GBSSI 유전자 3'UTR 영역의 발현 억제 dsRNAi 벡터를 이용한 아밀로스함량 조절 벼 개발

Variation of Amylose Content Using dsRNAi Vector by Targeting 3'-UTR Region of GBSSI Gene in Rice

  • 박향미 (농촌진흥청 국립식량과학원) ;
  • 최만수 (농촌진흥청 국립식량과학원) ;
  • 천아름 (농촌진흥청 국립식량과학원) ;
  • 이정희 (농촌진흥청 국립식량과학원 춘천출장소) ;
  • 김명기 (농촌진흥청 국립식량과학원) ;
  • 김연규 (농촌진흥청 국립식량과학원) ;
  • 신동범 (농촌진흥청 국립식량과학원) ;
  • 이장용 (농촌진흥청 국립농업과학원) ;
  • 김율호 (농촌진흥청 국립식량과학원)
  • 투고 : 2010.11.10
  • 발행 : 2010.12.31

초록

본 연구에서는 RNAi 기작을 이용하여 식미에 중요한 영향을 미치는 아밀로스 함량을 다양화하기 위해 GBSSI 유전자의 3'-UTR 부위를 targeting하여 dsRNA를 생성시킬 수 있는 운반체를 제작하고, 벼에 형질전환 하였다. 작성된 형질전환체들을 대상으로 $I_2$-KI 용액 반응과 아밀로스 함량을 분석한 결과, $I_2$-KI 용액에 대한 반응은 waxy 타입으로 나타났으나 아밀로스 함량은 찰벼와 저아밀로스 벼 사이에 해당되는 범위를 보였다. 원품종과 형질전환체 간의 아밀로펙틴 사슬 분포의 차이를 비교한 결과, 단쇄에 해당되는 A1과 B1 사슬의 분포는 감소한 반면, 중쇄에 해당되는 B2와 장쇄인 B3 사슬의 분포는 다소 증가하였으며, B3 사슬의 분포비율은 사용된 품종에 따라 약간의 차이를 보였다. 배유 단면의 전자현미경적 구조를 비교한 결과, 원품종에 비해 형질전환체의 전분립 크기가 작아지고 쪼개짐의 형태가 완만한 굴곡을 보였다. 이러한 결과를 바탕으로, RNAi 기술을 이용하여 다양한 아밀로스 함량이 조절된 형질전환 벼를 개발하기 위해서는 targeting 부위를 결정하는 것이 하나의 중요한 전략이 될 수 있음을 확인하였다.

The amylose content of starch is a major factor in the texture of cooked cereal grains. Therefore, down-regulation of amylose synthesis is one of the alternative method to improve eating quality of rice. We developed transgenic rice plants designed to suppress granule-bound starch synthase I(GBSSI) gene using RNA interference(RNAi) technology. Transgenic plants with RNAi vector containing the 3'-UTR region of GBSSI showed a lower amylose content in rice endosperm than that of wild-type. The range of amylose content was 5.9~9.0% in the transgenic plants, whereas that of wild-type was 17.7~18.0%. Transgenic rices showed the decrease of short chain and the increase of long chain by analyzing chain length distribution of amylopectin in the endosperm. In the SEM micrographs, we found that compound starch granules in whole grains of the wild-type rice were readily split during fracturing, while the starch granules in RNAi-transgenic lines showed small voluminous, non-angular rounded bodies.

