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Seed Storage Reserve Analysis
种子贮存储备分析

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Abstract

One of the major goals of plant research is to improve crop yield, by for instance increasing seed oil or protein content. Besides this, extensive research is done to change seed fatty acid (FA) composition in order to make vegetable oils more suitable for specific purposes. To determine the effect of genetic changes on seed FA composition, oil, protein and sugar content it’s important to use standardised protocols to compare results between different research groups. Here we describe standardised methods for the analysis of seed FA composition, oil, protein and sugar content.

Materials and Reagents

  1. Dry oilseed rape (Brassica napus) seed samples
  2. KCl (Sigma-Aldrich, catalog number: 31248-500G )
  3. Methanolic-HCl (Sigma-Aldrich, catalog number: 33050-U )
  4. Hexane (Sigma-Aldrich, catalog number: 10549380 )
  5. Toluene (Sigma-Aldrich, catalog number: 650579 )
  6. Pentadecanoin (Nu-Chek, catalog number: N-15-A )
  7. Heptadecanoin (Nu-Check, catalog number: N-17-A )
  8. Fatty acid standard (Supelco 37 component FAME mix) (Sigma-Aldrich, Supelco, catalog number: 47885-U )
  9. 6 ml screw cap glass vial (Schott AG, VWR, catalog number: 391-0140 ).
  10. Organic solvent resistant caps for 6 ml screw cap vials (SKS-Science, catalog number: 240409 ).
  11. GC column (BPX70) (SGE Analytical Science, catalog number: 0 54600 )
  12. 98% ethanol (Sigma-Aldrich, catalog number: 459844 )
  13. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  14. 80% ethanol (v/v) diluted in distilled water
  15. 50% ethanol (v/v) diluted in distilled water
  16. 0.1 M NaOH (4 g/L) in distilled water
  17. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016 )
  18. HEPES (Sigma-Aldrich, catalog number: H3375 )
  19. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  20. Adenosine-5'-triphosphate (ATP) (Roche Diagnostics, catalog number: 10 127 531 001 )
  21. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  22. Nicotinamide adenine dinucleotide phosphate (NADP) (Roche Diagnostics, catalog number: 10 240 35 4001 )
  23. Glucose-6-Phosphate Dehydrogenase grade II (G6PDHII) (Roche Diagnostics, catalog number: 10 737 232 001 ) (80 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
  24. Hexokinase (HK) (Roche Diagnostics, catalog number: 11 426 362 001 ) (120 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
  25. Phosphoglucose isomerase (PGI) (Roche Diagnostics, catalog number: 10 128 139 001 ) (60 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
  26. Invertase (INV) (Sigma-Aldrich, catalog number: I4504 ) (as much as possible to dissolve in 200 μl of Buffer B)
  27. 60 mg/ml ATP in distilled water
  28. 36 mg/ml NADP in distilled water
  29. Buffer A (see Recipes)
  30. Buffer B (see Recipes)
  31. Reagent mix 1 (see Recipes)

Equipment

  1. Gas chromatograph (GC) (Agilent, model: 7890A )
  2. MinispecMQ20 (Bruker Corporation)
  3. Robotic sample handling system (Rohasys BV)
  4. Centrifuges
    For the "Sucrose and protein content determination" procedure: Eppendorf Centrifuge 5415 R (Eppendorf, catalog number: 022621408 )
    For the "Determination of seed FA content and composition" procedure: Eppendorf Centrifuge 5810 R (Eppendorf, catalog number: 5811 000.010 )
  5. Vortex-Genie 2 Shaker (Scientific Industries, Cole-Parmer, catalog number: UY-04724-05 )
  6. 2 ml Eppendorf tube (Sigma-Aldrich, catalog number: T2795 but not important )
  7. Microplate 96 wells (Sigma-Aldrich, catalog number: M4561 but not important )

