Genome-wide identification of the fatty acid desaturases gene family in four Aspergillus species and their expression profile in Aspergillus oryzae

Fatty acid desaturases play a key role in producing polyunsaturated fatty acids by converting single bonds to double bonds. In the present study, a total of 13, 12, 8 and 8 candidate fatty acid desaturases genes were identified in the Aspergillus oryzae, Aspergillus flavus, Aspergillus fumigatus and Aspergillus nidulans genomes through database searches, which were classified into five different subfamilies based on phylogenetic analysis. Furthermore, a comprehensive analysis was performed to characterize conserved motifs and gene structures, which could provide an intuitive comprehension to learn the relationship between structure and functions of the fatty acid desaturases genes in different Aspergillus species. In addition, the expression pattern of 13 fatty acid desaturases genes of A. oryzae was tested in different growth stages and under salt stress treatment. The results revealed that the fatty acid desaturases genes in A. oryzae were highly expressed in adaptive phase growth and up-regulated under salt stress treatment. This study provided a better understanding of the evolution and functions of the fatty acid desaturases gene family in the four Aspergillus species, and would be useful for seeking methods to improve the production of unsaturated fatty acids and enhance efforts for the genetic improvement of strains to adapt to the complex surrounding environment. Electronic supplementary material The online version of this article (10.1186/s13568-018-0697-x) contains supplementary material, which is available to authorized users.


Introduction
Unsaturated fatty acids, which contain one or more double bonds, are the major structural components of cell membranes. Therefore, they play significant roles in maintaining cell structure and the membrane fluidity, which are involved in development, energy metabolism and stress response (Pereira et al. 2003). Unsaturated fatty acids are synthesized by individual fatty acid desaturases via introducing double bonds into the hydrocarbon chains of fatty acids (Chi et al. 2011;Shanklin and Cahoon the fatty acid desaturases domain. The fatty acid desaturases domain was the essential domain of the fatty acid desaturases gene family. However, some researches revealed that the cytochrome b5 domain play a key role in the synthesis of unsaturated fatty acids as an electron donor to activate desaturase (Pereira et al. 2003). Zhang et al. reported that the cytochrome b5 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans (Zhang et al. 2005). Although fatty acid desaturases orthologs of different organisms share some obvious structure characteristics, the structural and functional features were distinctive among plants, animals and fungi. Most studies of fatty acid desaturases gene family focused on plants and animals. For example, Liu et al. characterized 19 genes encoding fatty acid desaturases and analyzed their expression profiles in Gossypium raimondii under low temperature (Liu et al. 2015). Xue et al. cloned and characterized fatty acid desaturases gene family from Salvia hispanica and Perilla frutescens (Xue et al. 2018).
Compared with unsaturated fatty acids production in animals and plants, microbes possess many advantages in the production of polyunsaturated fatty acids. For instance, unsaturated fatty acids production in microbes has a short production cycle and is unaffected by sites, climates and seasons. Besides, it is suitable to exploit new functional lipid using diverse strains and culture medium. Furthermore, lower eukaryotes contain diverse fatty acid desaturases to produce polyunsaturated fatty acids (PUFA). Therefore, in recent years, microbial fatty acid desaturases have attracted great attention of researchers. Sakuradani et al. isolated and cloned ∆6 desaturase gene from Mortierella alpina IS-4. Then the ∆6 desaturase gene was expressed in Aspergillus oryzae (A. oryzae) and the results showed that the content of gamma linolenic acid (GLA) in total fatty acids was up to 25.2% (Sakuradani et al. 1999). Sakuradani et al. improved arachidonic acid (ARA) production by generating mutants with lower desaturation activity derived from Mortierella alpine (Sakuradani et al. 2004). On the other hand, researches related to the function of fatty acid desaturases from fungus on stress response are also a hotspot. Cheawchanlertfa et al. revealed that the upregulated expression of desaturase genes from Mucor rouxii was responded to low temperature (Cheawchanlertfa et al. 2011). The fatty acid desaturases genes in A. oryzae were up-regulated in response to salinity stress (He et al. 2017a). In brief, the fatty acid desaturases genes in fungi are responsible for multiple biological processes, from development and industrial production to adaption to the surrounding environment. However, systematic investigations of the fatty acid desaturases gene family at the whole-genome level was absent in fungi, especially in Aspergillus species, as Aspergillus species always encounter complex environments, artificially or non-artificially introduced (He et al. 2018).
With the price reduction of genome sequencing and the development of sequencing technology, the genome sequences were increasing available to provide opportunities for identifying important gene families at the whole-genome level. Millerozyma farinosa formerly known as Pichia farinosa, unlike Saccharomyces cerevisiae (containing a sole unsaturated fatty acid), contained multiple unsaturated fatty acids. It is a salt-tolerant and osmo-tolerant diploid yeast, of which the full genome sequence was completed in 2012 (Leh et al. 2012). The genome sequencing of A. oryzae, A. flavus, A. fumigatus and A. nidulans were completed earlier (Machida et al. 2005;Payne et al. 2008). On the other hand, the increasing availability of transcriptome data provides unprecedented opportunities to study the expression patterns of the members of gene family. For example, Dou et al. (2014) analyzed the expression profiles of WRKY gene family in different tissues of cotton through the transcriptome data.
Therefore, based on the related genomic data, we respectively identified fatty acid desaturases gene family members from two yeasts, including S. cerevisiae and M. farinosa, and four Aspergillus species, including A. oryzae, A. flavus, A. fumigatus and A. nidulans. In addition, a comprehensive analysis was performed to characterize conserved motifs and gene structures. Then, according to the transcriptome analysis of A. oryzae, the expression patterns of A. oryzae fatty acid desaturases gene family under salt stress and different growth periods were studied, and verified by qRT-PCR. The results of this study are propitious to comprehend the relationship between structure and functions of the fatty acid desaturases genes.