키워드

과제정보

연구 과제 주관 기관 : 국립식량과학원

참고문헌

  1. Cai XL, Wang ZY, Xing YY, Zhang JL, Hong MM. 1998. Aberrant splicing of intron 1 leads to the heterogeneous 5'-UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J 14:459- 465. https://doi.org/10.1046/j.1365-313X.1998.00126.x
  2. Choi HC. 2002. Current status and perspectives in varietal improvement of rice cultivars for high-quality and valueadded products. Korean J. Crop Sci. 47(s):15-32.
  3. Chuang C-F, Meyerowitz EM. 2000. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985-4990. https://doi.org/10.1073/pnas.060034297
  4. Frizzi A, Caldo RA, Morrell JA, Wang M, Lutfiyya LL, Brown WE, Malvar TM, Huang S. 2010. Compositional and transcriptional analyses of reduced zein kernels derived from the opaque2 mutation and RNAi suppression. Plant Mol Biol 73:569-585. https://doi.org/10.1007/s11103-010-9644-1
  5. Gibson, TS, Solah VA, McCleary BV. 1997. A procedure to measure amylose in cereal starches and flours with concanavalin A. Journal of Cereal Science 25:111-119. https://doi.org/10.1006/jcrs.1996.0086
  6. Gunja-Smith Z, Marshall JJ, Mercier C, Smith E, Whelan WJ. 1970. A revision of the Meyer-Bernfeld model of glycogen and amylopectin. FEBS Lett 12:101-106. https://doi.org/10.1016/0014-5793(70)80573-7
  7. Hirano HY, Sano Y. 1991. Molecular characterization of the waxy locus of rice (Oryza sativa L.). Plant Cell Physiol 32:989-997.
  8. Hizukuri S. 1986. Polymodel distribution of the chain lengths of amylopectins and its significance. Carbohydr Res 147:342-347. https://doi.org/10.1016/S0008-6215(00)90643-8
  9. Hizukuri S, Kaneko T, Takeda Y. 1983. Measurement of the chain length of amylopectin and its relevance to the origin of crystalline polymorphism of starch granules. Biochem Biophys Acta 760:188-191. https://doi.org/10.1016/0304-4165(83)90142-3
  10. Hori Y, Fujimoto R, Sato Y, Nishio T. 2007. A novel wx mutation caused by insertion of a retrotransposon-like sequence in a glutinous cultivar of rice (Oryza sativa). Theor App Genet 115:217-224. https://doi.org/10.1007/s00122-007-0557-6
  11. Inouchi N, Hibiu H, Li T, Horibata T, Fuwa H, Itani T. 2005. Structural and properties of endosperm starch from cultivated rice of Asia and other countries. J Appl Glycosci 52:239-246. https://doi.org/10.5458/jag.52.239
  12. Inukai T, Sako A, Hirano HY, Sano Y. 2000. Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus. Genome 43:589-596. https://doi.org/10.1139/g00-015
  13. Isshiki M, Morino K, Nakajima M, Okagaki RJ, Wessler SR, Izawa T, Shimamoto K. 1998. A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron. Plant J 15:133-138. https://doi.org/10.1046/j.1365-313X.1998.00189.x
  14. Juliano BO. (editor) 1985. Rice chemistry and technology. American Association of Cereal Chemists USA 774p.
  15. Kim YH, Lee JY, Sohn SI, Lee BK, Lee MS, Hong MY, Kim JK, Cho JH. 2007. Efficient plant transformation method mediated by Agrobacterium tumefaciens. Korea Patent. 10-0736209.
  16. Kimura T, Otani M, Noda T, Ideta O, Shimada T, Saito A. 2001. Absence of amylose in sweetpotato (Ipomoea batatas (L.) Lam.) following the introduction of granulebound starch synthase I cDNA. Plant Cell Rep 20:663-666.
  17. Kuipers AGJ, Jacobsen E, Visser RGF. 1994. Formation and deposition of amylase in the potato tuber starch granule are affected by the reduction of granule-bound starch synthase gene expression. Plant cell 6:43-52. https://doi.org/10.1105/tpc.6.1.43
  18. Kuipers AGJ, Soppe WJJ, Jacobsen E, Visser RGF. 1995. Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato. Mol Gen Genet 246:745-755. https://doi.org/10.1007/BF00290722
  19. Miki D, Shimamoto K. 2004. Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol. 45(4): 490-495. https://doi.org/10.1093/pcp/pch048
  20. Mitsuhara I, Shirasawa SN, Iwai T, Nakamura S, Honkura R, Ohashi Y. 2002. Release from poet-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for non inheritance of the silencing. Genetics 160:343-352.
