Skip to main content
SpringerLink
Log in

Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The fatty acid composition in the seed oil was significantly modified following the introduction of transgenes. To further enhance the desirable characteristics of rapeseed oil, it would be beneficial to develop a new approach for the simultaneous silencing of two or more target genes. Our goals in the current study were to (1) increase oleic acid to more than 75%, (2) reduce polyunsaturated fatty acids (PUFA) to about 10% and erucic acid to zero, and (3) accomplish these changes in a single-transformation event. In a single transformation, two fragments amplified from the fatty acid Δ12-desaturase 2 (BnaFAD2) and fatty acid elongase 1 (BnaFAE1) genes of Brassica napus were linked together to form a fusion fragment. The fusion fragment was then used to assemble unique intron-spliced hairpin interfering constructs. In the transgenic plant FFRP4-4, the expression of BnaFAD2 and BnaFAE1 genes was completely inhibited. The composition of oleic acid in FFRP4-4 rose to 85%, PUFA dropped to 10% and erucic acid was undetectable. All hybrid F1 seeds obtained from the reciprocal crossing of FFRP4-4 and GX-parents (with different genetic backgrounds) contained more than 80% oleic acid, about 10% PUFA and very low, or undetectable, erucic acid. The results confirmed that the fusion fragment silencing construct can simultaneously and effectively silence the target genes on a consistent basis. The strategy provides a useful tool for detecting gene function and advancing genetic engineering techniques for the improvement of agricultural crops.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Ackman RG (1990) Canola fatty acids—an ideal mixture for health, nutrition and food use. In: Shahidi F (ed) Canola and rapeseed: Production, Chemistry, Nutrition, and Processing Technology. Van Nostrand Reinhold, New York, pp 81–98

    Google Scholar 

  • Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151

    Article  PubMed  CAS  Google Scholar 

  • Bade JB, Damm B (1995) Gene transfer to plants. In: Potrykus I, Spangenberg G (eds) Springer lab manual. Springer, Berlin, pp 30–38

    Google Scholar 

  • Browse J, Spychalla J, Okuley J, Lightner J (1998) Altering the fatty acid composition of vegetable oils. In: Harwood JL (ed) Plant lipid biosynthesis: fundamentals and agricultural applications. Cambridge University Press, New York, pp 131–153

    Google Scholar 

  • Burton JW, Miller JF, Vick BA, Scarth R, Holbrook CC (2004) Altering fatty acid composition in oil seed crops. Adv Agron 4:273–306

    Article  CAS  Google Scholar 

  • Chen W, Li JF, Dong YS, Li GZ, Cun SX, Wang JQ (2006) Obtaining new germplast of Brassica napus with high oleic acid content by RNA interference and marker-free transformation of Fad2 gene. J Plant Physiol Mol Biol 32(6):665–671

    CAS  Google Scholar 

  • Damude HG, Kinney AJ (2008) Enhancing plant seed oils for human nutrition. Plant Physiol 147:962–968

    Article  PubMed  CAS  Google Scholar 

  • Downey R, Craig B (1964) Genetic control of fatty acid biosynthesis in rapeseed (Brassica napus L.). J Am Oil Chem 41:475–478

    Article  CAS  Google Scholar 

  • Ellerström M, Stålberg K, Ezcurra I, Rask L (1996) Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription. Plant Mol Biol 32:1019–1027

    Article  PubMed  Google Scholar 

  • Eskin NAM, McDonald BE, Przybylski R, Malcolmson LJ, Scarth R, Mag T, Ward K, Adolph D (1996) Canola oil. In: Hui YH (ed) Edible oil and fat products: oil and oil seeds. Wiley, New York, pp 1–96

    Google Scholar 

  • FAO (2008) Global Market Analysis. Oilseeds, oils and meals. food outlook. Available at Food and Agriculture Organization of the United Nations. http://www.fao.org/docrep/011/ai474e/ai474e07.htm

  • Finnegan J, McElroy D (1994) Transgene inactivation: plant fight back. Nat Biotechnol 12:883–888

    Article  Google Scholar 

  • Gutiérrez-Nava ML, Aukerman MJ, Sakai H, Tingey SV, Williams RW (2008) Artificial trans-acting siRNAs confer consistent and effective gene silencing. Plant Physiol 147:543–551

