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Chitin Extraction and Content Measurement in Magnaporthe oryzae
稻瘟病菌中的甲壳素提取和含量测定

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Abstract

Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example.

Keywords: Chitin(甲壳素), Cell wall(细胞壁), GlcNAc(GlcNAc), Pathogenicity(致病性), Magnaporthe oryzae(稻瘟病菌)

Background

Chitin is an important component of the cell wall in fungal pathogens and is well known as a pathogen associated molecule pattern (PAMP). Determining the chitin content of a fungal species is important for studying fungal biology and host-pathogen interactions. The Morgan-Elson method based on colorimetric approach has been adapted in yeast to measure cellular chitin levels (Leloir and Cardini, 1953; Bulik et al., 2003; Baker et al., 2007). However, there is no specific protocol established for Magnaporthe oryzae, which is the causal agent of rice blast, the most important fungal disease in the world. Here, we describe a reliable and simple protocol which was modified from the Morgan-Elson method to test the chitin content of M. oryzae (Song et al., 2010).

Materials and Reagents

  1. Microtube
  2. Microtiter plates (Corning, Costar®, catalog number: 42592 )
  3. Miracloth (EMD Millipore, catalog number: 475855 )
  4. Cells of Magnaporthe oryzae
  5. Potassium hydroxide (KOH) (Sangon Biotech, catalog number: A610441 )
  6. 10x phosphate buffered saline (PBS, 1.35 M NaCl, 47 mM KCl, 100 mM Na2HPO4, 20 mM NaH2PO4, pH = 7.4) (Beyotime, catalog number: ST476 )
  7. Streptomyces plicatus chitinase (Sigma-Aldrich, catalog number: C6137 )
  8. Sodium borate (Sangon Biotech, catalog number: A100390 )
  9. GlcNAc (Sigma-Aldrich, catalog number: PHR1432-1G )
  10. Sodium hydrogen (Na2HPO4) (Sangon Biotech, catalog number: A501727 )
  11. Citric acid (Sangon Biotech, catalog number: A501702 )
  12. p-dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: D2004 )
  13. Hydrochloric acid (HCl)
  14. Acetic acid (Sangon Biotech, catalog number: A501931 )
  15. Biotin
  16. Pyridoxin
  17. Thiamine
  18. Riboflavin
  19. p-aminobenzoic acid
  20. nicotinic acid
  21. ZnSO4·7H2O
  22. H3BO3
  23. MnCl2·4H2O
  24. FeSO4·7H2O
  25. CoCl2·6H2O
  26. CuSO4·5H2O
  27. Na2MnO4·2H2O
  28. Na4EDTA
  29. NaNO3
  30. KCl
  31. MgSO4·7H2O
  32. KH2PO4
  33. D-glucose
  34. Peptone
  35. Yeast extract
  36. Casamino acid
  37. Agar
  38. McIlvaine’s buffer (see Recipes)
  39. Ehrlich’s solution (see Recipes)
  40. Vitamin solution (see Recipes)
  41. Trace elements (see Recipes)
  42. 20x nitrate salts (see Recipes)
  43. CM medium (see Recipes)

Equipment

  1. Freeze dryer (Marin Christ German)
  2. Vortex mixer
  3. Water bath (SHEL Lab, model: W6M-2 )
  4. Centrifuge (Eppendorf centrifuge) (Eppendorf, model: 5418 )
  5. Incubator shaker (Crystal Technology & Industries, model: IS-RSD3 )
  6. pH/ATU electrode (Sartorius, German)
  7. Microplate reader (Molecular Devices, model: VersaMax ELISA )
  8. PCR instrument (Takara Bio, model: TP600 )
  9. Eppendorf micropipette (1,000 μl, 100 μl, 10 μl)
  10. Dark glass bottle

Software

  1. SPSS 2.0 (Chicago, IL, USA)

Procedure

  1. All of the strains are cultured on solid CM medium for 7 days at 28 °C. The agar culture is cut into 1 x 1 mm squares and the squares are cultured in liquid CM for another 2 days.
  2. Filtered through one layer of Miracloth to collect mycelium from liquid CM. Then the mycelium are quickly lyophilized by a freeze dryer for 24 h. 5 mg mycelium is mixed with 1 ml 6% KOH in each 2 ml microtube using a vortex mixer and then incubated in a water bath at 80 °C for 90 min.
  3. Samples are centrifuged at 16,000 x g for 10 min, and the suspension is discarded.
  4. Each pellet is washed with 1 ml 1x PBS (which is diluted from 10x PBS) for three times and then centrifuged at 16,000 x g for 5 min to discard the suspension.
  5. Each pellet is resuspended with 0.5 ml of McIlvaine’s buffer (see Recipes) (Baker et al., 2007). 100 μl chitinase is added into each sample. And then samples are incubated at 37 °C for 16 h in the dark, at 220 rpm in an incubator shaker.
  6. Chitinase-treated samples are mixed with equal volume of 0.27 M sodium borate (pH = 9.0) and incubated at 100 °C for 10 min in a PCR instrument.
  7. After being cooled down to room temperature, 200 μl of each sample is added to 1 ml Ehrlich’s solution (see Recipes) and then incubate at 37 °C for 30 min in an incubator shaker.
  8. 100 μl of each sample is transferred into a well of a microtiter plate with low-evaporation and the absorbance is measured at 585 nm by a microplate reader. Standard curves are prepared from stocks of 0.1 to 2.0 mM (0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM) GlcNAc.