Procedure

  1. Determination of seed FA content and composition
    1. Aliquot 0.4-2 mg of seeds into a 6 ml screw cap glass vial.
    2. Add 10 µl pentadecanoin or heptadecanoin standard (1 µg/µl stock in toluene).
      The reason to choose these FAs as standards is that they are not presents in Arabidopsis seeds and do not overlap with other fatty acid methyl esters (FAMEs) during GC analysis.
    3. Add 1 ml of 1 N methanolic-HCl.
    4. Heat for 1.5 h at 80 °C in order to produce FAMEs.
    5. Add 200 µl of hexane (100%).
    6. Add 1.5 ml of 0.8% KCl and vortex for 10 sec.
      It’s important to add the hexane and KCl in this order to avoid artefacts.
    7. Centrifuge for 2 min at 2,000 rpm at room temperature in order to induce a phase separation.
    8. Pipet of 150 µl of the upper phase and transfer to a GC vial.
    9. Run on GC column using the following parameters: 150 °C for 0.1 min, ramp up to 190 °C at 4 °C per min.
    10. Analyse data and determine FA content per seed.
    11. Retention times of different FAs can be determined by consulting literature or running standards.
      Figure 15 of the Arabidopsis book chapter about Acyl-Lipid metabolism shows the result of the GC analysis of Arabidopsis seed oil (Li-Beisson et al., 2010).
      On the Sigma-Aldrich website the separation of the Supelco 37 component FAME mix on different GC columns is described (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Supelco/Bulletin/t196907.pdf).

  2. Determination of seed oil and moisture content by low-resolution time domain NMR spectroscopy
    1. Construct oil and moisture calibration according to the ISO 10565:1998 standard (http://www.iso.org/iso/catalogue_detail.htm?csnumber=26317). Use nine approximately 0.5 gr seed samples (between 5% and 10% moisture content and between approximately 30% and 55% oil content (r2 = 0.99).
    2. Measure oil and moisture content of 0.5 grams of dry seeds.
      Normalize seed percentage oil content using 9% water content as reference.

  3. Extraction for sucrose and protein content determination
    1. Take samples of 10-20 intact seeds (minimum) in safe lock 2 ml Eppendorf tube.
    2. Add 250 μl of 80% ethanol.
    3. Vortex for 30 sec.
    4. Heat samples at 80 °C for 20 min.
    5. Centrifuge at 23,000 g for 5 min at room temperature.
    6. Transfer the supernatant (S1) in another 2 ml Eppendorf tube on ice.
    7. Add 150 μl of 80% ethanol.
    8. Vortex for 30 sec.
    9. Heat samples at 80 °C for 10 min.
    10. Centrifuge at 23,000 g for 5 min at room temperature.
    11. Transfer the supernatant (S2) in the corresponding tubes on ice.
    12. Add 250 μl of 50% ethanol.
    13. Vortex for 30 sec.
    14. Heat samples at 80 °C for 10 min.
    15. Centrifuge at 23,000 x g for 5 min at room temperature.
    16. Transfer the supernatant (S3) in the corresponding tubes on ice.
    17. Store the tubes containing S1+S2+S3 at -20 °C.
    18. Add 400 μl 0.1 M NaOH to the pellet.

  4. Sugar and protein content determination (method optimized for microplate 96 wells)
    1. Sugar content determination
      1. In each well dispense:
        50 μl of ethanolic extract (depending of your estimated concentration)
        The protein concentration depends on the species from which the samples are isolated. The amount of protein in the seeds of your species of interest can be checked in the literature.
      2. 160 μl of Reagent mix 1
      3. Read OD at 340 nm for 10 min (until OD stabilizes).
      4. Add 1 μl of HK per well.
      5. Read OD at 340 nm until OD stabilizes (usually 25 min).
      6. Add 1 μl of PGI per well.
      7. Read OD at 340 nm until OD stabilizes (usually 25 min).
      8. Add 1 μl of INV per well.
      9. Read OD at 340 nm until OD stabilizes (usually 1.5 h).
      10. Calculations.
        Calibration curve for sucrose (in duplicate): 200, 100, 50, 25, 12.5, 6.25 μg/ml (in buffer)
        or use the formula: μmol NADPH= ΔOD/(2.851*6.222)
        1: Pathlength for microplate
        2: NADPH molar extinction coefficient
    2. Protein content determination
      1. Heat the samples at 95 °C for 30 min.
      2. Cool down and centrifuge at 23,000 x g for 5 min at room temperature.
      3. Use 5-10 μl of the supernatant.
      4. Follow protocol from Thermo Scientific™ Pierce™ BCA™ Protein Assay (23227).
      5. NB: Dilute the standards as appropriate in 0.1 M NaOH.