    Article  CAS  Google Scholar 

  • Hardin-Fanning F (2008) The effects of a Mediterranean-style dietary pattern on cardiovascular disease risk. Nurs Clin North Am 43:105–115

    Article  PubMed  Google Scholar 

  • Harvey B, Downey R (1963) The inheritance of erucic acid content in rapeseed (Brassica napus L.). Can J Plant Sci 44:104–111

    Article  Google Scholar 

  • James DW, Lim E, Keller J, Plooy I, Ralston E, Dooner HK (1995) Directed tagging of the Arabidopsis fatty acid elongation1 (FAE1) gene with the maize transposon activator. Plant Cell 7:309–319

    Article  PubMed  CAS  Google Scholar 

  • Kinney AJ (1994) Genetic modification of the storage lipids of plants. Curr Opin Biotechnol 5:144–151

    Article  CAS  Google Scholar 

  • Knutzon DS, Thompson GA, Radke SE, Johnson WB, Knauf VC, Kridl JC (1992) Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci USA 89:2624–2628

    Article  PubMed  CAS  Google Scholar 

  • Lauridsen C, Nielsen JH, Henckel P, Sørensen MT (1999) Antioxidative and oxidative status in muscles of pigs fed on rapeseed oil, vitamin E and copper. J Anim Sci 77:105–115

    PubMed  CAS  Google Scholar 

  • Liu Q, Singh SP, Green AG (2002) High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129:1732–1743

    Article  PubMed  CAS  Google Scholar 

  • McVetty PBE, Scarth R (2002) Breeding for improved oil quality in Brassica oilseed species. J Crop Prod 5(1):345–369

    Article  CAS  Google Scholar 

  • Misra S (1990) Transformation of Brassica napus L. with a ‘Disarmed’ octopine plasmid of Agrobacterium tumefaciens: molecular analysis and inheritance of the transformed phenotype. J Exp Bot 41:269–275

    Article  CAS  Google Scholar 

  • Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using agrobacterium vectors. Plant Cell Rep 8(4):238–242

    Article  CAS  Google Scholar 

  • Østergaard L, King G (2008) Standardized gene nomenclature for the Brassica genus. Plant Methods 4:10

    Article  PubMed  CAS  Google Scholar 

  • Peerbolte R, Leenhouts K, Hooykass-van Slogteren GMS, Wullems GJ (1986) Clone from a shoort tobacco crown gall tumorll: irregular T-DNA structures and organization T-DNA, methylation and conditional expression of opine genes. Plant Mol Biol 7:285–299

    Article  CAS  Google Scholar 

  • Przybylski R (2005) Canola oil:physical and chemical properties. Available at Canola Council of Canada. https://canola-council.merchantsecure.com/canola_resources/product43.aspx

  • Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685–694

    Article  CAS  Google Scholar 

  • Röbbelen G, Nitsch A (1975) Genetical and physiological investigations on mutants for polyenoic fatty acids in rapeseed, B. napus L. I. Selection and description of new mutants. Z Pflanzenzüchtg 75:93–105

    Google Scholar 

  • Rücker B, Röbbelen G (1995) Development of high oleic acid winter rapeseed. In: Proceedings of 9th international rapeseed congress, Cambridge, UK, pp 389–391

  • Scarth R (1995) Developments in the breeding of edible oil in Brassica napus and B. rapa. In: Proceedings of 9th international rapeseed congress: rapeseed today and tomorrow, Cambridge, UK, pp 377–382

  • Scarth R, Tang JH (2006) Modification of Brassica oil using conventional and transgenic approaches. Crop Sci 46:1225–1236

    Article  CAS  Google Scholar 

  • Scarth R, McVetty PBE, Rimmer SR, Stefansson BR (1988) Stellar low linolenic-high linoleic acid summer rape. Can J Plant Sci 68:509–511

    Article  CAS  Google Scholar 

  • Schierholt A, Rücker B, Becker HC (2001) Inheritance of high oleic acid mutations in winter oilseed rape (Brassica napus L.). Crop Sci 41:1444–1449