Data analysis

Standard curves are prepared from stocks of 0.1 to 2.0 mM GlcNAc. Each result is presented at least three replicated measurements. The significance of differences between treatments is statistically evaluated using SDs and one-way analysis of variance (ANOVA) in SPSS 2.0 (Chicago, IL, USA). Data for two specific different treatments are compared statistically using ANOVA, followed by an F-test if the ANOVA result is significant at P < 0.05 or P < 0.01.

Notes

  1. Both McIlvaine’s buffer and Ehrlich’s solution need to be prepared just before use.
  2. In step 1, 25 squares are enough and the volume of the liquid CM is 70 ml.
  3. In step 2, samples are incubated in a water bath at 80 °C for 90 min. Samples need to be vortexed every 15 min.
  4. In step 6, after being extracted at 100 °C for 10 min, all the samples should be cooled on ice to room temperature immediately.
  5. In step 6, after being extracted at 100 °C for 10 min in the PCR instrument, the solution is clear and transparent
  6. In step 7, the Ehrlich’s solution is clear and does not need to be centrifuged.
  7. If the samples are difficult to suspend, break the pellet by an injector or a micropipette.

Recipes

  1. McIlvaine’s buffer
    0.2 M Na2HPO4
    0.1 M citric acid (pH = 6.0)
  2. Ehrlich’s solution
    10 g p-dimethylaminobenzaldehyde in 12.5 ml concentrated HCl (37%) and 87.5 ml glacial acetic acid
  3. Vitamin solution (100 ml)
    0.01 g Biotin
    0.01 g Pyridoxin
    0.01 g Thiamine
    0.01 g Riboflavin
    0.01 g p-aminobenzoic acid
    0.01 g nicotinic acid
    Add ddH2O to 100 ml and store in a dark glass bottle at 4 °C
  4. Trace elements (100 ml)
    2.2 g ZnSO4·7H2O
    1.1 g H3BO3
    0.5 g MnCl2·4H2O
    0.5 g FeSO4·7H2O
    0.17 g CoCl2·6H2O
    0.16 g CuSO4·5H2O
    0.15 g Na2MnO4·2H2O
    5 g Na4EDTA
    Add ddH2O to 100 ml and adjust the pH to 5.8, store at 4 °C
  5. 20x nitrate salts (1 L)
    120 g NaNO3
    10.4 g KCl
    10.4 g MgSO4·7H2O
    30.4 g KH2PO4
    Add ddH2O to 1 L and store at 4 °C
  6. CM medium
    10 g D-glucose
    2 g peptone
    1 g yeast extract
    1 g casamino acid
    1 ml vitamin solution
    1 ml trace elements
    50 ml 20x nitrate salts
    Add ddH2O to 1 L
    For solid media add 15 g agar and autoclave at 121 °C for 20 min

Acknowledgments

This research was supported by the key program of Natural Science Foundation of China (Grant No. 31530063, ZZ), National Science Foundation for Distinguished Young Scholars of China (Grant No. 31325022 to ZZ), Natural Science Foundation of China (Grant No. 31271998, ZZ), and the especially appointed professorship (Jiangsu, China).

References

  1. Baker, L. G., Specht, C. A., Donlin, M. J. and Lodge, J. K. (2007). Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot Cell 6(5): 855-867.
  2. Bulik, D. A., Olczak, M., Lucero, H. A., Osmond, B. C., Robbins, P. W. and Specht, C. A. (2003). Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell 2(5): 886-900.
  3. Leloir, L. F. and Cardini, C. E. (1953). The biosynthesis of glucosamine. Biochim Biophys Acta 12(1-2): 15-22.
  4. Song, W., Dou, X., Qi, Z., Wang, Q., Zhang, X., Zhang, H., Guo, M., Dong, S., Zhang, Z., Wang, P. and Zheng, X. (2010). R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS One 5(10): e13193.