Identification of fatty acid desaturases genes
The genomic and protein sequences of M. farinosa CBS 7064, A. oryzae 304, A. flavus NR3357, A. fumigatus Af293, and A. nidulans FGSC A4 were downloaded from the National Center for Biotechnology Information (NCBI). The fatty acid desaturases protein sequences of S. cerevisiae (ScFAD) and M. farinosa (MfFAD) were retrieved from Saccharomyces Genome Database (Cherry et al. 2012). To identify all candidate fatty acid desaturases genes in A. oryzae, A. flavus, A. fumigatus and A. nidulans, ScFAD and MfFAD proteins were employed as query sequences to search genome database using BLAST program with a threshold e-value of 1e−10 (Altschul et al. 1997). Then the identity and cover region (more than 50%) were used as a filter criteria to eliminate improper fatty acid desaturases genes. Subsequently, the Pfam database was used for domain analysis to ensure that the selected sequences were non-redundant sequences to ultimately identify candidate fatty acid desaturases gene family members (Finn et al. 2014).

Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignments of fatty acid desaturases proteins in A. oryzae, S. cerevisiae, M. farinosa, A. flavus, A. fumigatus and A. nidulans were performed using Clustal X version 2.0 with the default parameters (Larkin et al. 2007). MEGA 5.0 was further applied to construct an unrooted Neighbor-Joining phylogenetic tree with pairwise deletion option and poisson correction model. Bootstrap analysis with 1000 replicates was used to examine the statistical reliability (Saitou and Nei 1987;Tamura et al. 2011). The Figtree program (v1.4.3) was used to visualize it.

Analysis of conserved motifs and gene structures
To identify the conserved motifs of each fatty acid desaturases gene in the six species, deduced fatty acid desaturases protein sequences were subjected to MEME version 4.12.0 (http://meme-suite .org/tools /meme), with the default parameters except the number of motifs was chosen 5 (Bailey et al. 2009). The logo of motifs was produced by weblogo (http://weblo go.berke ley.edu/logo.cgi).
To illustrate exon-intron organization for each fatty acid desaturases gene, coding sequences (CDSs) and corresponding genomic sequences of fatty acid desaturases genes in the six species, downloaded from NCBI database, were compared on the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn) (Guo et al. 2007).

Expression analysis of AoFAD genes in different growth stages and under salt stress treatment
The genome-wide transcriptome data of A. oryzae in different growth stages and salt stress treatment were obtained from NCBI SRA databases under Bioproject Accession PRJNA407002 and PRJNA383095. The raw reads that contained adapters, reads containing unknown sequences 'N' with a rate more than 5% and low-quality bases which were identified based on CycleQ 30 were removed. After filtering, gene expression levels were normalized using the TopHat/Cufflinks pipeline with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) value ). An FPKM filtering cutoff of 1.0 in at least one of the collected samples was used to determine expressed transcripts. The heatmaps for expression profiles were generated with the Omic-Share Tools (http://www.omics hare.com/tools /Home/ Index /index .html). To further confirm the expression level of 13 fatty acid desaturases genes in A. oryzae 3042 (CICC 40092), quantitative real-time RT-PCR (qRT-PCR) experiments were performed. The genome-wide transcriptome data of A. oryzae were obtained at three stages of development (24, 48 and 72 h) and different conditions (cultivated in potato dextrose agar medium supplied with 0, 5, 10 and 15% NaCl). Three stages of development correspond to the adaptive phase, logarithmic phase, and stationary phase. And four conditions represent control, slight stress, moderate stress and severe stress, respectively. Samples under salt stress treatment were all harvested at 48 h. Total RNA of all collected samples was extracted using PrimeScript RTreagent kit (Takara, Dalian, China) following the instructions, in which our previous studies have been performed (He et al. 2017a). The specific primers for fatty acid desaturases genes in A. oryzae were listed in Additional file 1: Table S1. The qRT-PCR analysis was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA) in the BioRad CFX Connect Optics Module Real-time PCR System (Livak and Schmittgen 2001).