    CAS  Google Scholar 

  • Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133

    Article  PubMed  CAS  Google Scholar 

  • Shahidi F (1990) Rapeseed and canola: global production and distribution. In: Shahidi F (ed) Canola and rapeseed: Production, Chemistry, Nutrition, and Processing Technology. Van Nostrand Reinhold, New York, pp 3–13

    Google Scholar 

  • Sivaraman I, Arumugam N, Sodhi YS, Gupta V, Mukhopadhyay A, Pradhan AK, Burma PK, Pental D (2004) Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene. Mol Breed 13:365–375

    Article  CAS  Google Scholar 

  • Smith MA, Moon H, Chowrira G, Kunst L (2003) Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis. Planta 217:507–516

    Article  PubMed  CAS  Google Scholar 

  • Sovero M (1993) Rapeseed, a new oilseed crop for the United States. In: Janick J, Simon JE (eds) New crops. Wiley, New York, pp 302–307

    Google Scholar 

  • Stålberg K, Ellerström M, Josefsson LG, Rask L (1993) Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco. Plant Mol Biol 23:671–683

    Article  PubMed  Google Scholar 

  • Stefansson BR, Downey RK (1995) Rapeseed. In: Slinkard AE, Knott DR (eds) Harvest of gold: the history of field crop breeding in Canada. University Extension Press, University of Saskatchewan, Saskatoon, pp 140–152

    Google Scholar 

  • Stoutjesdijk PA, Hurlestone C, Singh SP, Green AG (2000) High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous D12–desaturases. Biochem Soc Trans 28:938–940

    Article  PubMed  CAS  Google Scholar 

  • Stoutjesdijk PA, Singh SP, Liu, 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

  • Taylor DC, MacKenzie SL, McCurdy AR, McVetty PBE, Giblin EM, Pass EW, Stone SJ, Scarth R, Rimmer SR, Pickard MD (1994) Stereospecific analyses of seed triacylglycerols from high-erucic acid Brassicaceae: detection of erucic acid at the sn-2 position in Brassica oleracea L. genotypes. J Am Oil Chem Soc 71:163–167

    Article  CAS  Google Scholar 

  • Töpfer R, Martini N, Schell J (1995) Modification of plant lipid synthesis. Science 268:681–686

    Article  PubMed  Google Scholar 

  • Vilkki JP, Tanhuanpää PK (1995) Breeding of high oleic acid spring turnip rape in Finland. In: Proceedings of 9th international rapeseed congress, Cambridge, UK, pp 386–388

  • Warner K, Mounts TL (1993) Frying stability of soybean and canola oils with modified fatty acid compositions. J Am Oil Chem Soc 60:983–988

    Article  Google Scholar 

  • Wong R, Patel JD, Grant I, Parker J, Charne D, Elhalwagy M, Sys E (1991) The development of high oleic canola. In: Abstracts, 8th international rapeseed congress, Saskatoon, Canada, pp 53

  • Wu G, Wu YH, Xiao L, Li XD, Lu CM (2008) Zero erucic acid trait of rapeseed (Brassica napus L.) results from a deletion of four base pairs in the fatty acid elongase 1 gene. Theor Appl Genet 116:491–499

    Article  PubMed  CAS  Google Scholar 

  • Zhang HJ, Xiao G, Tan TL, Li X, Guan CY (2008) High oleate material of rapeseed (Brassica napus) produced by EMS treatment. Sci Agric Sin 41(12):4016–4022

    CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National High Technology and Development Program of China (2008AA10Z152), and by the Development Plan of the State Key Fundamental Research of China (2006CB101603).

Author information

Authors and Affiliations

  1. Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Institute of Oil Crops, Hunan Agricultural University, Changsha, 410128, China

    Qi Peng, Yan Hu, Ran Wei, Chunyun Guan & Chunlin Liu

  2. Development Biology Department, Life Science and Technology College, Hunan Agricultural University, Changsha, 410128, China

    Qi Peng, Yuan Zhang & Ying Ruan

Authors
  1. Qi Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yan Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ran Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chunyun Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chunlin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ying Ruan or Chunlin Liu.

Additional information

Communicated by R. Schmidt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Peng, Q., Hu, Y., Wei, R. et al. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds. Plant Cell Rep 29, 317–325 (2010). https://doi.org/10.1007/s00299-010-0823-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-010-0823-y

Keywords

Search

Navigation