Recipes

  1. Buffer A
    1 M HEPES/KOH
    30 mM MgCl2 (pH 7.0)
  2. Buffer B
    0.1 M HEPES/KOH
    3 mM MgCl2 (pH 7.0)
  3. Reagent mix 1
    15.5 ml Buffer B
    480 μl ATP
    480 μl NADP
    200 μl G6PDHII

Acknowledgments

This work was supported by the Biotechnology and Biological Sciences Research Council (Institute Strategic Program grant and grant no. BB/E022197/1). The protocol for the determination of seed FA composition was adapted from Browse et al. (1986).

References

  1. Baud, S., Boutin, J.-P., Miquel, M., Lepiniec, L. and Rochat, C. (2002). An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40(2): 151-160.
  2. Browse, J., McCourt, P. J. and Somerville, C. R. (1986). Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152(1): 141-145.
  3. Li-Beisson, Y., Shorrosh, B., Beisson, F., Andersson, M. X., Arondel, V., Bates, P. D., Baud, S., Bird, D., Debono, A., Durrett, T. P., Franke, R. B., Graham, I. A., Katayama, K., Kelly, A. A., Larson, T., Markham, J. E., Miquel, M., Molina, I., Nishida, I., Rowland, O., Samuels, L., Schmid, K. M., Wada, H., Welti, R., Xu, C., Zallot, R. and Ohlrogge, J. (2010). Acyl-lipid metabolism. Arabidopsis Book 8: e0133.
  4. van Erp, H., Kelly, A. A., Menard, G. and Eastmond, P. J. (2014). Multigene engineering of triacylglycerol metabolism boosts seed oil content in Arabidopsis. Plant Physiol 165(1): 30-36.

简介

植物研究的主要目标之一是通过例如增加种子油或蛋白质含量来改善作物产量。 除此之外,还进行了广泛的研究以改变种子脂肪酸(FA)组成,以使植物油更适合于特定目的。 为了确定遗传变化对种子FA组成,油,蛋白质和糖含量的影响,使用标准化方案来比较不同研究组之间的结果是重要的。 在这里我们描述标准化的方法,用于分析种子FA组成,油,蛋白质和糖含量。

材料和试剂

  1. 干油菜子(
    Brassica napus )种子样品
  2. KCl(Sigma-Aldrich,目录号:31248-500G)
  3. 甲醇-HCl(Sigma-Aldrich,目录号:33050-U)
  4. 己烷(Sigma-Aldrich,目录号:10549380)
  5. 甲苯(Sigma-Aldrich,目录号:650579)
  6. 十五烷酸(Nu-Chek,目录号:N-15-A)
  7. 十七烷酸(Nu-Check,目录号:N-17-A)
  8. 脂肪酸标准(Supelco 37组分FAME混合物)(Sigma-Aldrich,Supelco,目录号:47885-U)
  9. 6ml螺旋盖玻璃小瓶(Schott AG,VWR,目录号:391-0140)
  10. 用于6ml螺旋盖小瓶(SKS-Science,目录号:240409)的有机溶剂耐受性盖。
  11. GC柱(BPX70)(SGE Analytical Science,目录号:054600)
  12. 98%乙醇(Sigma-Aldrich,目录号:459844)
  13. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  14. 80%乙醇(v/v)稀释在蒸馏水中
  15. 用蒸馏水稀释的50%乙醇(v/v)
  16. 0.1M NaOH(4g/L)在蒸馏水中的溶液
  17. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:484016)
  18. HEPES(Sigma-Aldrich,目录号:H3375)
  19. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  20. 腺苷-5'-三磷酸(ATP)(Roche Diagnostics,目录号:10 127 531 001)
  21. 蔗糖(Sigma-Aldrich,目录号:84097)
  22. 烟酰胺腺嘌呤二核苷酸磷酸(NADP)(Roche Diagnostics,目录号:10 240 35 4001)
  23. 葡萄糖-6-磷酸脱氢酶II级(G6PDHII)(Roche Diagnostics,目录号:10 737 232 001)(80μl,在200μl缓冲液B中离心并重悬沉淀)
  24. 己糖激酶(HK)(Roche Diagnostics,目录号:11 426 362 001)(120μl,在200μl缓冲液B中离心并重悬沉淀)
  25. 磷酸葡萄糖异构酶(PGI)(Roche Diagnostics,目录号:10 128 139 001)(60μl,在200μl缓冲液B中离心并重悬沉淀)
  26. 转化酶(INV)(Sigma-Aldrich,目录号:I4504)(尽可能溶解于200μl缓冲液B中)
  27. 60 mg/ml ATP在蒸馏水中的溶液
  28. 36mg/ml NADP的蒸馏水溶液
  29. 缓冲区A(参见配方)
  30. 缓冲液B(参见配方)
  31. 试剂混合物1(见配方)

