Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Inhibition of Starch Biosynthesis by Antisense Expression of cDNAs Encoding ADP-Glucose Pyrophosphorylase Small Subunit in Sweetpotato

고구마에서 ADP-Glucose Pyrophosphorylase Small Subunit cDNA의 Antisense 발현에 의한 전분생합성 저해

  • Min, Sung-Ran (Plant Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Bae, Jung-Myung (School of Life Sciences and Biotechnology, Korea University) ;
  • Harn, Chee-Hark (Biotechnology Institute, Nongwoo Bio Co., Ltd.) ;
  • Jeong, Won-Joong (Plant Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Young-Bok (Dept. of Horticulture, Chungnam National University) ;
  • Liu, Jang-Ryol (Plant Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • 민성란 (한국생명공학연구원 식물유전체연구센터) ;
  • 배정명 (고려대 생명공학원) ;
  • 한지학 ((주)농우바이오 생명공학연구소) ;
  • 정원중 (한국생명공학연구원 식물유전체연구센터) ;
  • 이영복 (충남대 농업생명과학대학 원예학과) ;
  • 유장렬 (한국생명공학연구원 식물유전체연구센터)
  • Published : 2007.12.31
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Abstract

Embryogenic calluses derived from shoot apical meristem explants of sweetpotato were subjected to particle bombardment to generate transgenic plants for antisense expression of cDNAs encoding two different AGPase small subunit (ibAGP1 and ibAGP2). Plants were generated via somatic embryogenesis. PCR and Southern analysis demonstrated that the incorporation of ibAGP1 and ibAGP2 into the genome in an antisense orientation. Immunoblot analysis confirmed reduced levels of AGPase small subunit in transgenic plant leaves. Plants with both ibAGP1 and ibAGP2 produced a lower level of the protein than plants with ibAGP1 alone. iodine test demonstrated that transgenic plant leaves and storage root accumulated reduced amounts of starch. Iodine staining of leaf tissues indicated that transgenic plants accumulated less amount of starch than control. In accordance with western blot analysis, plants with both ibAGP1 and ibAGP2 accumulated a lower amount of starch than plants with ibAGP1 alone. Both transgenic plants exhibited a severely retarded growth, resulting in bare survival. It is suggested that disrupted expression of the gene encoding AGPase small subunit is lethal to the growth of sweetpotato contrast to other species including potato.

고구마 유래 ibAGP1, ibAGP2 유전자가 antisense 방향으로 각각 도입된 벡터를 섞어서 particle bombardment 방법으로 고구마 배발생 캘러스에 형질전환을 수행하였다. 선발배지에서 형성된 배발생 캘러스로부터 체세포 배발생 과정을 거쳐 식물체로 재분화된 개체에서 PCR과 Southern 분석으로 고구마의 염색체 게놈에 ibAGP1, ibAGP2 유전자가 도입되었음을 확인하였고, western blot을 통해 형질전환체의 AGPase small subunit 효소 생산이 전반적으로 대조구에 비해 감소하였다. 또한, 형질전환체의 잎과 괴근 절편체의 전분염색 결과에서도 대조구에 비해 부분적으로 전분염색이 이루어짐을 알 수 있었다. 특히, ibAGP1, ibAGP2 유전자가 동시에 들어 있는 식물체 잎에서는 ibAGP1만 들어있는 것에 비해 전분합성이 더욱 감소됨을 확인하였다. 고구마의 경우 감자를 비롯한 다른 식물들과는 달리 외형적인 생장자체를 심하게 저해하였으며 괴근이 형성되지 않아 ibAGP1, 2의 발현 여부가 식물체의 생장에 절대적인 영향을 미침을 시사한다.