  21. Nishi A, Nakamura Y, Tanaka N, Satoh H. 2001. Biochemical and genetic analysis of the effects of amyloseextender mutation in rice endosperm. Plant Physiol 127: 459-472. https://doi.org/10.1104/pp.010127
  22. Otani M, Hamada T, Katayama K, Kitahara K, Kim SH, Takahata Y, Suganuma T, Shimada T. 2007. Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants. Plant Cell Rep 26:1801-1807. https://doi.org/10.1007/s00299-007-0396-6
  23. Padmanaban S, Lin Xiaoying, Perera I, Kawamura Y, Sze H. 2004. Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growild-typeh as revealed by RNAi. Plant Physiology 134:1514-1526. https://doi.org/10.1104/pp.103.034025
  24. Satoh H, Nishi A, Yamashita Y, Takemoto Y, Tanaka Y, Hosaka Y, Sakurai A, Fujita N, Nakamura Y. 2003. Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm. Plant Physiol 133:1111-1121. https://doi.org/10.1104/pp.103.021527
  25. Sohn SH, Rhee Y, Hwang DJ, Lee SY, Lee JR, Lee YH, Shin YS, Jeung JU, Kim MK. 2010. Molecular characterization of granule-bound starch synthase (GBSSI) gene of Waxy locus mutants in Japonica rice (Oryza sativa L.). Korean J Breed Sci 42(1):1-10.
  26. Sohn SI, Kim YH, Cho JH, Kim JK, Lee JY. 2006. An efficient selection scheme for agrobacterium-mediated cotransformation of rice using two selectable marker genes hpt and bar. Korean J Breed 38:173-179.
  27. Song J, Kim JH, Kim DS, Lee CK, Youn JT, Kim SL, Suh SJ. 2008. Physicochemical Properties of Starches in Japonica Rices of Differenct Amylose Content. Korean J Crop Sci 53(3):285-291.
  28. Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG. 2002. hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723-1731. https://doi.org/10.1104/pp.006353
  29. Suzuki K, Nakamura S, Satoh H, Ohtsubo K. 2006. Relation between chain-length distributions of waxy rice amylopectins and physical properties of rice grains. Journal of Applied Glycoscience. 53:227-232. https://doi.org/10.5458/jag.53.227
  30. Takemoto-Kuno Y, Suzuki K, Nakamura S, Satoh H, Ohtsubo K. 2006. Soluble starch synthase I effects differences in amylopectin structure between indica and japonica rice varieties. J Agric Food Chem 54:9234-9240. https://doi.org/10.1021/jf061200i
  31. Umeda M, Ohtsubo H, Ohtsubo E. 1991. Diversification of the rice Waxy gene by inserion of mobile DNA elements into introns. Jpn J Genet 66:569-586. https://doi.org/10.1266/jjg.66.569
  32. Umemoto T, Yano M, Satoh H, Shomura A, Nakamura Y. 2002. Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties. Theor Appl Genet 104:1-8. https://doi.org/10.1007/s001220200000
  33. Wanchana S, Toojinda T, Tragoonrung S, Vanavichit A. 2003. Duplicated coding sequence in the waxy allele of tropical glutinous rice (Oryza sativa L.). Plant Sci 165: 1193-1199. https://doi.org/10.1016/S0168-9452(03)00326-1
  34. Wang ZY, Zheng FQ, Shen GZ, Gao JP, Snustad P, Li MG, Zhang JL, Hong MM. 1995. The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene. Plant J 7:613-622. https://doi.org/10.1046/j.1365-313X.1995.7040613.x
  35. Waterhouse PM, Helliwell CA. 2003. Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4:29-38. https://doi.org/10.1038/nrg982
  36. Wei C, Qin F, Zhu L, Zhou W, Chen Y, Wang Y, Gu M, Liu Q. 2010. Microstructure and ultrastructure of high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J Agric Food Chem 58:1224-1232. https://doi.org/10.1021/jf9031316
  37. Wu Y, Messing J. 2010. RNA interference-mediated change in protein body morphology and seed opacity through loss of different zein proteins. Plant Physiology 153:337- 347. https://doi.org/10.1104/pp.110.154690
  38. Zeng D, Yan M, Wang Y, Liu X, Qian Q, Li J. 2007. Du1, encoding a novel Prp1 protein, regulates starch biosynthesis through affecting the splicing of $Wx^b$ pre-mRNAs in rice (Oryza sativa L.). Plant Mol Biol 65:501-509. https://doi.org/10.1007/s11103-007-9186-3