaf samples could not be re-used to recover remaining attached trichomes. Contaminating leaf pieces from a 1 min shear were larger and easily to separate manually from the detached trichomes within the sieve, rendering a purified intact trichome preparation. The method was also successful at purifying trichomes from soybean (Glycine max) and tomato (Solanum lycopersicum).

Keywords: Trichome (毛状体), Brassica (甘蓝), Isolation (隔离), Villosa (长毛猪笼草), Vertex (顶点)

Materials and Reagents

  1. Fresh trichome-bearing leaf tissue from Brassica villosa (drepanensis cv.) (from two month old up-to two year leaves), soybean cv. B220, and tomato cv. Roma
    Note: Depending on the plant species and the subsequent type of analysis after trichome isolation, any tissue with non-glandular trichomes will likely suffice. However, tissues with denser trichome coverage will provide a greater yield of detached trichomes.
  2. Liquid nitrogen
    Note: Training in the safe use of liquid nitrogen is imperative.
  3. Sterile Millipore purified water
  4. Ice
  5. 0.4 % trypan blue (see Recipes)

Equipment

  1. Gloves to resist freezing temperatures (Cryo-Gloves) (VWR International, catalog number: 32885-736 )
  2. Polyfoam insulated box with ice
  3. 1-ml pipette with plastic pipette tips
  4. 50 ml BD FalconTM conical plastic centrifuge tubes (BD, catalog number: 14-432-22 ) or plastic 250 ml centrifuge bottles (Thermo Fisher Scientific, catalog number: EW-06105-20 )
  5. BD-Falcon nylon mesh cell strainer (40 μm pore size) (BD Biosciences, catalog number: 08-771-1 )
  6. Blunt end forceps to remove contaminating debris from the strainer (Seton, catalog number: AA969 )
  7. Genie 2 vortex mixer (Thermo Fisher Scientific) having round mixer head (for tubes) and flat mixer head (for larger bottles)
  8. -80 °C freezer
  9. 4 °C refrigerator
  10. FlexidryTM unit (FTS Systems, catalog number: 4283 )
  11. Controlled environment greenhouse or growth cabinet.
    Note: Plant growth conditions depend on the nature of subsequent analyses. For example, chemical composition of plant tissuses can change substantially with altered light intensity and temperature (16 h light/8 h dark, at least 400 µE.m-2.s-1, 20/17 °C recommended, but light intensity from 700-1,400 µE.m-2.s-1 will mimic outside daylight).
  12. Axiovert 100 microscope (ZEISS) with 10x magnification

