In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology

Abstract

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

Keywords: Camelina sativa; fatty acid desaturase; fatty acid profile; seed oil content; structure; subcellular location.

Figure 1

Synteny analysis of fatty acid desaturase genes in A. thaliana and C. sativa. The map was developed with TBtools software [49]. AtChr and Csa represent the chromosomes in A. thaliana and C. sativa, respectively. Gene names are arranged according to their position in the chromosomes, outside the circle.

Figure 2

Evolutionary relationships of fatty acid desaturases. The tree was inferred using the Neighbor-Joining method [50]. The evolutionary distances were computed using the p-distance method [51] and are in the units of the number of amino acid differences per site. The analysis involved 87 amino acid sequences. Desaturase families were clustered into five classes, indicated by different colors. Numbers indicate the bootstrap value (%). Evolutionary analyses were conducted in MEGAX [52].

Figure 3

Gene structure of fatty acid desaturases in A. thaliana and C. sativa. Numbers indicate the intron phase.

Figure 4

Expression pattern of fatty acid desaturase genes in C. sativa.

Figure 5

Subcellular location of CsaFAD7 and CsaFAD8. The CsaFAD7 and CsaFAD8 coding sequences were fused with EGFP and transiently transformed into A. thaliana mesophyll protoplast. GFP, the green fluorescent detection channel signal. Chloroplast-auto, the chlorophyll autofluorescence. Bar = 10 μm.

Figure 6

Transgenic screen of CsaFAD7 and CsaFAD8 seed-specific overexpression lines and fatty acid composition. (a) CsaFADs overexpression cassettes structure. LB, left border; RB, right border; Gly term, Glycinin terminator; NOS term, nopaline synthase terminator. (b) Screening of transgenic lines according to red fluorescence DsRed3 marker. BF, bright field; 520 nm light, seedlings and seeds were activated by wavelength 520 nm light. (c,d) Fatty acid composition in CsaFAD7 and CsaFAD8 overexpression lines. T3 generation dry mature seeds were collected for GC analysis with 5 times repeat for each line. Asterisk represents a significant difference (p < 0.01, n = 5).

Figure 7

Oil content and seed morphology of CsaFAD7 and CsaFAD8 transgenic lines. (a) Oil content of CsaFAD7 and CsaFAD8 overexpression lines dry seeds. DW, dry weight. T3 generation dry mature seeds were collected for GC analysis with 5 repeats for each line. Asterisk represents a significant difference (p < 0.01, n = 5). (b) Thousand seed weight of CsaFAD7 and CsaFAD8 overexpression lines dry seeds. The dry mature seed size was measured with 5 repeats for each line. Asterisk represents a significant difference (p < 0.01, n = 5). Vector control, pBinGlyRed3 transgenic lines under wild type background as control. (c) Seed appearance of CsaFAD7, CsaFAD8 overexpression lines, and WT dry seeds.