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Plant Cell Reports

, Volume 35, Issue 12, pp 2523–2537 | Cite as

Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens

  • Kyeong-Ryeol Lee
  • Yongjik Lee
  • Eun-Ha Kim
  • Seul-Bee Lee
  • Kyung Hee Roh
  • Jong-Bum Kim
  • Han-Chul Kang
  • Hyun Uk KimEmail author
Original Article

Abstract

Key message

We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens.

Abstract

Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

Keywords

Perilla frutescens var. frutescens FAD2 FAD3 FAD7 Microsomal oleate 12-desaturase ω-3 Fatty acid desaturase 

Abbreviations

PUFA

Polyunsaturated fatty acid

FA

Fatty acid

ER

Endoplasmic reticulum

PC

Phosphatidylcholine

UTR

Untranslated region

FAME

Fatty acid methyl ester

Nt

Nucleotide

Bp

Base pair

Aa

Amino acid

PG

Phosphatidylglycerol

DAG

Diacylglycerol

PA

Phosphatidic acid

TMDs

Transmembrane domains

Notes

Acknowledgments

The perilla seeds used in this study were kindly provided by Dr. Myung-Hee Lee of the Dept. of the Southern Area Crop Science, National Institute of Crop Science, in Miryang, Republic of Korea. This study was conducted with the support of the Research Program for Agricultural Science & Technology Development (Project No. PJ01007504), the National Institute of Agricultural Science, Rural Development Administration, and the Next-Generation BioGreen 21 Program (SSAC, Grant No. PJ01108101), Republic of Korea as well as the faculty research fund (Project No. 20160163) of Sejong University.

Compliance with ethical statement

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2016_2053_MOESM1_ESM.pptx (887 kb)
Supplementary Fig. S1 Multiple sequence alignment of two FAD2 genes from perilla leaves and developing seeds tissues. Black box and dotted box indicate PfrFAD2-1 and PfrFAD2-2 specific sequence, respectively. Multiple sequence alignment was performed using DNASTAR® MegAlign (Ver. 8.1.4) (PPTX 887 kb)
299_2016_2053_MOESM2_ESM.pptx (588 kb)
Supplementary Fig. S2 Multiple sequence alignment of two FAD3 genes from diverse perilla cultivar. Black box and dotted box indicate PfrFAD3-1 and PfrFAD3-2 specific sequence, respectively. Exceptionally, there is a single nucleotide polymorphism at 300 bp position in F2 and F4. 13 sequences except PfrFAD3-1 and PfrFAD3-2 were from perilla cultivar as follows. A1,2, Dayudeulkkae; B2, K131012; C1,3, K135903; D4, K131017; E5,6, Anyu; F2,3,4, K135858; G2,3, K126202. These cultivars were obtained from National Agrobiodiversity Center, National Institute of Agricultural Sciences, Republic of Korea. Multiple sequence alignment was performed using DNASTAR® MegAlign (Ver. 8.1.4) (PPTX 588 kb)
299_2016_2053_MOESM3_ESM.pptx (49 kb)
Supplementary Fig. S3 Relative expression levels of PfrFAD2, PfrFAD3, PfrFAD7-1, and PfrFAD7-2 genes from leaves of perilla treated or untreated at low temperature (15˚C) for two weeks. Unattached any letter in front of gene name and LT-gene name indicate untreated and treated low temperature, respectively. Experiments were performed in triplicate and error bars indicate standard deviations (PPTX 48 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Agricultural BiotechnologyNational Institute of Agricultural Science, Rural Development AdministrationJeonjuRepublic of Korea
  2. 2.Division of Integrative Biosciences and BiotechnologyPohang University of Science and TechnologyPohangRepublic of Korea
  3. 3.Department of Bioindustry and Bioresource Engineering, Plant Engineering Research InstituteSejong UniversitySeoulRepublic of Korea

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