Identification of genes associated with the biosynthesis of unsaturated fatty acid and oil accumulation in herbaceous peony 'Hangshao' (Paeonia lactiflora 'Hangshao') seeds based on transcriptome analysis

Abstract

Background: Paeonia lactiflora 'Hangshao' is widely cultivated in China as a traditional Chinese medicine 'Radix Paeoniae Alba'. Due to the abundant unsaturated fatty acids in its seed, it can also be regarded as a new oilseed plant. However, the process of the biosynthesis of unsaturated fatty acids in it has remained unknown. Therefore, transcriptome analysis is helpful to better understand the underlying molecular mechanisms.

Results: Five main fatty acids were detected, including stearic acid, palmitic acid, oleic acid, linoleic acid and α-linolenic acid, and their absolute contents first increased and then decreased during seed development. A total of 150,156 unigenes were obtained by transcriptome sequencing. There were 15,005 unigenes annotated in the seven functional databases, including NR, NT, GO, KOG, KEGG, Swiss-Prot and InterPro. Based on the KEGG database, 1766 unigenes were annotated in the lipid metabolism. There were 4635, 12,304, and 18,291 DEGs in Group I (60 vs 30 DAF), Group II (90 vs 60 DAF) and Group III (90 vs 30 DAF), respectively. A total of 1480 DEGs were detected in the intersection of the three groups. In 14 KEGG pathways of lipid metabolism, 503 DEGs were found, belonging to 111 enzymes. We screened out 123 DEGs involved in fatty acid biosynthesis (39 DEGs), fatty acid elongation (33 DEGs), biosynthesis of unsaturated fatty acid (24 DEGs), TAG assembly (17 DEGs) and lipid storage (10 DEGs). Furthermore, qRT-PCR was used to analyze the expression patterns of 16 genes, including BBCP, BC, MCAT, KASIII, KASII, FATA, FATB, KCR, SAD, FAD2, FAD3, FAD7, GPAT, DGAT, OLE and CLO, most of which showed the highest expression at 45 DAF, except for DGAT, OLE and CLO, which showed the highest expression at 75 DAF.

Conclusions: We predicted that MCAT, KASIII, FATA, SAD, FAD2, FAD3, DGAT and OLE were the key genes in the unsaturated fatty acid biosynthesis and oil accumulation in herbaceous peony seed. This study provides the first comprehensive genomic resources characterizing herbaceous peony seed gene expression at the transcriptional level. These data lay the foundation for elucidating the molecular mechanisms of fatty acid biosynthesis and oil accumulation for herbaceous peony.

Keywords: Differentially expressed gene; Fatty acid biosynthesis; Paeonia lactiflora ‘Hangshao’; Transcriptome; qRT-PCR.

Fig. 1

Oil content and absolute content of five main fatty acids in seeds of Paeonia lactiflora ‘Hangshao’. a Seeds in the collecting period; b Oil content in the developing seed; c Absolute content of five main fatty acids in the developing seed. DAF, days after flowering

Fig. 2

Analysis of DEGs in seeds of Paeonia lactiflora ‘Hangshao’. a Gene analysis of DEGs in different stages; b Cluster analysis of DEGs in different stages

Fig. 3

Analysis of DEGs in significantly enriched KEGG pathways. a Number of DEGs in the top 25 significantly enriched KEGG pathways; b Number of DEGs in the l4 lipid metabolism KEGG pathways; * represents a significantly enriched KEGG pathway

Fig. 4

Analysis of DEGs obtained from the intersection of three groups. a Venn diagram between DEGs from three groups; b Functional distribution of intersection DEGs annotated by GO; c Functional distribution of intersection DEGs annotated by KEGG

Fig. 5

Cluster analysis of DEGs in the lipid metabolism pathway of Paeonia lactiflora ‘Hangshao’ seeds. a Hierarchical clustering dendrogram of DEGs in lipid metabolism. The red highlighting indicates genes that were highly expressed, whereas the blue highlighting indicates genes with low expression. b The four cluster groups with different gene expression patterns

