Korean Journal of Breeding Science (한국육종학회지)

The Korean Society of Breeding Science (한국육종학회)
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Development of Perilla frutescens with Low Levels of Alpha-Linolenic Acid by Inhibition of a delta 15 desaturase Gene

Delta 15 desaturase 유전자 억제에 의해 알파리놀렌산 함량이 낮은 들깨 육성

  • Kim, Kyung-Hwan (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Lee, Kyeong-Ryeol (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Kim, Jung-Bong (Dept. of Agro-food Resources, National Institute of Agricultural Sciences, RDA) ;
  • Lee, Myoung Hee (Dept. of Southern Area Crop Science, National Institute of Crop Science, RDA) ;
  • Lee, Eungyeong (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Kim, Nyunhee (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Lee, Hongseok (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Kim, Song Lim (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Baek, JeongHo (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Choi, Inchan (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA) ;
  • Ji, Hyeonso (Dept. of Agricultural Biotechnology, National Institute of Agricultural Sciences, RDA)
  • 김경환 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이경렬 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김정봉 (농촌진흥청 국립농업과학원 농식품자원부) ;
  • 이명희 (농촌진흥청 국립식량과학원 남부작물부) ;
  • 이은경 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김년희 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이홍석 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김송림 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 백정호 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 최인찬 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 지현소 (농촌진흥청 국립농업과학원 농업생명자원부)
  • Received : 2018.10.30
  • Accepted : 2018.11.07
  • Published : 2018.12.01
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Abstract

Perilla is an oilseed crop cultivated in Korea since ancient times. Due to the high ${\alpha}-linolenic$ acid content in perilla, perilla seed oil can easily become rancid. ${\alpha}-Linolenic$ acid is synthesized by two enzymes, endoplasmic reticulum-localized ${\Delta}15$ desaturase (FAD3) and chloroplast-localized ${\Delta}15$ desaturase (FAD7) in vivo. In order to lower the ${\alpha}-linolenic$ acid content of the seed oil without disturbing plant growth, we tried to suppress the expression of only the FAD3 gene using RNA interference, whilst maintaining the expression of the FAD7 gene. Seventeen transgenic plants with herbicide ($Basta^{TM}$) resistance were obtained by Agrobacterium-mediated transformation using hypocotyls of perilla plants. The transgenic plants were firstly confirmed by treatment with 0.3% (v/v) $Basta^{TM}$ herbicide, and the expression of FAD3 was measured by Northern blot analysis. The ${\alpha}-linolenic$ acid content was 10-20%, 30-40%, and 60% in two, seven, and three of the twelve $T_1$ transgenic perilla plants which had enough seeds to be analyzed for fatty acid composition, respectively. Analysis of the fatty acid composition of $T_2$ progeny seeds from $T_1$ plants with the lowest ${\alpha}-linolenic$ acid content showed that the homozygous lines had 6-10% ${\alpha}-linolenic$ acid content and the heterozygous lines had 20-26% ${\alpha}-linolenic$ acid content. It is expected that the reduction in ${\alpha}-linolenic$ acid content in perilla seed oil will prevent rancidity and can be utilized for the production of high-value functional ingredients such as high ${\gamma}-linolenic$ acid.

들깨는 우리나라에서 재배되어온 대표적인 유지작물로 지질의 함량 중 알파리놀렌산의 함량이 60% 전후로 불포화도가 높아서 산패가 쉽게 일어나는 문제가 있다. 알파리놀렌산은 소포체 유래의 ${\Delta}15$ desaturase (FAD3)와 엽록체 유래의 ${\Delta}15$ desaturase (FAD7)에 의해서 합성된다. 엽록체 유래의 FAD7 유전자 발현의 손상없이 종자의 알파리놀렌산 함량을 낮추기 위해 소포체 유래 FAD3 유전자를 RNAi기법을 이용하여 발현을 억제하였다. 재배종인 엽실들깨의 배축을 이용하여 아그로박테리움 매개 형질전환법으로 제초제(바스타) 저항성을 가진 형질전환체 17개체를 획득하였다. 형질전환체는 0.3% (v/v) 바스타제초제를 이용하여 선발하였으며 Northern blot으로 FAD3 유전자의 발현이 억제되는 것을 확인하였다. 온실에서 수확한 12개체 종자 지방산 함량을 분석한 결과 알파리놀렌산 함량이 10-20% 2개체, 30-40% 7개체, 대조구와 비슷한 60%대 3개체를 획득하였다. 형질전환체의 $T_2$ 종자의 분리비와 지방산 조성을 분석한 결과 동형접합체 계통에서 6-10% 알파리놀렌산 함량을 보였으며 이형접합계통은 20-26% 알파리놀렌산 함량을 나타내어 동형으로 고정시 FAD3 유전자 발현이 상당히 강력히 억제됨을 확인할 수 있었다. 들기름의 지방산 중 알파리놀렌산 함량의 감소는 들깨의 산패를 방지하고 감마리놀렌산 등의 고가의 건강기능성 지방산 생산에 활용할 수 있을 것으로 기대된다.

