Cloning and analysis of the full-length cDNA of AsFAD2 gene family
We cloned the full-length cDNAs of twenty-one AsFAD2 genes from different A. sphaerocephala tissues using RT-PCR and RACE methods based on transcriptome sequence data (Additional file 8: Table S8). However, because of the short lengths of the core fragments and low expression levels of these genes in the tissue the full-length cDNAs of AsFAD2-3, -17, -18, -25, and -26 genes were not obtained. The size of full-length cDNAs for the twenty-one AsFAD2 genes varied between 1320-1728 bp, whereas the length of 5′ UTRs and 3′ UTRs were between 27-373 bp and 87-279 bp, respectively, and the predicted protein sizes were between 371 and 429 amino acids. The theoretical molecular masses and isoelectric points of predicted proteins were about 43.50-49.13 and 6.22-8.83, respectively. According to grand average of hydropathicity (GRAVY) analysis, AsFAD2-2, -7, -14, and -23 genes encoded hydrophobic proteins, whereas the other genes encoded hydrophilic proteins, as they had positive and negative GRAVY values differently. The predicted transmembrane number was between 3 and 6. Plant-mPLoc analysis predicted that the twenty-one AsFAD2 genes were located in the ER.
Sequence identity of multiple members of AsFAD2 gene family
Sequence similarity among the coding regions of twenty-one AsFAD2 genes at the amino acid level was presented in Additional file 9: Figure S1. The result showed that the pairwise similarity of AsFAD2-1 and AsFAD2-12, AsFAD2-16 and AsFAD2-19 were identical with the similarity level of 100.00%, whereas there was only one amino acid different in the pairwise similarity of AsFAD2-5 and AsFAD2-16/19, AsFAD2-6 and AsFAD2-24, AsFAD2-7 and AsFAD2-14, namely, the the similarity levels among these amino acid sequences were 99.74%. Thus, the AsFAD2-1, AsFAD2-5, AsFAD2-6, and AsFAD2-7 were selected for further study. The putative amino acid sequences of sixteen AsFAD2 genes were significantly different, and the similarity level range from 36.54% to 97.85%.
Phylogenetic and motif analysis of encoded proteins of AsFAD2 gene family
To elucidate phylogenetic relationship of the AsFAD2 gene family, the deduced polypeptide sequences of the selected sixteen AsFAD2 genes (Additional file 10: Table S9) were aligned with FAD2 sequences of other plants, including oil plants, model plants, and some plant with divergent FAD2 fatty acid modifying enzymes (Fig. 1). Phylogenetic analysis showed that the sixteen AsFAD2 were divided into seven groups. AsFAD2-1 was clustered with other seed expressed FAD2s, such as sunflower HaFAD2-1 and safflower CtFAD2-1. AsFAD2-10 was clustered together with other constitutively expressed FAD2s, such as sunflower HaFAD2-2, HaFAD2-3, and safflower CtFAD2-2. AsFAD2-23 were clustered together with fatty acid acetylenases and hydroxylases from other plants. AsFAD2-9 and CtFAD2-9, AsFAD2-2, -5, -6, -15 and CtFAD2-8, and AsFAD2-11 and CtFAD2-7 were positioned next to each other, respectively, in the same branch. AsFAD2-4, -8, and -21 were clustered with fatty acid conjugases from Calendula officinalis. AsFAD2-7, -13, -20, and -22 proteins were clustered together with fatty acid acetylenases and epoxygenases from several plant species.
The alignment of putative AsFAD2 polypeptides together with selected plant orthologs was shown in Additional file 11: Figure S2. The AsFAD2 polypeptides contained C-terminal aromatic amino acid-rich motifs. For example, AsFAD2-1, AsFAD2-2, and AsFAD2-4 had YKNKM, FKNKL and WFKK, respectively. Additionally, AsFAD2 family proteins contained three highly conserved histidine-rich motifs. Motifs of FAD2 protein sequences of sixteen A. sphaerocephala, one Arabidopsis thaliana and one Nicotiana tabacum were analyzed (Fig. 2). The detailed information of twenty putative conserved motifs were shown in Additional file 12: Figure S3. These proteins all had nine conserved motifs, including motif 1, 2, 3, 4, 6, 7, 8, 9, and 11. The motif composition of the AsFAD2 family proteins was relatively conserved. AsFAD2-2, -5, -6 and -15 were clustered together to be a branch (Fig.1), and they all had fourteen identical motifs, AsFAD2-5 and AsFAD2-15 contained motif 19. AsFAD2-9 and AsFAD2-2 were next to each other, and had same motifs. The motif composition of AsFAD2-23 was different from other AsFAD2s. AsFAD2-10, AtFAD2, AsFAD2-1 and NtFAD2 were clustered together to be a branch (Fig.1). The motifs of AtFAD2, AsFAD2-1 and NtFAD2 were completely identical. AsFAD2-10 lacked motif 16. AsFAD2-4, -8, -11 and -21 were located next to each other and formed a branch (Fig.1), AsFAD2-4 and AsFAD2-21 had same motifs. In contrast, AsFAD2-8 contained motif 14 and lacked motif 12, AsFAD2-11 had motif 12. AsFAD2-7, -13, -20 and -22 were situated next to each other and formed a branch (Fig.1), and they all had fifteen same motifs.