Identification of fatty acid desaturases genes in the six species
The candidate fatty acid desaturases genes were identified from the A. oryzae, A. flavus, A. fumigatus and A. nidulans genome using the Blast programs with the query sequences of S. cerevisiae and M. farinosa fatty acid desaturases genes. Subsequently, the retrieved sequences were submitted to the Pfam databases to confirm the presence of conserved domains. A total of 13, 12, 8 and 8 candidate fatty acid desaturases genes were identified in the A. oryzae, A. flavus, A. fumigatus and A. nidulans genomes, respectively (Table 1) of fatty acid desaturases genes in the six species was provided in Table 1.

Classification and phylogenetic analysis of the fatty acid desaturases genes
To evaluate the phylogenetic relationships among the fatty acid desaturases gene members, all the genes from the six species were aligned separately by Neighboring-Joining method to generate an un-rooted phylogenetic tree. As shown in the phylogenetic tree (Fig. 1), the fatty acid desaturases genes in these six species were divided into two groups, which were named I and II group.
According to the homology of the fatty acid desaturases genes, groups I and II were respectively further divided into I-A, I-B and II-A, II -B-1, II-B-2. Group I was composed of 31 fatty acid desaturases genes, while group II contained 23 fatty acid desaturases genes. Phylogenetic analysis of fatty acid desaturases genes in the six species revealed considerable diversification and conservation of the fatty acid desaturases gene family in fungi. In the phylogenetic tree, ScFAD, fatty acid desaturase in S. cerevisiae, was clustered with MfFAD1 and MfFAD2 into one branch, which belongs to group I-B. Every two fatty acid desaturases genes of M. farinosa shared one subbranch, which suggested that the fatty acid desaturases genes of M. farinosa had a strong conservation. From the phylogenetic tree, the 12 fatty acid desaturases genes from A. flavus were all clustered with A. oryzae into a subbranch, which revealed the close relationship between the A. oryzae and A. flavus, while AoFAD9 grouped closely with MfFAD1, 2, 3 and 4. In addition, fatty acid desaturases genes between A. fumigatus and A. nidulans appeared to be more closely than the two other Aspergillus. These results can not only illustrate the relationship between the Aspergillus species and yeast, but also provide a potential method to distinguish A. oryzae and A. flavus.

Conserved motifs analysis of the fatty acid desaturases genes
Conserved motifs in the 54 fatty acid desaturases proteins were identified using the MEME program. A total of five conserved motifs were identified in the fatty acid desaturases proteins and their consensus sequence information was listed in Table 2. The logo of five conserved motifs identified in the fatty acid desaturases proteins were shown in Additional file 1: Figure S2. According to the phylogenetic tree and conserved motifs (Fig. 2), we could know that the same group of fatty acid desaturases genes had substantially consistent conserved motifs, which indicated there might be similar genetic functions. The fatty acid desaturases domains of most fatty acid desaturases gene members in group I-A contain motif 1 and motif 4, while most of group I-B fatty acid desaturases domains in group I-B all were consisted of motif 1, motif 2 and motif 3. The fatty acid desaturases domains were only composed of motif 1 in groups II-A and II-B-1. However, the fatty acid desaturases domains of subgroup II-B-2 were mainly consisted of motif 1, motif 2 and motif 5. The fatty acid desaturases domains consisting of different motifs in subgroup II-B-1 and subgroup II-B-2, suggested that functional differentiation might occur in the group II-B fatty acid desaturases genes. Besides, we found that motif 1, motif 3, motif 4 and motif 5 were contained in the fatty acid desaturases domains, while motif 2 was the part of Cyt-b5 domains, excepting the motif 2 in AoFAD9. The results revealed the conservation of motifs among various species.