设备

  1. 气相色谱仪(GC)(Agilent,型号:7890A)
  2. MinispecMQ20(Bruker Corporation)
  3. 机器人样品处理系统(Rohasys BV)
  4. 离心机
    对于"蔗糖和蛋白质含量测定"程序:Eppendorf离心机5415R(Eppendorf,目录号:022621408)
    对于"种子FA含量和组成的确定"程序:Eppendorf Centrifuge 5810 R(Eppendorf,目录号:5811000.010)
  5. Vortex-Genie 2振荡器(Scientific Industries,Cole-Parmer,catalo g number:UY-04724-05)
  6. 2ml Eppendorf管(Sigma-Aldrich,目录号:T2795,但不重要)
  7. 微孔板96孔(Sigma-Aldrich,目录号:M4561,但不重要)

程序

  1. 种子FA含量和组成的测定
    1. 将0.4-2毫克种子等分到6毫升螺旋盖玻璃小瓶中
    2. 加入10μl十五烷酸或十七烷醇标准品(1μg/μl储备在甲苯中) 选择这些FAs作为标准的原因是它们不存在于拟南芥种子中,并且在GC分析期间不与其它脂肪酸甲酯(FAME)重叠。
    3. 加入1ml 1N甲醇-HCl
    4. 在80℃下加热1.5小时以产生FAME。
    5. 加入200μl己烷(100%)
    6. 加入1.5ml的0.8%KCl并涡旋10秒 重要的是按此顺序加入己烷和KCl以避免假象。
    7. 在室温下以2,000rpm离心2分钟以诱导相分离。
    8. 移取150μl上层相并转移至GC小瓶
    9. 使用以下参数在GC柱上运行:150℃0.1分钟,以4℃/分钟升温至190℃。
    10. 分析数据并确定每个种子的FA含量。
    11. 不同FA的保留时间可以通过咨询文献或运行标准来确定 关于酰基脂质代谢的拟南芥书章的图15显示了拟南芥种子油的GC分析的结果(Li-Beisson等人 >,2010)。
      在Sigma-Aldrich网站上,描述了不同GC柱上的Supelco 37组分FAME混合物的分离(http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Supelco/Bulletin/t196907.pdf)。

  2. 通过低分辨率时域NMR光谱法测定种子油和水分含量
    1. 根据ISO 10565:1998标准构建油和水分校准( http://www .iso.org/iso/catalogue_detail.htm?csnumber = 26317 )。 使用九个约0.5gr种子样品(含水量在5%和10%之间,含油量在约30%和55%之间(r 2 = 0.99)。
    2. 测量0.5克干燥种子的油和水分含量。
      使用9%的水含量作为参考,归一化种子百分比油含量
  3. 提取蔗糖和蛋白质含量测定
    1. 取10-20完整种子(最小)的样品在安全锁2 ml Eppendorf管
    2. 加入250μl的80%乙醇
    3. 涡旋30秒。
    4. 在80℃下加热样品20分钟
    5. 在室温下以23,000g离心5分钟
    6. 转移上清(S1)在另一个2毫升的Eppendorf管在冰上
    7. 加入150μl的80%乙醇。
    8. 涡旋30秒。
    9. 在80℃下加热样品10分钟
    10. 在室温下以23,000g离心5分钟
    11. 转移上清(S2)在相应的管在冰上
    12. 加入250μl50%乙醇。
    13. 涡旋30秒。
    14. 在80℃下加热样品10分钟
    15. 在室温下以23,000×g离心5分钟
    16. 转移上清(S3)在相应的管在冰上
    17. 将含有S1 + S2 + S3的试管存放在-20°C
    18. 向沉淀中加入400μl0.1M NaOH

