Genome-wide Identification of the CsFAD Gene Family in Camelina sativa and Functional Characterization of the Key Gene CsFAD3.3 Involved in α-linolenic Acid Biosynthesis

doi:10.13560/j.cnki.biotech.bull.1985.2025-1058

Abstract

Abstract:

Objective Camelina sativa (L.) Crantz is a cruciferous oilseed crop with great development potential and its seed oil is rich in unsaturated fatty acids. Fatty acid desaturases (FADs) are key enzymes that catalyze the biosynthesis of unsaturated fatty acids. The identification of the CsFAD gene family in camelina and the functional characterization of the key gene CsFAD3.3 involved in α-linolenic acid (ALA) biosynthesis may provide theoretical support for improving the oil quality of camelina seeds. Method Genome-wide data combined with bioinformatics approaches were used to identify the CsFAD gene family in camelina and to analyze chromosomal distribution, phylogenetic relationships, gene structures, and conserved motifs. Transcriptome data and RT-qPCR were employed to examine tissue-specific expression patterns. Subcellular localization assays were conducted to determine the localization of CsFAD3.3, and transgenic camelina and tobacco were generated for functional validation. Result A total of 54 CsFAD genes were identified, unevenly distributed across 20 chromosomes. Phylogenetic analysis classified them into six subfamilies, consistent with conserved structures and motif features. Expression profiling showed that CsFAD genes exhibited diverse expression patterns across different subgroups but similar patterns within the same subfamily. Notably, members of the CsFAD3 subfamily, especially CsFAD3.3, were predominantly expressed in seeds. Subcellular localization confirmed that CsFAD3.3 was located in the endoplasmic reticulum. Functional assays demonstrated that overexpression of CsFAD3.3 significantly increased ALA content in camelina seeds. Moreover, both the transient expression in N. benthamiana leaves and the stable transformation in N. tabacum seeds demonstrated that CsFAD3.3 could significantly enhance ALA accumulation in recipient leaves and transgenic seeds, respectively. Conclusion CsFAD3.3 is localized in the endoplasmic reticulum and functions as a key rate-limiting enzyme for ALA biosynthesis in camelina. Overexpression of CsFAD3.3 significantly increased ALA content in camelina seeds, tobacco leaves, and tobacco seeds, confirming its functional conservation.

Key words: Camelina sativa (L.) Crantz, FAD gene family, CsFAD3.3 gene, expression pattern analysis, functional validation, α-linolenic acid

GAO Hui-ling, JIA Zheng-rong, XUE Jin-ai, SONG Ya-nan, JIA Xiao-yun, LI Run-zhi. Genome-wide Identification of the CsFAD Gene Family in Camelina sativa and Functional Characterization of the Key Gene CsFAD3.3 Involved in α-linolenic Acid Biosynthesis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1058.

Figures/Tables 11

Fig. 1 Phylogenetic tree, motif, conserved domain and gene structure of CsFAD gene family in camelina

Fig. 2 Phylogenetic tree of CsFAD gene family in camelina

Fig. 3 Chromosome mapping of CsFAD gene family in camelina

Fig. 4 Intraspecific synteny analysis of CsFAD genes in camelina

Fig. 5 Synteny analysis of FAD genes between Camelina sativa, Arabidopsis thaliana (A), and Brassica rapa (B)

Fig. 6 Cis-element analysis in camelina CsFAD promoter sequences

Fig. 7 Expression heatmap of CsFAD family genes across various tissues and seed developmental stages in camelina

Fig. 8 Expression profiles of CsFAD3.1, CsFAD3.2, and CsFAD3.3 in different tissues and seed developmental stages (A) as well as dynamic changes in oil content during seed development of camelina (B)The expression patterns of CsFAD3 genes in roots, stems, leaves, petals, and seeds at 10, 20, 30, and 40 days after flowering (DAF) were analyzed by RT-qPCR. CsActin was used as the internal reference gene, and the relative expression levels were calculated using the 2-ΔΔCt method. Data represent the mean ± standard deviation (SD) from three biological replicates (n=3). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to evaluate significant differences among groups, and different lowercase letters indicate statistically significant differences (P<0.05), the same below

Fig. 9 Subcellular localization of the CsFAD3.3 protein

Fig. 10 Analysis of total lipid content (A) and fatty acid composition (B) in camelina seeds overexpressing CsFAD3.3

Fig. 11 Functional validation of CsFAD3.3 through transient expression in tobacco leaves and stable overexpression in tobacco seedsA, B: Total lipid content (A) and fatty acid composition (B) in tobacco leaves with transient expression of CsFAD3.3. C, D: Total lipid content (C) and fatty acid composition (D) in seeds of stable transgenic tobacco

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