GBSS SDS-PAGE (GBSS) 60 KD (60 KD) (waxy protein) GBSS. (amylose) (amylopectin) (Wx locus) (encode) (granule-bound starch synthase GBSS) (Wx-protein)

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1 70:65-72 (2001) 65 1 GBSS SDS-PAGE (GBSS) SDS-PAGE EITB 60 KD (60 KD) (waxy protein) GBSS (amylose) (amylopectin) (19,30) (27) (Wx locus) (encode) (granule-bound starch synthase GBSS) (Wx-protein) (14) (26,32) (8) (23) (29) (9) (24) (2,5) GBSS (cross-reaction) cdna GBSS (24,31) GBSS (9,24,29) (Buckwheat) (Polygonaceae) (Fagopyrium) 60%

2 66 (17,20,21) (16) 25% 25% 14~28% (26) 30% (4) (1,12) SDS-PAGE (GBSS) Echt and Schwartz (10) 50 SDS-extraction buffer (55 mm Tris ph % SDS 5% β-me 10% glycerol) ,000 xg 15 Echt and Schwartz (10) SDS-extraction buffer 12,000 xg SDS-extraction buffer 12,000 xg Echt and Schwartz (10) 30 mg SDS-extraction buffer ,000 xg 15 SDS-PAGE SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Laemmli (15) 1. Separating gel (12.5%) M Tris -HCl ph % SDS 12.5% acrylamide/bis (30:0.8) 0.075% (NH4)2S2O8 0.05% TEMED (N,N,N,N -tetramethylethylenediamine) 2. Stacking gel (5%) M Tris -HCl ph %

3 GBSS 67 SDS 5% acrylamide/bis (30:0.8) 0.075% (NH4)2S2O % TEMED 3. Running buffer 25 mm Tris 192 mm glycine 0.1% SDS 4. (ATTO PATENT. P.) 30 ma( ) Coomassie blue Stain solution (0.2% Coomassie brilliant blue R % methanol 7% acetic acid) Destain solution (20% methanol 7% acetic acid) Tsang et al. (28) EITB (Enzyme-linked Immunoelectrotransfer Blot) Sambrook (25) NC (nitrocellulose membrane) blot buffer (0.025 M Tris M glycine 20% methanol ph 8.35) 10 ph blot buffer NC PBST [0.01 M PBS (phosphate-buffer saline) (0.15M NaCl 0.01 M Na2HPO4/ NaH2PO4 ph 7.0) 0.3% Tween 20 ] NC 2 PBST 5% 0.01% Sigma Antiform A NC 2 NC TBST 5% 0.01% Antiform A (anti-wx protein antiserum 1/ 2,500 ) (6) PBST 5% 0.01% Sigma Antiform A NC 4 PBST 5% 0.01% Sigma Antiform A ( 2,000 ) PBST NC 3 NC buffer 3 (100 mm Tris -HCl 100 mm NaCl 50 mm MgCl2 ph 9.5) ph NC (BCIP/NBT tablets SIGMA FAST Sigma) 10 ml TE buffer (10 mm Tris -HCl 1 mm EDTA ph 8.0) NC SDS-PAGE SDS-PAGE KD ( ) 60 KD SDS-PAGE GBSS GBSS SDS-PAGE 60 KD ( )

4 68 SDS-PAGE Fig. 1. SDS-PAGE analysis of endosperm protein extract from buckwheat and sorghum seeds A B C D E F G H I M (KD) 60 SDS-PAGE Fig. 2. SDS-PAGE analysis of endosperm starch graule-bounded proteins from buckwheat and sorghum seeds.

5 GBSS 69 ( ) 60 KD Fig. 3. EITB analysis of endosperm starch graule-bounded proteins from buckwheat and sorghum seeds. 4-15um 60% water-binding capacity value 103.7% (blue value) ~68 25% (16,21) SDS-PAGE GBSS 60 KD (60 KD) Echt Schwartz (10) 60 KD