Fig. 6

Proposed gene networks involved in unsaturated fatty acid biosynthesis and oil accumulation in Paeonia lactiflora ‘Hangshao’ seeds. The expression levels (represented by the Log₂ FPKM) of the possible candidates are highlighted by color scales (blue to red scale) in Paeonia lactiflora ‘Hangshao’ seeds at different development stages (30, 60 and 90 DAF). BC: biotin carboxylase; BCCP: biotin carboxyl carrier protein; α-CT: carboxyl transferase subunit alpha; β-CT: carboxyl transferase subunit beta; MACT: malonyl-CoA-acyl carrier protein transacylase; KASIII: 3-oxoacyl-ACP synthase III; KAR: 3-oxoacyl-ACP reductase; HAD: 3-hydroxyacyl-ACP dehydratase; EAR: enoyl-ACP reductase I; KASII: 3-oxoacyl-ACP synthase II; FATB: fatty acyl-ACP thioesterase B; FATA: fatty acyl-ACP thioesterase A; SAD: stearoyl-ACP desaturase; FAD: fatty acid desaturase; PCH: palmitoyl-CoA hydrolase; LACS: long-chain acyl-CoA synthetase; PC: phosphatidylcholine LPC: lysophosphatidylcholine; LPCAT: lysophosphatidylcholine acyltransferase; PLA2: phospholipase A2; KCS: 3-ketoacyl-CoA synthase; KCR: very-long-chain 3-oxoacyl-CoA reductase; HCD: very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase; ECR: very-long-chain enoyl-CoA reductase; GPAT: glycerol-3-phosphate acyltransferase; LPAAT: 1-acyl-sn-glycerol-3-phosphate acyltransferase; PAP: phosphatide phosphatase; DGAT: diacylglycerol O-acyltransferase; PDAT: phospholipid:diacylglycerol acyltransferase; PDCT: phosphatidylcholine:diacylglycerol cholinephosphotransferase; OB: oil body; OLE: oleosin; CLO: caleosin. * represented DEGs in all three groups. This model was developed based on the transcriptome data obtained in this study and information from Li et al. [17], Tian et al. [24] and Yang et al. [28]

Fig. 7

Comparative and phylogenetic analysis and FPKM value of 17 genes putatively encoding BCCP in Paeonia lactiflora ‘Hangshao’. a Protein domain analysis of 17 BCCP: MEME prediction resulet; b Amino acid sequence alignment of BCCP family in plant species; c Phylogenetic analysis of the BCCP proteins in Paeonia lactiflora ‘Hangshao’ and other plant speices. Other plant species including Gossypium hirsutum (Gh), Gossypium arboreum (Ga), Theobroma cacao (Tc), Juglans regia (Jr), Pistacia vera (Pv), Glycine soja (Gs), Glycine max (Gm), Malus domestica (Md), Vitis vinifera (Vv), Vitis riparia (Vr), Camellia sinensis (Cs-XM 02822298), Prunus persica (Pp), Prunus mume (Pm), Prunus dulcis (Pd), Arabidopsis thaliana (At), Vernicia fordii (Vf), Jatropha curcas (Jc), Ricinus communis (Rc), Citrus sinensis (Cs-XM 006482671), Quercus suber (Qs), Quercus lobata (Ql), Nelumbo nucifera (Nn), Helianthus annuus (Ha), Salvia miltiorrhiza (Sm), Sesamum indicum (Si), Olea europaea var. sylvestris (Oe) and Nicotiana tabacum (Nt). d FPKM value of 17 BCCP in Paeonia lactiflora ‘Hangshao’ from transcriptome database

Fig. 8

qRT-PCR validation of the expression levels of DEGs related to unsaturated fatty acid biosynthesis and oil accumulation for developmental seeds of Paeonia lactiflora ‘Hangshao’ The column chart shows the qRT-PCR validations in 5 periods; the broken line graph shows the transcriptome sequencing in 3 periods.