  4. 糖和蛋白质含量测定(方法优化微孔板96孔)
    1. 糖含量测定
      1. 在每个井中分配:
        50μl乙醇提取物(根据您的估计浓度)
        蛋白质浓度取决于样品分离的物种。 您可以在文献中检查您感兴趣物种的种子中的蛋白质含量
      2. 160μl试剂混合物1
      3. 在340nm处读取OD 10分钟(直到OD稳定)
      4. 每孔加入1μlHK
      5. 在340nm处读取OD,直到OD稳定(通常25分钟)
      6. 每孔加入1μlPGI
      7. 在340nm处读取OD,直到OD稳定(通常25分钟)
      8. 每孔加入1μlINV。
      9. 在340nm读OD,直到OD稳定(通常1.5小时)。
      10. 计算。
        蔗糖的校准曲线(一式两份):200,100,50,25,12.5,6.25μg/ml(在缓冲液中)
        或使用下式:μmolNADPH =ΔOD/(2.85 1 * 6.22 2
        1 :微孔板的路径长度
        2 :NADPH摩尔消光系数
    2. 蛋白含量测定
      1. 将样品在95℃下加热30分钟
      2. 冷却并在室温下以23,000×g离心5分钟
      3. 使用5-10μl的上清液。
      4. 按照来自Thermo Scientific TM Pierce TM BCA TM蛋白测定(23227)的方案
      5. 注意:在0.1 M NaOH中适当稀释标准品

食谱

  1. 缓冲区A
    1 M HEPES/KOH
    30mM MgCl 2(pH 7.0)
  2. 缓冲区B
    0.1 M HEPES/KOH
    3mM MgCl 2(pH 7.0)
  3. 试剂混合1
    15.5 ml缓冲液B
    480μlATP
    480μlNADP
    200μlG6PDHII

致谢

这项工作是由生物技术和生物科学研究委员会(研究所战略计划赠款和拨款号BB/E022197/1)支持。用于测定种子FA组成的方案改编自Browse等(1986)。

参考文献

  1. Baud,S.,Boutin,J.-P.,Miquel,M.,Lepiniec,L。和Rochat,C。(2002)。 拟南芥(Arabidopsis thaliana)生态型WS中种子发育的综合概述。植物生理生物化学(Plant Physiol Biochem)40(2):151-160。
  2. Browse,J.,McCourt,P.J.and Somerville,C.R。(1986)。 在组合消化后确定叶脂质的脂肪酸组成,并从新鲜组织形成脂肪酸甲酯。/a> Anal Biochem 152(1):141-145。
  3. Li-Beisson,Y.,Shorrosh,B.,Beisson,F.,Andersson,MX,Arondel,V.,Bates,PD,Baud,S.,Bird,D.,Debono,A.,Durrett,TP,Franke ,RB,Graham,IA,Katayama,K.,Kelly,AA,Larson,T.,Markham,JE,Miquel,M.,Molina,I.,Nishida,I.,Rowland,O.,Samuels, Schmid,KM,Wada,H.,Welti,R.,Xu,C.,Zallot,R。和Ohlrogge,J。(2010)。 酰基脂质 代谢。 Arabidopsis Book 8:e0133。
  4. van Erp,H.,Kelly,AA,Menard,G.and Eastmond,PJ(2014)。三酰基甘油代谢的多基因工程促进拟南芥中的种子油含量。 植物生理学165(1):30-36。
  • English
  • 中文翻译
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Erp, H. v., Menard, G. and Eastmond, P. J. (2014). Seed Storage Reserve Analysis. Bio-protocol 4(20): e1263. DOI: 10.21769/BioProtoc.1263.
  2. van Erp, H., Kelly, A. A., Menard, G. and Eastmond, P. J. (2014). Multigene engineering of triacylglycerol metabolism boosts seed oil content in Arabidopsis. Plant Physiol 165(1): 30-36.
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