高粱根中独脚金内酯的提取和测定

Kaori Yoneyama; Xiaonan Xie; Takahito Nomura; Koichi Yoneyama

doi:10.21769/BioProtoc.1763

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Peer-reviewed

Extraction and Measurement of Strigolactones in Sorghum Roots

高粱根中独脚金内酯的提取和测定

KY Kaori Yoneyama

XX Xiaonan Xie

TN Takahito Nomura

KY Koichi Yoneyama email

发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1763 浏览次数: 10304

评审: Arsalan DaudiLaia ArmengotAnonymous reviewer(s)

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Abstract

Strigolactones (SLs) are carotenoid-derived signaling chemicals containing two lactone moieties in their structures and induce seed germination of root parasitic plants, Striga and Orobanche spp. In the rhizosphere, SLs are essential host recognition signals not only for root parasitic plants but also for arbuscular mycorrhizal fungi. In plants, SLs play important roles as plant hormones regulating shoot and root architecture. Plants produce only trace amounts of chemically unstable SLs, which makes it difficult to determine SL contents in plant tissues. Here, we describe how to extract and quantify sorgomol and 5-deoxystrigol, major SLs produced in sorghum roots.

Keywords: Strigolactone (独角金内酯)

LC-MS/MS (LC-MS/MS)

Sorgomol (sorgomol)

5-deoxystrigol (5-deoxystrigol)

Quantification (量化)

Materials and Reagents

50 ml Screw cap bottles (MonotaRO Co., Duran, catalog number: 371-05-20-52 )
Filter paper (90 mm) (MISUMI Corporation, Toyo Roshi Kaisha Ltd, catalog number: 00011090 )
pH indicator paper (Merck Millipore Corporation, catalog number: 109526 )
Spin column (Merck Millipore Corporation, catalog number: UFC3 0HV 000 )
Vials and caps (Chromacol, catalog number: 1030-41201 and 1030-42473 ) for LC-MS/MS analysis
Sorghum (Sorghum bicolor) plants grown under P or N deficiency
Note: Striga-tolerant sorghum cultivars may be obtained from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). We examined several sorghum “Hybrid” cultivars for their SL production, and there were essentially no quantitative and qualitative differences in SL production among them.
Ethyl acetate (KANTO CHEMICAL, catalog number: 14029-70 )
500 pg of d₆-5-deoxystrigol (Ueno et al., 2010) added to each sample in our experiments
Note: Internal standards if available.
Anhydrous MgSO₄ (KANTO CHEMICAL, catalog number: 25035-00 ) or Na₂SO₄ (Kanto Chemical, catalog number: 37280-00 )
Acetonitrile (KANTO CHEMICAL, catalog number: 01031-2B )
0.2 M K₂HPO₄ (see Recipes)
Acetonitrile with 0.1% acetic acid for LC-MS/MS (see Recipes)
Water with 0.1% acetic acid for LC-MS/MS (see Recipes)

Equipment

Funnel (45 mm diameter) (MonotaRO Co., AGJ, catalog number: 233-09-11-04 )
Erlenmeyer flasks (50 ml) (Sansyo, Iwaki, catalog number: 4980FK50 )
Separatory funnel (100 ml) (Iwaki, catalog number: 6402FS100R )
Evaporator flasks (300 ml, 50 ml) (Sibata, catalog number: 371-13-68-34 and 371-13-68-21 )
Pasteur pipette (Thermo Fisher Scientific, catalog number: 13-678-20C )
Mini-benchtop centrifuge (IKA, catalog number: 969-65-03-01 )
LC-MS/MS (AB Sciex, model: QTRAP 5500 )
UHPLC system (Shimadzu Corporation, model: Nexera X2 )
18 column (ϕ 2.1 x 150 mm, 2.6 μm) (Phenomenex, model: Kinetex C18 )

Procedure

English

中文翻译

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Yoneyama, K., Xie, X., Nomura, T. and Yoneyama, K. (2016). Extraction and Measurement of Strigolactones in Sorghum Roots. Bio-protocol 6(6): e1763. DOI: 10.21769/BioProtoc.1763.