Procedure

  1. Healthy trichome-bearing leaves of B. villosa, soybean, and tomato were selected after growth in a greenhouse.
    Note: For B. villosa, up to 2 year old plant leaves can be used.
  2. Harvested leaves were cut into different sizes based on the size of the vessel used for the isolation of trichome (see Video 1 below).
    Note: One 3 cm x 3 cm leaf piece if using a 50 ml tube and two 10 cm x 4 cm strips if using 250 ml centrifuge bottles. The choice of tube and tissue size is dependent on the amount of trichome tissue needed for subsequent analyses (see recommendations below). For any leaf size used, the maximum purity trichome preparation achieved is ~20% of the total trichomes present on the leaf piece.
  3. Maximum 30% of the tube or bottle was filled with liquid N2 and capped (loosely screwed on) to prevent overflow during mixing (without tube breakage from N2 vapour pressure).
  4. Using gloves, tubes were mixed for 1 min in liquid N2 using a Genie 2 vortex mixer at maximum speed (level 8). One min vortexing maximizes the recovery of detached trichomes while minimizing the disruption of leaf tissue into small contaminating fragments that are difficult to remove later.
  5. The tubes were then kept fully open on ice to maximize liquid N2 evaporation.
  6. Large leaf pieces were manually removed from the tubes using forceps and retained as a trichome-stripped leaf control sample.
  7. Released trichomes adhering to the inside walls of the tube were gently suspended by swirling in 2 ml of sterile Millipore purified water.
  8. Water initially frozen in the tube (because of liquid N2) was kept on ice for 3-4 min to thaw.
  9. Thawed water from the tubes having trichomes (and also minute pieces of leaves) was sieved through a BD-Falcon nylon mesh cell strainer into a fresh 50 ml tubes.
  10. Minute pieces of leaves on the cell strainer netting were again manually removed using forceps.
  11. The strainer was then inverted over the same fresh tube.
  12. Trichomes were dislodged into the tube by gently sweeping them (i.e. rinsing the strainer with a pipette) using an additional 1-2 ml of Millipore water (see Video 1 below).
    Note: Cell strainers could be used up to 8-10 times before the pores became clogged. Intensive careful cleaning will help lengthen strainer use for additional 2-3 times, but new ones are preferred after pore clogging, and further cleaning will result in a hole in the strainer.
  13. Freshly prepared trichome batches (in water) and trichome-dislodged leaves (control) were immediately stored at 4 °C separately until all tissue sections were processed.
  14. 1 ml of water containing trichomes was stained fresh by adding 0.5 ml of 0.4 % trypan blue. Contents were incubated for 3-4 h at room temperature (in the light or dark) without shaking in a petri-dish in preparation for microscopy.
  15. An Axiovert 100 microscope (10x magnification) was used to confirm the proportion of un-ruptured trichome cells and the quality of the preparation.
    Notes:
    1. The trichome isolation protocol has no washing step, but to obtain the image of trichomes (Figure 1) we conducted a washing step using water for improving the appearance of the trichomes.
    2. Intact cells are unstained; disrupted cells are stained blue; a poor quality preparation would contain more disrupted trichomes and/or a greater proportion of contaminating leaf tissue fragments compared with Figure 1.
  16. The samples were then flash frozen in liquid N2, stored at -80 °C for 4-5 h, and freeze dried overnight in a FlexidryTM unit.
  17. The method can be used for purifying non-glandular trichomes from other plant species (Figure 1).
  18. Suggested leaf sample size for isolating B. villosa trichomes for various purposes (leaves with 300-500 µm trichome length, ~4,000 trichomes/cm2 and 1 mg of dried trichomes isolation from 100 g of fresh leaf tissue [FL]): (a) for electron microscopic structural analysis (100/cm2); (b) for RNA isolation (300 g FL); (c) for biochemical extraction (500 g FL); for HPLC-UV analysis (500 g FL); for NMR structural analysis (800-1,000 g FL); for metal content analysis by inductively coupled plasma mass spectroscopy (500 g FL).


    Figure 1. High quality trichome preparations at 10x magnification after isolation from a Brassica villosa b Soybean (Glycine max) c Tomato (Solanum lycopersicum). Arrows indicate contaminating leaf tissue fragments or broken trichomes.

Representative data

Video 1. Detailed procedure for trichome isolation from Brassica villosa

Recipes

  1. 0.4 % trypan blue
    40 mg of trypan blue dissolved in 100 ml Millipore-filtered water

Acknowledgments

This research was supported by grants from the Canola Council of Canada, Sask-Canola, the Saskatchewan Agriculture Development Fund, and funding from Agriculture and Agri-Food Canada.

References

  1. Aziz, N., Paiva, N. L., May, G. D. and Dixon, R. A. (2005). Transcriptome analysis of alfalfa glandular trichomes. Planta 221(1): 28-38.
  2. Koona, P. and Jackai, L. (2004). The potential of pod-shaving in studies of the role of trichomes in Vigna resistance to the pod-bug Clavigralla tomentosicollis Stål (Hemiptera: Coreidae). International Journal of Tropical Insect Science 24(04): 298-303.
  3. Levin, D. A. (1973). The role of trichomes in plant defense. Q Rev Biol: 3-15.
  4. Mathur, J. (2006). Trichome cell morphogenesis in Arabidopsis: a continuum of cellular decisions. This review is one of a selection of papers published in the Special Issue on Plant Cell Biology. Botany 84:604-612.
  5. Nayidu, N. K., Tan, Y., Taheri, A., Li, X., Bjorndahl, T. C., Nowak, J., Wishart, D. S., Hegedus, D. and Gruber, M. Y. (2014). Brassica villosa, a system for studying non-glandular trichomes and genes in the Brassicas. Plant Mol Biol 85(4-5): 519-539.
  6. Zhang, X. and Oppenheimer, D. G. (2004). A simple and efficient method for isolating trichomes for downstream analyses. Plant Cell Physiol 45(2): 221-224.