简介

壳多糖是由β(1→4)连接的N-乙酰葡糖胺(GlcNAc)残基组成的线性多糖。在真菌中,几丁质是细胞壁的重要成分。在这里,我们提供了使用稻瘟病菌(Metaporthe oryzae)来测量真菌细胞的壳多糖含量的方案。

背景 壳多糖是真菌病原体细胞壁的重要成分,是众所周知的病原体相关分子模式(PAMP)。确定真菌物种的几丁质含量对于研究真菌生物学和宿主 - 病原体相互作用是重要的。基于比色法的Morgan-Elson方法已经在酵母中调节以测量细胞几丁质水平(Leloir和Cardini,1953; Bulik等人,2003; Baker等人, ,,2007)。然而,没有针对稻米稻瘟病菌确定的具体方案,这是世界上最重要的真菌病的稻瘟病的致病因子。在这里,我们描述了一种可靠和简单的方案,该协议由Morgan-Elson方法修改,以测试M的甲壳质含量。 oryzae (Song 等人,2010)。

关键字:甲壳素, 细胞壁, GlcNAc, 致病性, 稻瘟病菌

材料和试剂

  1. Microtube
  2. 微量滴定板(Corning,Costar ®,目录号:42592)
  3. Miracloth(EMD Millipore,目录号码:475855)




  4. 氢氧化钾(KOH)(Sangon Biotech,目录号:A610441)
  5. 10倍磷酸缓冲盐水(PBS,1.35M NaCl,47mM KCl,100mM Na 2 HPO 4,20mM NaH 2 PO 3 > 4, pH = 7.4)(Beyotime,目录号:ST476)
  6. 链霉菌 plicatus几丁质酶(Sigma-Aldrich,目录号:C6137)
  7. 硼酸钠(Sangon Biotech,目录号:A100390)
  8. GlcNAc(Sigma-Aldrich,目录号:PHR1432-1G)
  9. 氢钠(Na 2 HPO 4)(Sangon Biotech,目录号:A501727)
  10. 柠檬酸(Sangon Biotech,目录号:A501702)
  11. 二甲基氨基苯甲醛(Sigma-Aldrich,目录号:D2004)
  12. 盐酸(HCl)
  13. 乙酸(Sangon Biotech,目录号:A501931)
  14. 生物素
  15. 吡哆素
  16. 硫胺素
  17. 核黄素
  18. 对氨基苯甲酸
  19. 烟酸
  20. ZnSO 4·7H 2 O
  21. 3 <3> 3
  22. MnCl 2·4H 2 O
  23. FeSO 4·7H 2 O O
  24. CoCl 2·6H 2 O ○
  25. CuSO 4·5H 2 O
  26. Na 2 MnO 4·2H 2 O O
  27. Na 4 EDTA
  28. NaNO 3
  29. KCl
  30. MgSO 4·7H 2 O→/ /
  31. KH 2 PO 4
  32. D-葡萄糖
  33. 蛋白胨
  34. 酵母提取物
  35. Casamino酸
  36. 琼脂
  37. McIlvaine的缓冲区(见配方)
  38. Ehrlich的解决方案(见配方)
  39. 维生素溶液(见食谱)
  40. 微量元素(参见食谱)
  41. 20x硝酸盐(见食谱)
  42. CM介质(见配方)

设备

  1. 冷冻干燥机(Marin Christ German)
  2. 涡街搅拌机
  3. 水浴(SHEL实验室,型号:W6M-2)
  4. 离心机(Eppendorf离心机)(Eppendorf,型号:5418)
  5. 孵化器振荡器(Crystal Technology&amp; Industries,型号:IS-RSD3)
  6. pH / ATU电极(Sartorius,德语)
  7. 酶标仪(Molecular Devices,型号:VersaMax ELISA)
  8. PCR仪(Takara Bio,型号:TP600)
  9. Eppendorf微量移液管(1000μl,100μl,10μl)
  10. 黑色玻璃瓶

软件

  1. SPSS 2.0(Chicago,IL,USA)

程序

  1. 所有菌株在固体CM培养基上于28℃培养7天。将琼脂培养物切成1×1mm的正方形,并将方块在液体CM中再培养2天
  2. 通过一层Miracloth过滤,从液体CM收集菌丝体。然后通过冷冻干燥器将菌丝体快速冻干24小时。使用涡旋混合器将5mg菌丝体与2ml微量管中的1ml 6%KOH混合,然后在80℃的水浴中温育90分钟。
  3. 将样品以16,000xg离心10分钟,弃去悬浮液。
  4. 每个沉淀物用1ml 1x PBS(其从10x PBS稀释)洗涤三次,然后以16,000xg离心5分钟以丢弃悬浮液。
  5. 每个沉淀物用0.5ml McIlvaine的缓冲液(参见Recipes)重新悬浮(Baker等人,2007)。每个样品中加入100μl几丁质酶。然后将样品在37℃下在黑暗中以220rpm在培养箱振荡器中孵育。
  6. 将几丁质酶处理的样品与等体积的0.27M硼酸钠(pH = 9.0)混合,并在PCR仪器中在100℃下孵育10分钟。
  7. 冷却至室温后,将200μl每个样品加入1ml Ehrlich's溶液(参见食谱),然后在37℃下在培养箱中培养30分钟。
  8. 将100μl的每个样品转移到微量滴定板的孔中,并用微量酶读数器在585nm下测量吸光度。标准曲线由0.1至2.0mM(0.1mM,0.5mM,1.0mM,1.5mM和2.0mM)GlcNAc的原液制备。