Expression analysis of AsFAD2 gene family in A. sphaerocephala
The transcript levels of the sixteen AsFAD2 genes in different A. sphaerocephala tissues were detected using qRT-PCR. It displayed that the expression patterns of AsFAD2 gene family were diverse, and they may play different functions roles in different tissues and organs. The numbers and relative expression levels of AsFAD2 genes increased significantly in the seed swelling and germination, especially the expression levels of AsFAD2-2, -15, -20 increased significantly (Fig. 3a-c). The expression level of AsFAD2-15 was the highest in roots (Fig. 3d). AsFAD2-15 and AsFAD2-20 showed high expression levels in stems and leaves (Fig. 3d-f). In flower buds and flowers, the expression levels of AsFAD2-20 and AsFAD2-13 were the highest (Fig. 3g-h), respectively, compared to other AsFAD2s. AsFAD2-1 was strongly expressed in developing seeds, but had low expression levels in other tissues, belonging to gene of the seed-type expression. AsFAD2-10 was expressed in all the checked tissues, belonging to gene of the constitutive expression. AsFAD2-1 and AsFAD2-10 may play an important role in the formation of high linoleic acid in A. sphaerocephala seeds (Fig. 3i-j).
According to the expression pattern of AsFAD2 genes in various A. sphaerocephala organs, eleven genes with high expression in leaves were selected to analyze their response to salt stress (Fig. 5). Forty-five day-old seedlings were treated with 50 and 200 mM NaCl for 7 days, and the relative expression of the AsFAD2 genes in leaves was compared with that in untreated control plants. At 50 mM NaCl, the expression of AsFAD2-1 and -10 were downregulated significantly, whereas that of AsFAD2-2, -15, and -22 were upregulated, and that of the other genes were unchanged compared to control. At 200 mM NaCl, the expression of AsFAD2-2 and -5 genes were increased significantly, whereas that of AsFAD2-7 was decreased significantly, and that of the other genes showed no difference compared to control. Overall, mRNA expression of eleven AsFAD2 genes was not significantly changed with NaCl treatment.
Subcellular Localization of AsFAD2 proteins
Based on phylogenetic relationship and tissue expression patterns, seven AsFAD2 genes were selected for subcellular localization analysis, including AsFAD2-1, -9, -10, -11, -15, -20, and -23. The results showed that seven AsFAD2 cDNA-encoded proteins were localized to network-like organelles, the strong GFP and RFP signals were observed in the epidermal cells of tobacco leaves, and the both fluorescent signals could be overlapped and displayed as yellow fluorescent signals, indicating that the selected seven AsFAD2s were transiently expressed in the ER of tobacco leaf epidermal cells (Fig. 5). It was speculated that the other AsFAD2 proteins could also be located in the ER.
Functional analysis of AsFAD2 genes in yeast
Sixteen AsFAD2 family members were expressed in the S. cerevisiae INVSc1, and the fatty acid compositions of the yeasts were analyzed (Fig. 6 and Additional file 13: Table S10). The results indicated that dienoic fatty acids, including palmitolinoleic acid (C16:2) and LA (C18:2), were not produced in the yeast with the empty pYES2 vector (Fig. 6a). However, C18:2 content was respectively 18.58%, 16.54% and 3.29% of total fatty acids in the transformed yeast expressing the AsFAD2-1 (Fig. 6b), AsFAD2-10 (Fig. 6c), and AsFAD2-23 (Fig. 6d), the conversion ratio of C18:1 to C18:2 were 60.07%, 57.49% and 12.78%, respectively (Additional file 13: Table S10). In addition, C16:2 was detected in the transformed yeast strains expressing AsFAD2-1 and AsFAD2-10, C16:2 content was respectively 18.10% and 9.95%, and the conversion ratio were 36.41% and 18.82%, respectively (Additional file 13: Table S10). However, no corresponding fatty acid product was detected in yeast cells expressing other genes (Additional file 13: Table S10).