6 70 (10) 60 KD GBSS GBSS I. cdna II p ( ) 30(2): Wx cdna ( ) (Wx ) 29(2): Clark, J. R., M. Robertson and C. C. Ainsworth Nucleotide sequence of a wheat (Triticum aestivum L.) cdna clone encoding the waxy protein. Plant Mol. Biol. 16: Dry, I., A. Smith, A. Edwards, M. Bhattacharyya, P. Dunn and C. Martin Characterization of cdnas encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organ of pea and potato. Plant J. 2(2): Echt C. S. and D. Schwartz Ev idence for the inclusion of controlling elements within the structure gene at waxy locus in maize. Genetics: 99: Hsieh J. S Genetic studies on the Wx gene of sorghum (Sorghum bicolor L. Moench). Bot. Bull. Academia Sinica 29: Hong J. H., K. Ikeda, I. Kreft and K. Yasumoto Near-infrared diffuse reflestance spectroscopic analysis of the amount of moisture, protein, starch, amylose, and tannin in buckwheat Flours. J. Nutr. Sci. Vitaminol. 42: Juliano, B. O Rice: chemistry and technology. St. Paul, Minnesota. U. S. A. p Klösgen, R. B., A. Gierl, Z. Schwarz-Sommer and H. Saedler Molecular analysis of the waxy locus of Zea mays. Mol. Gen. Genet. 203: Laemmli, U. K Cleavage of structure protein during assembly and lipoprotein samples. Anal. Biochem. 87: Li W., R. Lin and H. Corke Physiochemical properties of common and tatary buckwheat starch. Cereal Chemistry 74(1):

7 GBSS Marshall, H. G. and Y. Pomeranz Buckwheat: description, breeding, production, and utilization. Advance in Cereal Science and Technology. 5: Martin C. and A. M. Smith Starch synthesis. The Plant Cell 7: Morell M. K., S. Rahman, S. L. Abrahams and R. Apples The biochemistry and molecular biology of starch synthesis in cereal. Aust. J. Plant Physiol. 88: Oomah B. D. and G. Mazza Flavonoids antioxidative activities in buckwheat. J. Agric. Food Chem. 44: Pomeranz Y Buckwheat: Structure, Composition, and Utilization. CRC Critical Reviews in Food Science and Nutrition 19: Richard G. F. V. and E. Jacobsen Towards modifying plants for altered starch content and composition. TIBTECH 11: Rohde, W., D. Becker and F. Salamini Structural analysis of the waxy locus from Hordeum vulgare. Nucl. Acids Res. 16: Salehuzzaman, S. N. I. M., E. Jacobsen and R. G. F. Visser Isolation and characterization of a cdna encoding granule-bound starch synthase in cassave (Manihot esculenta Crantz) and its antisense expression in potato. Plant Mol. Biol. 23: Sambrook J., E. F. Fritsch and T. Maniatis Molecular cloning. Cold Spring Harber Lab. Press. 26. Sano Y Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68: Smith A. M., K. Denyer and C. Martin The synthesis of the starch granule. Annu. Rev. Plant Mol. Biol. 48: Tsang, C. W., G. E. Bers and K. Hancock Enzyme-linked immunoelectrotranfer blot enzyme-mediated immunoassay. Plenum Press, New York and London. p van der Leij, F. R., R. G. F. visser, A. S. Ponstein, E. Jacobsen and W. J. Feenstra Sequence of the structure gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele. Mol. Gen. Genet. 228: Visser. R. G. F. and E. Jacobsen Towards modifying plants for altered starch content and composition. TIBTECH 11: Vos-Scheperkeuter, G. H., W. DeBoer, R. G. Visser, W. J. Feenstra and B. Witholt Identification of granule-bound starch synthase in potato tubers. Plant Physiol. 82: Wang, Z. Y., Z. Wu, Y. Xing, F. Zheng, X. Guo, W. Zhang and M. Hong Nucleotide sequence of rice waxy gene. Nucl. Acids Res. 18: 5898.

8 72 The Preliminary Studies on the GBSS Proteins of Buckwheat (Fagopyrium esculentum L. Moench) 1 Long-Zen Chang 2, Gwo-Jhin Wang 3 and Jaw-Shu Hsieh 3 ABSTRACT Seed proteins, starch proteins and granule -bound starch synthase (GBSS) proteins were extracted and purified from buckwheat seeds. By using SDS-PAGE and EITB analytical methods, the protein was extracted from seeds of non-waxy sorghum as a check. The results showed that there existed one clearly and densely colored band with molecular weight of 60 KD in eight of common buckwheat cultivars. It was also noticed that there existed a high degree of homology of the 60 KD protein between sorghum and buckwheat. The GBSS protein extracted from buckwheat seeds could be cle arly recognizable by the electrophoretical analysis of antiserum (produced by the endosperm starch bounded protein of sorghum as an antigen) by EITB method. It suggested that GBSS protein presented in buckwheat seeds. Key words: Buckwheat, GBSS protein, Wx- protein, EITB. 1 Contribution No from Taichung DAIS, COA. 2 Assistant Researcher, Crop Improvement Department of Taichung DAIS, COA. 3 Former Graduate Student and Professor of Agronomy Department of National Taiwan University.