数据分析

标准曲线由0.1至2.0mM GlcNAc的原料制备。每个结果呈现至少三个重复测量。在SPSS 2.0(Chicago,IL,USA)中使用SDs和单因素方差分析(ANOVA)对治疗间差异的意义进行统计学评估。使用ANOVA对两个特定不同治疗的数据进行统计比较,随后如果ANOVA结果在P

0.05或 0.01。


笔记

  1. McIlvaine的缓冲区和Ehrlich的解决方案需要在使用前准备。
  2. 在步骤1中,25个正方形足够,液体CM的体积为70ml
  3. 在步骤2中,将样品在80℃的水浴中孵育90分钟。样品需要每15分钟涡旋一次
  4. 在步骤6中,在100℃下提取10分钟后,所有样品应立即在冰冷至室温。
  5. 在步骤6中,在PCR仪器中在100℃下提取10分钟后,溶液清澈透明
  6. 在步骤7中,Ehrlich的解决方案是清晰的,不需要离心
  7. 如果样品难以悬浮,用注射器或微量移液管破碎颗粒。

食谱

  1. McIlvaine的缓冲区
    0.2M Na 2 HPO 4
    0.1M柠檬酸(pH = 6.0)
  2. Ehrlich的解决方案
    10g 12.5%浓HCl(37%)和87.5ml冰醋酸的p-二甲基氨基苯甲醛
  3. 维生素溶液(100ml)
    0.01克生物素
    0.01g吡哆素
    0.01克硫胺素
    0.01 g核黄素
    0.01g对氨基苯甲酸 0.01克烟酸
    将ddH 2 O加入到100ml中,并保存在4°C的深色玻璃瓶中
  4. 微量元素(100 ml)
    2.2g ZnSO 4·7H 2 O
    1.1g H 3 BO 3
    0.5g MnCl 2·4H 2 O
    0.5g FeSO 4·7H 2 O
    0.17g CoCl 2·6H 2 O
    0.16g CuSO 4·5H 2 O
    0.15g Na 2 MnO 4·2H 2 O 5g Na 4 EDTA
    将ddH 2亚O加入到100ml中,将pH调节至5.8,在4℃下储存
  5. 20x硝酸盐(1升)
    120g NaNO 3
    10.4克KCl
    10.4g MgSO 4·7H 2 O→/ / 30.4g KH 2 PO 4
    将ddH 2 O添加到1 L,并保存在4°C
  6. CM媒体
    10g D-葡萄糖
    2克蛋白胨
    1克酵母提取物
    1克酪蛋白氨基酸
    1 ml维生素溶液
    1毫升痕量元素
    50ml 20x硝酸盐盐
    将ddH 2 O添加到1 L
    对于固体培养基,加入15g琼脂并在121℃下高压灭菌20分钟

致谢

这项研究得到了中国自然科学基金重点项目(授权号31530063,ZZ),国家杰出青年科学基金(授权号ZZ)31325022,中国自然科学基金(批准号) 31271998,ZZ)和特别授权的教授(江苏,中国)。

参考文献

  1. Baker,LG,Specht,CA,Donlin,MJ and Lodge,JK(2007)。&nbsp; 壳多糖,几丁质的脱乙酰形式,对于新型隐球酵母中的细胞壁完整性是必需的。真核细胞 6(5): 855-867。
  2. Bulik,DA,Olczak,M.,Lucero,HA,Osmond,BC,Robbins,PW and Specht,CA(2003)。&nbsp; 酿酒酵母中的甲壳素合成是为了补充具有葡糖胺和细胞壁应力的生长培养基。 Eukaryot Cell 2(5):886-900。
  3. Leloir,LF和Cardini,CE(1953)。生物合成葡萄糖胺。生物化学Biophys Acta 12(1-2):15-22。
  4. Song,W.,Dou,X.,Qi,Z.,Wang,Q.,Zhang,X.,Zhang,H.,Guo,M.,Dong,S.,Zhang,Z.,Wang, Zheng,X.(2010)。&nbsp; R-SNARE同系物MoSec22是米曲霉米曲霉的分生型,细胞壁完整性和发病机制所必需的。 PLoS One 5(10):e13193。
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引用:Liu, X. and Zhang, Z. (2017). Chitin Extraction and Content Measurement in Magnaporthe oryzae. Bio-protocol 7(5): e2164. DOI: 10.21769/BioProtoc.2164.
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