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In vitro CLE Peptide Bioactivity Assay on Plant Roots

植物根部CLE肽的生物活性的体外实验

Shiyan Chen Shiyan Chen
Xiaohong Wang Xiaohong Wang

发布: 2015年12月20日第5卷第24期 DOI: 10.21769/BioProtoc.1689 浏览次数: 11020

评审: Tie LiuFang XuArsalan Daudi

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Jan 2015

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Abstract

Plant CLAVATA3/ESR (CLE)-related proteins play diverse roles in plant growth and development including regulating the development of root meristem. The mature functional forms of CLE peptides are typically 12-13 amino acids (aa) in length that are derived from the conserved C-termini of their precursor proteins. Genes encoding small secreted peptides sharing similarity to plant CLE proteins have recently been cloned from plant-parasitic nematodes, pests that infect many important crops. It is demonstrated that exogenous application of synthetic 12-14 aa CLE peptides corresponding to the CLE domain of their precursor proteins can suppress plant root growth. This protocol is to evaluate the bioactivity of CLE peptides originated from plant-parasitic nematodes by measuring the growth of plant roots or the size of root apical meristem (RAM) after CLE peptide treatment. Plants used in the study included Arabidopsis and potato.

Keywords: CLE peptide (CLE肽) search Plant-parasitic nematode (植物寄生线虫) search Root apical meristem (根顶端分生组织) search Potato root growth (马铃薯根生长) search

Materials and Reagents

  1. Micropore tape (Thermo Fisher Scientific, catalog number: 19-027-761 )
  2. Sterile square petri dishes (VWR International, catalog number: 60872-310 )
  3. Sterile 6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
  4. 1.7 ml microcentrifuge tubes (Laboratory Products Sales, catalog number: L211511 )
  5. 50 ml conical centrifuge tubes
  6. Microscope slides (Laboratory Products Sales, catalog number: M130400 )
  7. Sterile steel blades (Staples, catalog number: 111322 )
  8. Arabidopsis thaliana (Columbia-0) seeds (https://www.arabidopsis.org/abrc/index.jsp)
  9. Potato true seeds (Solanum tuberosum) (http://www.ars-grin.gov/nr6/)
  10. Potato plant (Solanum tuberosum cv. Désirée) (http://www.ars-grin.gov/nr6/)
  11. CLE peptides (> 70% purity) (Selleck Chemicals LLC) (www.selleckchem.com)
  12. Bleach (6% sodium hypochlorite) (Clorox Company)
  13. Sterile distilled water
  14. Agarose (Thermo Fisher Scientific, catalog number: BP160-100 )
  15. Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
  16. Thiamine hydrochloride (C12H17ClN4OS.HCl) (Sigma-Aldrich, catalog number: T1270 )
  17. MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
  18. Inositol (Thermo Fisher Scientific, catalog number: AC122261000 )
  19. 2-(4-Morpholino)ethane Sulfonic Acid (MES) (Thermo Fisher Scientific, catalog number: BP300-100 )
  20. Sodium Phosphate Monobasic Monohydrate (NaH2PO4.H2O) (Thermo Fisher Scientific, catalog number: S369-500 )
  21. Sucrose (Thermo Fisher Scientific, catalog number: S5-12 )
  22. Gelrite (Thermo Fisher Scientific, catalog number: CAS 71010-52-1 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: CAS 71010-52-1 ”.
  23. Agar (Thermo Fisher Scientific, catalog number: BP1423-500 )
  24. Propagation medium (1 L) (see Recipes)
  25. ½ MS medium (1 L) (see Recipes)
  26. 0.1% agarose (see Recipes)
  27. Sodium phosphate buffer (see Recipes)

Equipment

  1. Sterile forceps and scalpel (sterilized by heat treatment using a Bunsen burner)
  2. 360° Multi-functional tube rotator (Thermo Fisher Scientific, Barnstead Thermolyne, model: Labquake Shaker Rotisserie )
  3. Growth chamber (Percival Scientific, model: I-66LLVL )
  4. Biosafety cabinet (NuAire, model: Class II Type A/B3 )
  5. Olympus BX-50 microscope
  6. Digital CCD camera (QImaging, model: Retiga EXi )

Software

  1. MetaMorph imaging analysis software
    (http://core.phmtox.msu.edu/Scheduling/ItemDocs/9/MetaMorph_Software_Basic_Analysis_Guide.pdf)

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Chen, S. and Wang, X. (2015). In vitro CLE Peptide Bioactivity Assay on Plant Roots. Bio-protocol 5(24): e1689. DOI: 10.21769/BioProtoc.1689.
  2. Chen, S., Lang, P., Chronis, D., Zhang, S., De Jong, W. S., Mitchum, M. G. and Wang, X. (2015). In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism. Plant Physiol 167(1): 262-272.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 蛋白质

植物科学 > 植物生理学 > 植物生长

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