Page 318 - ebook

Identification and characterization of FATTY
ACID BIOSYNTHESIS 2 genes

Hyun-A Choi and Hyun Uk Kim *
Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 05006 Republic of Korea

Abstract
Perilla is one of the oilseed crops cultivated mainly in Korea and Asia. Perilla seed has 43% Fig. 2 Amino acid sequence alignment
triacylglycerol (TAG), which is composed of 64% of α-linolenic acid and 14 % of linoleic acid. These among FAB2 genes isolated from
polyunsaturated fatty acids (PUFAs) have the advantage of lowering cholesterol levels in humans.
FATTY ACID BIOSYNTHESIS 2 (FAB2) is responsible for forming a double bond in catalyzing 18:0- Arabidopsis, P.citridora, and
acyl carrier protein (ACP) to 18:1-ACP in the plastid. 18:1-ACP is transported to the endoplasmic P. frutescens.
reticulum (ER) and is used as a precursor to synthesize PUFAs. In this study, we identified the FAB2
gene from the wild-type diploid perilla (Perilla citriodora) and the cultivated tetraploid perilla (Perilla Green box indicates N-terminal chloroplast
frutescens var. frutescens) genomes. Sequence analysis revealed that P. citriodora has two copies of transit peptide. Red box shows conserved
PfrFAB2A and PfrFAB2B, which is different from Arabidopsis has one copy of FAB2. The histidine motifs. The asterisk shows the
P.frutescens has four copies of PfrFAB2A-1, PrFAB2A-2, PfrFAB2B-1, and PfrFAB2B-2, which is difference in amino acid sequence between
indicating that tetraploid perilla came from natural crossing between P.citrodora and unidentified FAB2A and FAB2B in Perilla. Green asterisk
diploid perilla. To test the functional activity of perilla FAB2 genes, we will conduct a indicates amino acids specific to Perilla
complementation test by transforming perilla FAB2 genes to Arabidopsis fab2 mutant. FAB2A and blue asterisk indicates amino
acids specific to Perilla FAB2B only, when
Introduction comparing 22 FAB2s of 9 species
(d)
(a)

Perilla frutescens is one of the oilseed crops cultivated mainly in Korea and Asia. Mostly leaves and (b)
seeds are consumed, and the seeds composed of 43% triacylglycerol (TAG) and 23% carbohydrate. (c)
TAG is consisted of 64% of α-linolenic acid and 14% of linoleic acid. This polyunsaturated fatty acids
have the advantage of helping metabolism and lowering cholesterol levels in humans.
Recently, people are getting more and more interested in health and Perilla seed oil. In line graphs
regard, the increase in domestic annual cultivation and consumption of perilla can be seen in the
graph and can be expected to increase further in the future.
Fig. 3 Confirmation of T-DNA insertion fab2 mutants by genotyping and its phenotype
This is a schematic diagram of the synthesis and
metabolism of fatty acid based lipids in Arabidopsis. (a) Schematic genomic DNA structure of fab2 mutants.
Among these genes, FAB2 (FATTY ACID (b) Genotyping results of fab2 mutants by PCR
BIOSYNTHESIS 2) are target protein of this (c) FAB2 gene expression by RT- PCR
research. FAB2 is responsible for providing 18:1 fatty (a) Plant phenotype of homozygous fab2 mutants compared to Arabidopsis WT(col-0)
acid moiety to chloroplast lipid for leaf and
triacylglycerol in seeds. (a)
The 18:1 moiety is transported to endoplasmic ( (c)
reticulum (ER) and it is used for precursor to c )
synthesize polyunsaturated fatty acids (PUFAs) of
triacylglycerol (TAG) in seed. In the absence of
FAB2, unsaturated fatty acids are not synthesized.
(b)
In this study, the purpose is that identification of
FAB2 gene from Perilla and regulation the
unsaturated and saturated fatty acid in Perilla and
Arabidopsis. Then, it will be helpful for industrial
applications in plant. (d)
Results
(d)
(a)
(b)

Fig. 4 Seed analysis of Arabidopsis fab2 homozygous mutants
To ensure fab2 mutant seed genotype, we identify seed data.
(a)Phenotype of Arabidopsis fab2 mutant seed
(c) (b)Seed length, width, size and Seed weight of Arabidopsis WT(col-0)
and homozygous mutants of AtFAB2.
(c) Analyze Seed Fatty acid composition by GC-MS method
(d) Analyze Leaf Fatty acid composition by GC-MS method
Ongoing study
➢ We will make pB2FWG2-PfrFAB2 vector and transfer PfrFAB2 gene into fab2 mutants of
Arabidopsis to generate transgenic lines
➢ We will analyze fatty acid composition in transgenic lines transferred PfrFAB2 gene
➢ We will analyze expression of FAB2s in tissue and seed development stage
Fig. 1 Schematic structure of FAB2 genes in Arabidopsis and Perilla
and phylogenetic tree References
(a)Genomic DNA structure of FAB2 in Arabidopsis thaliana ➢ KIM et al.(2016) Transcriptome analysis and identification of genes associated with ω-3
(b)Genomic DNA structure of FAB2 in Perilla citiodora (diploid perilla). fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens BMC Genomics 17:474.
.
.
(c) Genomic DNA structure of FAB2 in Perilla frutescens (cultivated tetraploid perilla).
PfrFAB2 exists in two copies unlike AtFAB2. ➢ Jonathan Lightner, Jingrui Wu, and John Browse. (1994) A Mutant of Arabidopsis with
(d) Phylogenetic analysis of amino acid sequences of FA biosynthesis genes. lncreased Levels of Stearic Acid. Plant Physiol. 106: 1443-1451
Sejong University, Seoul Korea