Keywords

Acknowledgement

Supported by : 국립농업과학원

References

  1. Alwine JC, Kemp DJ, Stark GR. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74: 5350-5354. https://doi.org/10.1073/pnas.74.12.5350
  2. Asif M. 2011. Health effects of omega-3,6,9 fatty acids: Perilla frutescens is a good example of plant oils. Orient Pharm Exp Med 11: 51-59. https://doi.org/10.1007/s13596-011-0002-x
  3. Bilyeu K, Gillman JD, LeRoy AR. 2011. Novel FAD3 mutant allele combinations produce soybeans containing 1% linolenic acid in the seed oil. Crop Sci 51: 259-264. https://doi.org/10.2135/cropsci2010.01.0044
  4. Ciftci ON, Przybylski R, Rudzinska M. 2012. Lipid components of flax, perilla, and chia seeds. Eur J Lipid Sci Tech 114: 794-800. https://doi.org/10.1002/ejlt.201100207
  5. Cloutier S, Ragupathy R, Niu Z, Dugid S. 2011. SSR-based linkage map of flax (Linim usitatissimum L) and mapping of QTLs underlying fatty acid composition traits. Mol Breeding 28: 437-451. https://doi.org/10.1007/s11032-010-9494-1
  6. Ecker JR, Davis RW. 1986. Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83: 5372-5376. https://doi.org/10.1073/pnas.83.15.5372
  7. Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. https://doi.org/10.1038/35888
  8. Holster MD, de Waele A, Depicker E, Messens M, van Montagu M, Schell J. 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163: 181-187. https://doi.org/10.1007/BF00267408
  9. Iba K, Gibson S, Nishiuchi T, Fuse T, Nishimura M, Arondel V, Hugly S, Somerville C. 1993. A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem 268: 24099-24105.
  10. Karimi M, Inze D, Depicker A. 2002. $GATEWAY^{TM}$ vectors for Agrobacterium-mediated plant transformation. Trend Plant Sci 7: 193-195. https://doi.org/10.1016/S1360-1385(02)02251-3
  11. Kim KH, Lee YH, Kim D, Park YH, Lee JY, Hwang YS, Kim YH. 2004. Agrobacterium-mediated genetic transformation of Perilla frutescens. Plant Cell Rep 23: 386-390. https://doi.org/10.1007/s00299-004-0825-8
  12. Lee KR, Lee Y, Kim E-H, Lee S-B, Roh KH, Kim J-B, Kang H-C, Kim HU. 2016. Functional identification of oleate 12-desaturase and ${\omega}$-3 fatty acid desaturase genes from Perilla frutescens var. frutescens. Plant Cell Rep 35: 2523-2537. https://doi.org/10.1007/s00299-016-2053-4
  13. Longvah T, Deosthale YG. 1991. Chemical and nutritional studies on hanshi (Perilla frutescens) a traditional oilseed from northeast India. J Am Oil Chem Soc 68: 781-784. https://doi.org/10.1007/BF02662172
  14. Mcconn M, Hugly S, Browse J, Somerville C. 1994. A Mutation at the Fad8 Locus of Arabidopsis Identifies a 2nd Chloroplast Omega-3 Desaturase. Plant Physiol 106: 1609-1614. https://doi.org/10.1104/pp.106.4.1609
  15. 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
  16. Napoli C, Lemieux C, Jorgensen R. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279-289. https://doi.org/10.2307/3869076
  17. Shin HS, Kim SW. 1994. Lipid composition of perilla seed. J Am Oil Chem Soc 71: 619-622. https://doi.org/10.1007/BF02540589
  18. Singer SD, Weselake RJ, Rahman H. 2014. Development and characterization of low ${\alpha}$-linolenic acid Brassica oleracea lines bearing a novel mutation in a 'class a' FATTY ACID DESATURASE 3 gene. BMC Genetics 15: 94-105.
  19. Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D. 1988. Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 14: 724-726.
  20. Spasibionek S. 2006. New mutants of winter rapeseed (Brassica napus L.) with changed fatty acid composition. Plant Breed 125: 259-267. https://doi.org/10.1111/j.1439-0523.2006.01213.x
  21. Tanhuanpaa PK, Vikki JP, Vikki JH. 1996. Mapping of a QTL for oleic acid concentration in spring turnip rape (Braccica rapa ssp. oleifera). Theor Appl Genet 92: 952-956. https://doi.org/10.1007/BF00224034
  22. Thambugala D, Duguid S, Loewen E, Rowland G, Booker H, You FM, Cloutier S. 2013. Genetic variation of six desaturase genes in flax and their impact on fatty acid composition. Theor Appl Genet 126: 2627-2641. https://doi.org/10.1007/s00122-013-2161-2
  23. Vrinten P, Hu Z, Munchinsky MA, Rowland G, Qiu X. 2005. Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiol 139: 79-87. https://doi.org/10.1104/pp.105.064451
  24. Wassom JJ, Mikkelinem V, Bohn MO, Rocheford TR. 2008. QTL for fatty acid composition of maize kernel oil in Illinois high oil x B73 backcross-drived lines. Crop Sci 48: 69-78. https://doi.org/10.2135/cropsci2007.04.0208