Gene structure analysis of the fatty acid desaturases genes
In order to gain further insight into the structural diversity of fatty acid desaturases genes, coding sequences (CDSs) and corresponding genomic sequences were investigated through the six species. In the present study, a detailed illustration of the gene structures was shown in Fig. 3. The fatty acid desaturases genes of A. oryzae and A. flavus, clustered into a subbranch, had similar gene structures. The minor difference of the fatty acid desaturases gene structures between A. oryzae and A. flavus is that most of AoFAD contained upstream and downstream while only AflFAD11 and AflFAD9 contained an upstream sequence. And most of fatty acid desaturases genes in the two species possessed one or two introns except for AoFAD9, which had seven introns. The fatty acid desaturases genes of S. cerevisiae and M. farinosa were lacked of introns. Furthermore, the fatty acid desaturases genes of M. farinosa, which were clustered into the same branch, had same gene structures, further indicating the conservation of the fatty acid desaturases genes in M. farinosa.

Expression of AoFAD genes during growth
Aspergillus oryzae undergoes morphological differentiation across the different growth stages, which always accompany with the change of gene expression profile as well as metabolic pathways and influence the process productivity. To characterize the patterns of fatty acid desaturases gene expression during the growth stages of A. oryzae, samples at 24, 48 and 72 h (corresponding to the adaptive phase, logarithmic phase, and stationary phase), containing three biological replicates (i.e. Ao_24_1, 2, 3), were harvested. The expression patterns of the fatty acid desaturases genes at different growth periods in A. oryzae were shown in Fig. 4. Eight fatty acid desaturases genes, including AoFAD3, AoFAD7, AoFAD4, AoFAD12, AoFAD10, AoFAD5, AoFAD6 and AoFAD11, showed the maximal expression in adaptive phase (Ao_24_1, 2, 3) and lower expression levels at logarithmic phase and stationary phase. AoFAD1, AoFAD2 and AoFAD9 were significantly up-regulated at 72 h, while the expression of AoFAD8 and AoFAD13 was obvious at 24 h and 72 h but not palpable at 48 h, indicating different roles of AoFAD with respect to the development of A. oryzae. To further confirm the expression profiles of AoFAD, six AoFAD genes were selected for qRT-PCR analysis. The results of qRT-PCR have strong consistency with those of transcriptome analysis (Additional file 1: Figure S1).

Expression of AoFAD genes under salt stress
Unsaturated fatty acids play critical roles in the tolerance to various abiotic stresses, such as salt stress, cold stress, etc. (Sakamoto and Murata 2002).Therefore, gene expression patterns for all the fatty acid desaturases genes of A. oryzae were also observed under different levels of salt concentration. Results showed that the salt stress caused changes of the AoFAD expression patterns in the form of up-regulation (Fig. 5). Of these AoFAD genes, the expression of AoFAD2, AoFAD3, AoFAD8 and AoFAD9 reached the highest under 5% NaCl treatment. Four fatty acid desaturases genes in A. oryzae, including AoFAD11, AoFAD1, AoFAD13 and AoFAD12, were highly expressed under 15% NaCl treatment. The expression level of AoFAD4, AoFAD5, AoFAD6, AoFAD7 and AoFAD10 was the highest under 10% NaCl treatment and was decreased under 15% NaCl treatment. The expression pattern of AoFAD genes under salt stress indicated that AoFAD are components of a complex transcriptional network regarding the salt stress and the mechanism of AoFAD genes involved in salt stress is complex and diversified. The coordination results of qRT-PCR were further confirmed the accuracy of analysis (Additional file 1: Figure S1).