简介

来自一种或多种表皮细胞的向外生长是众所周知的毛状体(植物毛细胞)(Levin,1973; Mathur,2006)。从富含毛藓的芸苔制备纯的完整无腺毛状体依赖于使毛状体足够坚硬,以便在未受损的状态下将它们从叶子上除去,这可以进一步用于结构,转录组,基因组和生化或化学分析。用油漆刷(如Zhang和Oppenheimer(2004)中所述)从芸苔属villosa( B。villosa )中移出柔性毛状体证明太柔和,剃刀刀片[如Zhang和Oppenheimer(2004)]导致毛状体细胞破裂。 Aziz等人(2005)报道了通过在液氮中剪切来分离苜蓿腺毛状体(N 2 sub)。在本研究中,使用类似的方法。通过用液体N 2处理组织以硬化柔性毛状体,接着用共同的涡旋混合器剪切1分钟的剪切力以从其叶床上除去毛状体来分离非腺体叶毛状体(Nayidu et al。 em> et al。,2014)。高达〜20%的毛状体从叶表面除去,并且大多数未破裂(通过台盼蓝没有染色证实;图1)。然后通过使组织/毛/水混合物通过筛子来纯化毛状体。然而,如果将叶组织搅拌较长时间,则在浸入液体N 2后叶组织也变得非常脆,并且分裂成非常小的碎片。因此,N 2处理的叶样品不能再次用于回收剩余的附着毛状体。来自1分钟剪切的污染叶片较大并且容易从筛内分离的毛状物手动分离,得到纯化的完整毛状体制剂。该方法在从大豆(大豆大豆)和番茄(大豆)中净化毛状体也是成功的。

关键字:毛状体, 甘蓝, 隔离, 长毛猪笼草, 顶点

材料和试剂

  1. 来自芸苔(drepanensis cv。)(从两个月大到两年的叶)的新鲜毛状叶组织,大豆cv。 B220和番茄cv。 罗马
    注意:根据植物种类和毛状体分离后的后续分析类型,具有非腺毛状体的任何组织可能就足够了。 然而,具有更致密的毛状体覆盖的组织将提供更大的分离毛状体的产量。
  2. 液氮
    注意:必须进行安全使用液氮的培训。
  3. 无菌Millipore纯水

  4. 0.4%台盼蓝(见配方)

设备

  1. 耐冻结温度的手套(Cryo手套)(VWR International,目录号:32885-736)
  2. 带有冰的聚苯乙烯绝缘盒
  3. 1毫升移液器用塑料移液器吸头
  4. 50ml BD Falcon TM锥形塑料离心管(BD,目录号:14-432-22)或塑料250ml离心瓶(Thermo Fisher Scientific,目录号:EW-06105-20)
  5. BD-Falcon尼龙网孔过滤器(40μm孔径)(BD Biosciences,目录号:08-771-1)
  6. 平头钳以从过滤器(Seton,目录号:AA969)去除污染的碎屑
  7. 具有圆形混合器头(用于管)和扁平混合器头(用于较大瓶)的Genie 2涡流混合器(Thermo Fisher Scientific)
  8. -80°C冰箱
  9. 4°C冰箱
  10. Flexidry TM 单元(FTS Systems,目录号:4283)
  11. 受控环境温室或生长柜。
    注意:植物生长条件取决于后续分析的性质。 例如,植物组织的化学组成可以随着改变的光强度和温度(16小时光/8小时黑暗,至少400μE )而发生实质性变化。 em > ,建议使用20/17°C,但光强度为700-1,400μE em>将模仿外面的日光)。
  12. Axiovert 100显微镜(ZEISS),放大10倍