Keywords

References

  1. Asemota HN (1995) A fast, simple, and efficient miniscale method for the preparation of DNA from tissues of yam (Dioscorea spp.). Plant Mol Biol Rep 1: 19-21 https://doi.org/10.1007/BF02712670
  2. Bae JM, Liu JR (1997) Molecular cloning and characterization of two novel isoforrns of the small subunit of ADP-glucose pyrophosphorylase from sweetpotato. Mol Gen Genet 254: 179-185 https://doi.org/10.1007/s004380050406
  3. Bae JM, Ham CH, Tae KH, Suh MC, Shin JS, Liu JR (2001) Partial male sterility induced in tobacco by overproduction of mRNA of sweetpotato small subunit ADP-glucose pyrophosphorylase. J Plant Physiol 158: 1273-1279 https://doi.org/10.1078/0176-1617-00615
  4. Choi SB, Kim KH, Kavakli IH, Lee SK, Okita TW (2001) Transcriptional expression chracterization and subcellular localization of ADP-glucose pryophosphorylase in the oil plant Perilla frutescens. Plant Cell Physiol 42: 146-153 https://doi.org/10.1093/pcp/pce019
  5. Gama MICS, Leite Jr RP, Cordeiro AR, Cantliffe DJ (1996) Transgenic sweetpotato plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tissue Organ Cult 46: 237-244 https://doi.org/10.1007/BF02307100
  6. Ghosh HP, Preiss J (1966) Adenisine diphosphate glucose pyrophosphorylase: a regulatory enzyme in the biosynthesis of starch in spinach leaf chloroplasts. J Biol Chem 241: 4491-4504
  7. Giroux MJ, Hannah LC (1994) ADPglucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize. Mol Gen Genet 243: 400-408
  8. Giroux MJ, Shaw J, Barry G, Cobb BG, Greene T, Okita T, Hannah LC (1996) A single mutation that increases maize seed weight. Proc Natl Acad Sci USA 93: 5824-5829
  9. Kwak MS, Min SR, Lee SM, Kim KN, Liu JR, Paek KH, Shin JS, Bae JM (2007) A sepal-expressed ADP-glucose pyrophosphorylase gene (NtAGP) is required for petal expansion growth in 'Xanthi' tobacco. Plant Physiol 145: 277-289 https://doi.org/10.1104/pp.107.102095
  10. Laemmli V (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  11. Lim S, Yang KS, Kwon SY, Paek KY, Kwak SS, Lee HS (2004) Agrobacterium-mediated genetic transformation and plant regeneration of sweetpotato (Ipomoea batatas). Korean J Plant Biotechnol 31: 267-271 https://doi.org/10.5010/JPB.2004.31.4.267
  12. Liu JR, Cantliffe DJ, Simonds SC, Yuan JF (1989) High frequency somatic embryogenesis from cultured shoot apical meristem dome of sweetpotato (Ipomoea batatas). SABRAO J 21: 93-101
  13. Min SR, Liu JR, Rho TH, Kim CH, Ju JI (1994) High frequency somatic embryogenesis and plant regenetation in tissue culture of Korean cultivar sweetpotato. Korean J Plant Tiss Cult 21: 157-160
  14. Min SR, Jeong WJ, Lee YB, Liu JR (1998) Genetic transformation of sweetpotato by particle bombardment. Korean J Plant Tiss Cult 25: 329-333
  15. Muller-Rober B, Sonnewald U, Willmitzer L (1992) Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J 11: 1229-1238
  16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  17. Newell CA, Lowe JM, Merryweather A, Rooke LM, Hamilton WDO (1995) Transformation of sweetpotato (Ipomoea bat alas (L.) Lam.) with Agrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin. Plant Sci 107: 215-227 https://doi.org/10.1016/0168-9452(95)04109-8
  18. Otani M, Mii M, Handa T, Kamada H, Shimada T (1993) Transformation of sweetpotato (Ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizoqenes. Plant Sci 94: 151-159 https://doi.org/10.1016/0168-9452(93)90016-S
  19. Otani M, Shimada T, Kimura T, Saito A (1998) Transgenic plant production from embryogenic callus of sweetpotato (Ipomoea hatatas (L) Lam) using Agrobacterium tumefaciens. Plant Biotechnol 15: 11-16
  20. Outlaw WC Jr, Tarczynski MC (1984) Guard cell starch biosynthesis regulated by effectors of ADP-glucose pyrophosphorylase. Plant Physiol 74: 424-429 https://doi.org/10.1104/pp.74.2.424
  21. Plaxton WC, Preiss J (1987) Purification and properties of non-proteolytically degraded ADP-glucose pyrophosphorylase from maize endosperm. Plant Physiol 83:105-112 https://doi.org/10.1104/pp.83.1.105
  22. Prakash CS, Varadarajan U (1992) Genetic transformation of sweetpotato by particle bombardment. Plant Cell Rep 11: 53-57
  23. Preiss J (1991) Biology and molecular biology of starch synthesis and its regulation. Oxford Surv Plant Mol Cell Biol 7: 59-114
  24. Prioul JL, Jeannette E, Reyss A, Gregory N, Giroux M, Hannah LC, Causse M (1994) Expression of ADPglucose pyrophosphorylase in maize (Zea mays L.) grain and source leaf during grain filling. Plant Physiol 104: 179-187 https://doi.org/10.1104/pp.104.1.179
  25. Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK, Talbert LE, Giroux MJ (2002) Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increase seed yield. Proc Natl Acad Sci USA 99: 1724-1729
  26. Smidansky ED, Martin JM, Hannah LC, Fischer AM, Giroux MJ (2003) Seed yield and plant biomass increase in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase. Planta 216: 656-664
  27. Southern EM (1975) Detection of specific sequences among DNA fragments. J Mol Biol 98: 503-517 https://doi.org/10.1016/S0022-2836(75)80083-0
  28. Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore GM (1992) Regulation of the amount of starch in tissue by ADP-glucose pyrophosphorylase. Science 258: 287-292 https://doi.org/10.1126/science.258.5080.287
  29. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacryamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354
  30. Weber H, Heim U, Borisjuk L, Wobus U (1995) Cell-type, specific, coordinate expression of two ADP-glucose pyrophorylase genes in relation to starch biosynthesis during seed development in Vicia faba L. Planta 195: 352-361