Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz

Abstract

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.

Keywords: Camelina sativa (L.) Crantz; fatty acid desaturase (FAD2-2); gene family; linolenic acid (C18:3); low temperature (4 °C).

Figure 1

Phylogenetic analysis of FAD proteins in Camelina sativa, Arabidopsis thaliana, and Glycine max. Neighbor-joining phylogeny of 36 FAD genes in the 3 species was analyzed by using MEGA 7.0 with 2000 times bootstrap replications. Tree scale is 0.1. FAD family was divided into four subfamilies, namely Omega-3, Omega-6, FAD4, and SLD, which are labeled in the outermost layer. Three different colors were selected to represent three species.

Figure 2

The motifs and structures of FAD gene family from Camelina sativa. MEME was used to analyze the protein sequence, and 10 motifs were predicted in FAD sequence. Conservative domains are represented by different colors, and the black lines are non-conservative domains. Scale bars represent 50 amino acids. Yellow represents CD sequences, black lines represent introns, and blue represents non-coding regions (UTR). Scale bars represent 500 bp.

Figure 3

TBtools was used to draw the maps of chromosome location. Uneven distribution of CsFAD genes on 14 chromosomes. Cluster I represent fatty acid desaturase (FAD) that can catalyze cis-double-bond fatty acids. Cluster II represents fatty acid desaturase (FAD) that can catalyze trans-double-bond fatty acids.

Figure 4

Expression profiles of CsFAD family genes in different Camelina tissues and organs: (A) the normalized expression levels of the hierarchical clustering of 24 CsFAD genes in 5 tissues (leaves, stem, flower, seeds 10 days after flowering (DAF), and seeds 20 days after flowering (DAF)). The transcription level was graded color scale from green to red. Expression data were obtained from the Camelina genome database. The normalized expression level of CsFAD genes is expressed in the form of log2 values; (B) qRT-PCR profiles of CsFAD genes in different Camelina tissues and organs. The expression level of the CsActin gene in Camelina was normalized as “1”. Vertical bars indicated the standard error of the mean. Data are shown as means ± SD of three replicates (n = 3 × 3).

Figure 5

Expression patterns of CsFAD genes in Camelina during low-temperature stress: (A) normalized expression levels of CsFAD genes in Camelina during low-temperature stress. qRT-PCR was used to investigate the expression levels of the CsFAD genes during low-temperature stress. Log2 fold change for low-temperature treatment was used to present expression change under low-temperature treatment. Expression levels are illustrated by graded color scale from green to red; (B) qRT-PCR profiles of CsFAD genes during low-temperature treatment. The low-temperature (4 °C) treatment lasted 0 h, 3 h, and 6 h. The expression level of CsFADs in control was normalized as “1”. Vertical bars indicate the standard error of the mean. Data are shown as means ± SD of three replicates (n = 3 × 3).

Figure 6

Subcellular localization of CsFAD2-2. Subcellular localization of CsFAD2-2 in Arabidopsis protoplasts. CsFAD2-2 was fused with CaMV35S: GFP to obtain the GFP–CsFAD2-2 fusion protein. The upper row shows the fluorescent signal from transforming CaMV35S: GFP vector. The bottom row shows the fluorescent signal from transforming CaMV35S: CsFAD2-2–GFP vector. Scale bars, 2 μm.

Figure 7

Determination of fatty acid in Pichia pastoris: Determination of fatty acid content in Pichia pastoris. Data are shown as means ± SD of three replicates (n = 3 × 3); Statistical significance analysis was carried out by performing Student’s t-test ( ** p < 0.01 and *** p < 0.001).