程序

  1. B的健康毛状体叶。 villosa,大豆和番茄在温室中生长后选择 注意:对于B. villosa,可以使用高达2年的植物叶子。
  2. 基于用于分离毛状体的容器的尺寸将收获的叶切成不同的尺寸(参见下面的视频1)。
    注意:如果使用50ml管,则使用一个3cm×3cm的叶片,如果使用250ml离心瓶,则使用两个10cm×4cm的条。管和组织大小的选择取决于随后分析所需的毛状体组织的量(参见下面的建议)。对于使用的任何叶片尺寸,获得的最大纯度毛状体制剂是存在于叶片上的总毛状体的约20%。
  3. 最大30%的管或瓶填充有液体N 2并盖上(松散地旋拧)以防止混合期间的溢流(没有管从N 2蒸汽压力破坏) 。
  4. 使用手套,使用Genie 2涡流混合器以最大速度(水平8)将管在液体N 2中混合1分钟。一分钟涡旋使分离的毛状体的恢复最大化,同时将叶组织的破坏最小化为稍后难以去除的小污染性碎片。
  5. 然后将管在冰上保持完全打开,以最大化液体N 2蒸发
  6. 使用镊子从管中手工除去大叶片,并保留为毛状体剥离的叶对照样品。
  7. 通过在2ml无菌Millipore纯化水中涡旋,轻轻悬浮粘附于管内壁的释放的毛状物。
  8. 将最初在管中冷冻的水(因为液体N 2)保持在冰上3-4分钟以解冻。
  9. 将来自具有毛状体(以及微小叶片)的管的解冻水通过BD-Falcon尼龙网孔过滤器筛分到新鲜的50ml管中。
  10. 再次使用镊子手动移除细胞滤网上的小片叶。
  11. 然后将过滤器在同一新鲜试管上倒置
  12. 通过使用另外的1-2ml Millipore水(参见下面的视频1)轻轻地将它们扫除(即,用移液管冲洗过滤器),将毛状体移入管中。
    注意:细胞过滤器可以在孔堵塞之前使用高达8-10次。强烈的细致清洁将有助于将过滤器的使用时间再延长2-3倍,但是在堵塞孔隙之后,最好使用新的过滤器,并且进一步清洁将在过滤器中产生一个孔。
  13. 将新鲜制备的毛状体批次(在水中)和毛状体脱落叶(对照)立即在4℃下储存,直到所有组织切片被加工。
  14. 通过加入0.5ml的0.4%台盼蓝将1ml含有毛的水染色为新鲜。将内容物在室温(光照或黑暗)下孵育3-4小时,而不在培养皿中摇动以准备显微镜检查。
  15. 使用Axiovert 100显微镜(10×放大率)来确认未破裂的毛状体细胞的比例和制剂的质量。
    注意:
    1. 毛状体分离方案没有洗涤步骤,而是获得 毛状体的图像(图1),我们使用水进行洗涤步骤 以改善毛状体的外观。
    2. 完整单元格 未染色破碎的细胞染成蓝色;质量差的准备 将含有更多的破坏的毛状体和/或更大比例的 污染叶片组织碎片与图1相比。
  16. 然后将样品在液N 2中快速冷冻,在-80℃下储存4-5小时,并在Flexidry TM单元中冷冻干燥过夜。 >
  17. 该方法可用于从其他植物物种中纯化非腺毛状体(图1)
  18. 建议的用于分离B的叶样本大小。 (具有300-500μm毛状体长度,〜4,000毛状/cm 2和1mg干燥毛状体从100g新鲜叶组织[FL]分离的叶) :(a)用于电子显微镜结构分析(100/cm 2); (b)用于RNA分离(300μgFL); (c)用于生化提取(500g FL);用于HPLC-UV分析(500g FL);用于NMR结构分析(800-1,000g FL);用于通过电感耦合等离子体质谱(500g FL)的金属含量分析

    图1.在从 芸苔属 分离后放大10倍的高品质毛状体制剂 最大大豆 )c蕃茄( Solanum lycopersicum 表示污染叶组织碎片或断裂的毛状体

代表数据

视频1.毛茛从
芸苔中分离的详细步骤
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食谱

  1. 0.4%台盼蓝
    将40mg台盼蓝溶于100ml微孔过滤的水中

致谢

这项研究得到了加拿大Canola委员会,Sask-Canola,萨斯喀彻温农业发展基金,以及加拿大农业和农业食品部的资助。

参考文献

  1. Aziz,N.,Paiva,N.L.,May,G.D.and Dixon,R.A。(2005)。 苜蓿腺毛状体的转录组分析 Planta 221( 1):28-38。
  2. Koona,P。和Jackai,L。(2004)。 荚果在研究毛状体作用的研究中的潜力Stål(半翅目:科里达科)。 国际热带昆虫科学杂志 24(04):298 -303。
  3. Levin,D.A。(1973)。 毛状体在植物防御中的作用 Q Rev Biol :3- 15。
  4. Mathur,J。(2006)。 拟南芥中的毛状细胞形态发生:连续的细胞决定。这个评论是发表在植物细胞生物学特刊的一系列论文之一。 植物学 84:604-612。
  5. Nayidu,N.K.,Tan,Y.,Taheri,A.,Li,X.,Bjorndahl,T.C.,Nowak,J.,Wishart,D.S.,Hegedus,D.and Gruber, Brassica villosa ,是一种用于研究非腺体毛状体和基因的系统 The Brassicas。 Plant Mol Biol 85(4-5):519-539。
  6. Zhang,X。和Oppenheimer,D.G。(2004)。 一种简单有效的分离下游分析毛状体的方法植物细胞 Physiol 45(2):221-224。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nayidu, N., Bonham-Smith, P. and Gruber, M. Y. (2014). Trichome Isolation and Integrity Test from Brassica villosa and Other Species. Bio-protocol 4(24): e1361. DOI: 10.21769/BioProtoc.1361.
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