Discussion
Studies have indicated that fatty acid desaturases is expressed in plants, animals and fungi, and plays an important role in the synthesis of polyunsaturated fatty acid (Garba et al. 2016;Murphy and Piffanelli 1998). Fatty acid desaturases genes in fungi are responsible for multiple biological processes, from development and industrial  production to adaption to the surrounding environment (Watanabe et al. 2010). Therefore, the study of the fatty acid desaturases genes is becoming a hot spot in the current biological research. So far, researchers have used DNA library, cDNA library and RT-PCR to clone fatty acid desaturases genes from plants, animals, bacteria, fungi and algae. For example, 29 and 21 fatty acid desaturases gene members were respectively identified from the fatty acid desaturases gene families of the soybean and Arabidopsis thaliana (Chi et al. 2011). Besides, there are some reports on the cloning and expression of fatty acid desaturases in microbes. For example, the sole fatty acid desaturases gene in Bacillus subtilis, named des, encoding Δ5-desaturase, was cloned by Ma and Liu (Ma and Liu 2010). The expression of three fatty acid desaturases genes in the Cytosolic cyanobacteria, named desA, desB and desD, was up-regulated under low temperature (Los et al. 1997). Aspergillus species has been intensively used for the production of traditional fermented foods and secondary metabolite, such as fumagillin. Therefore, the synthesis of unsaturated fatty acid appears to be essential and vital for the Aspergillus species to adapt to some complex environments and regulate the growth as well as development. However, systematic investigations of the fatty acid desaturases gene family at the whole-genome level was absent in Aspergillus species. Therefore, a comprehensive survey of fatty acid desaturases gene family in Aspergillus species was undertaken. The results in this study showed that all of the 54 fatty acid desaturases genes in the six fungi species contained Fig. 4 Expression of the AoFAD genes during growth. Ao_24_1, 2, 3 indicated the three biological reduplicates at 24 h (lag phase). Ao_48_1, 2, 3 indicated the three biological reduplicates at 48 h (logarithmic phase). Ao_72_1, 2, 3 indicated the three biological reduplicates at 72 h (stationary phase) FA_desaturase domain and most of the fatty acid desaturases genes had Cyt-b5 domain as well. These findings echo previous studies which studied the regulation of fatty acid synthesis pathways in the Caenorhabditis elegans. He et al. (2017b) reported that, in the desaturation of fatty acids, electrons are transferred to cytochrome b5 through cytochrome b5 reductase, which activates desaturase, and introduces unsaturated bonds to the unsaturated fatty acids at the specific location of the carbon chain of fatty acids (He et al. 2017b). When the fatty acid desaturases genes were absent of the Cyt-b5 domain, there need to be additional cytochrome b5 reductase to transfer electrons. From their result, we could infer that only FA_desaturase domain was specifically required for the activity of some fatty acid desaturases while some fatty acid desaturases were activated by FA_desaturase and Cyt-b5 domain. The other researches showed that some of the fatty acid desaturases were activated by FA_ desaturase and DUF3474 domain (He et al. 2017b). In our study, the fatty acid desaturases genes without the Cyt-b5 domain existed in Aspergillus species as well, which may be need additional cytochrome b5 reductase to transfer electrons. In addition, we found a fatty acid desaturases gene in A. nidulans (AnFAD5) which depends on FA_ desaturase and DUF3474 domain to activate. However, the functions of some domains identified in the fatty acid desaturases genes of Aspergillus species, such as DUF953, were not clear.
The relationship of A. oryzae and A. flavus is a controversial issue which has long been plagued with researchers. A very strong phylogenetic connection between A. oryzae and A. flavus has been clearly demonstrated by molecular methods, including isozyme analyses, DNA/ DNA hybridization studies and DNA sequencing (Chang Fig. 5 Expression of the AoFAD genes under salt stress. WT, NaCl_5, NaCl_10 and NaCl_15 indicated samples cultivated in PDA medium supplied with 0, 5, 10 and 15% NaCl, respectively et al. 2006;Geiser et al. 2000). Furthermore, the morphological characteristics and genomes of the two Aspergillus species were similar, which was thus difficult to distinguish. In this study, there were 13 fatty acid desaturases genes identified in A. oryzae, whereas 12 fatty acid desaturases genes were identified in A. flavus. From the phylogenetic tree, the 12 fatty acid desaturases genes from A. flavus were all clustered with A. oryzae into a sub-branch, which supported a close relationship between the A. oryzae and A. flavus. Additionally, AoFAD9 was grouped closely with MfFAD1, 2, 3 and 4, which could be considered as a method to distinguish A. oryzae and A. flavus.
In this study, analysis of the AoFAD expression profiles showed that the different concentration of salt stress caused changes of the AoFAD expression patterns in the form of up-regulation. The results revealed that AoFAD genes were assumed to be associated with salt stress, which has been mentioned in the previous studies (He et al. 2017a). The potential mechanism was that the increase of unsaturated fatty acids is beneficial to maintain membranes in an appropriate fluid state, which counteracts the fluidizing effect of salt stress. In fact, there are many studies which convey that the fatty acid desaturases genes have a closed relation with the salt stress in many species. For example, in the Arabidopsis thaliana, the FAD2 and FAD6 are essential for improving the early growth and salt tolerance of the seedlings while the antisense expression of FAD7 gene reduces plant tolerance to salt stress (Zhang et al. 2009(Zhang et al. , 2012. In addition, the overexpression of LeFAD3 gene can enhance the salt tolerance of early growth of the tomato seedlings (Wang et al. 2014). Our results, taken together with these earlier studies, imply that the fatty acid desaturases genes have an effect on salt stress.