Mol Biol Rep DOI 10.1007/s11033-014-3373-5

Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds Fatemeh Dehghan Nayeri • Kazem Yarizade

Received: 25 October 2013 / Accepted: 16 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows

F. Dehghan Nayeri (&)  K. Yarizade Agricultural Biotechnology Department, Engineering and Technology Faculty, Imam Khomeini International University, Qazvin, Iran e-mail: [email protected]

FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds. Keywords Bioinformatics  Desaturation  FAD2  Oilseeds  Unsaturated fatty acid

Introduction Oilseed crops are economically important because of their nutritional properties in human diet [1]. In addition to nutritional oil production, oilseeds are used in biodiesel production and industrial applications such as resin, plastic, varnish and paint [2, 3]. Generally, oil content of oilseeds can vary significantly among and within oilseed species even cultivars from under 4 % of dry weight (e.g. Triticum sativum) to over 60 % (e.g. Ricinus communis). It also varies by region due to climate and other environmental factors. In plants, fatty acids as the main group of seed storage lipid and the major source of energy are divided into saturated and unsaturated fatty acid species. Fatty acid desaturases have function in fatty acid metabolism and introduce double bonds into the hydrocarbon chains of fatty acids. Fatty acid desaturases are grouped into two classes including soluble acyl–acyl protein (ACP)- desaturases and membrane-bound fatty acid desaturases. The non-heme diiron ACP-desaturases have active site and catalyze the first step in poly unsaturated fatty acid biosynthesis. Membrane-bound desaturases contain membrane-spanning domains and histidine box motifs which are crucial for their activity. In plants, membrane-bound fatty acid desaturases are present in the plastid and the

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endoplasmic reticulum (ER). The electron donors for plastid desaturases are typically ferredoxins, while ER enzymes use cytochrome b5 (Cytb5) [4]. Desaturation process occurs in both plastidial and ER membranes through two different pathways [5]. Triglycerides in vegetable oils are mostly composed of five fatty acids including palmitate, stearate, oleate, linoleate and linolenate that differ in terms of acyl chain length and number of double bonds, leading to different physical properties [6]. The quality of vegetable oils is highly dependent on the content of unsaturated fatty acids. Oleic acid (C18:1, delta9) and linoleic acids (C18:2, delta-9,12) constitute two major unsaturated fatty acids and their proportions in the oilseeds determine the nutritional properties and oxidative stability of edible oils. In plants seed-specific FAD2 (delta12 fatty acid desaturase 2, microsomal oleate desaturase, microsomal oleoyl phosphatidylcholine desaturase) is responsible for the high content of linoleic acid in seed oil. Enzymatic protein of FAD2 converts oleic acid to linoleic acid in the ER membrane. This enzyme introduces a double bond at the delta-12 (omega-6) position of oleic acid to form linoleic acid [5]. A simplified pathway of linoleic acid biosynthesis has been shown in Fig. 1. FAD2 is a multifunctional enzyme such as bi-functional hydroxylase/desaturase and tri-functional acetylenase which can also catalyze the formation of both trans and cis double bonds at the delta-12 position of oleic acid. The key step in the production of polyunsaturated fatty acids through the delta-12 desaturation is catalyzed by FAD2 enzyme. Therefore, it has an essential role in the biological membrane systems, signaling, energy storage, thermal adaptation and resistance to biotic and abiotic stresses [5, 7]. The deduced amino acids of FAD2 genes displayed the typical three conserved histidine boxes, a characteristic of all membrane-bound desaturases, as the place for oxygen activation and substrate oxidation [8, 9]. Histidine-rich motifs form a part of the diiron center, where oxygen activation and substrate oxidation occur. These conserved motifs contain functional group of imidazole that donates a proton serve as a ligand and form coordinate bond with the metal ions such as Fe2?, Ca2?, Zn2?, and Cu2? in the active site of the enzyme. Replacement of one of the histidine residues weaken the ion binding and reduce catalytic activity of the enzyme. The histidine-rich region between transmembrane domains acts as a sensor of metal concentration and confers to plants resistance to toxic levels of metal ions. For example, induction of a protein with a

histidine-rich metal-binding domain has been found in plasma membrane fraction of young rice roots under salinity [10]. In Arabidopsis, only a single FAD2 gene exists, while in other crops at least one additional FAD2 gene is express. For example, two FAD2 genes have been identified in soybean, flax and olive, three genes in sunflower, four genes in cotton and 4–6 different FAD2 genes in rape [5, 11–13]. FAD2 genes are different in the nucleotide and amino acid sequences of the conserved region from other desaturases (FAD1, FAD3, FAD6, FAD7 and FAD8) and no common motifs were identified in all the members of these fatty acid desaturases [14]. A large family of FAD2 genes was cloned in safflower (Carthamus tinctorius L). This family has eleven nonallelic members with different expression patterns in different tissues of safflower. It appears that this gene family plays the major role in producing very high levels of linoleic acid present in the seed storage oils of the wild-type safflower [5]. In developing soybean seeds, FAD2 genes are considered to play an important role in controlling the oleic acid levels in developing soybean seeds and has been used as targets or candidate genes to produce high oleic acid levels in soybean [15, 16]. FAD2-2 genes encoding isoforms of the large and functionally diverse FAD2 gene family have been detected in developing seeds that suggested their major roles in storage oil desaturation in seed. FAD2-2 genes have been identified in several oilseed plants including safflower (Carthamus tinctorius), sunflower (Helianthus annuus), rapeseed (Brassica napus), sesame (Sesamum indicum), soybean (Glycine max), olive (Olea europaea), flax (Linum usitatissimum), castor bean (Ricinus communis), pumpkin (Cucurbita pepo), grape (Vitis labrusca), peanut (Arachis hypogaea), ironweed (Vernonia galamensis) and tung tree (Vernicia fordi). In virgin olive oil, FAD2-2 is the main gene that determines the linolenic acid (C18:3, delta9,12,15) content [17]. Polyunsaturated fatty acids linoleic acid and linolenic acid are storage compounds mainly in the form of triacylglycerol and structural components of membrane lipids which are synthesized by plants but not by most other higher eukaryotes [18, 19]. High concentration of linoleic and linolenic acids in oil undergo oxidative deterioration and cause rancidity of oils. For solving this problem, chemical hydrogenation has been employed to increase oleic acid content and reduce the amount of the

Fig. 1 Linolenic acid biosynthesis pathway. Conversion of oleic acid to linoleic acid is catalyzed by FAD2 enzyme [11]

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Mol Biol Rep Table 1 Sixteen FAD2 proteins selected from 14 oilseed species

Index

Species

Family

Common name

Protein accession no.

No. of transmembrane domains

1

Arachis hypogaea

Fabaceae

Peanut

ACZ06072

5

2

Arachis hypogaea

Fabaceae

Peanut

AER27517

4

3

Brassica napus

Brassicaceae

Rapeseed

ACP39503

5

4

Carthamus tinctorius

Asteraceae

Safflower

ADM48789

5

5

Cucurbita pepo

Cucurbitaceae

Pumpkin

AAS19533

4

6

Glycine max

Fabaceae

Soybean

AAB00860

5

7 8

Helianthus annuus Linum grandiflorum

Asteraceae Linaceae

Sunflower Red flax

AAL68982 AEQ28965

5 3

9

Linum usitatissimum

Linaceae

Flax

ACF49507

4

10

Olea europaea

Oleaceae

Olive

AAW63041

4

11

Olea europaea subsp. europaea

Oleaceae

European olive

ADZ39688

4

12

Ricinus communis

Euphorbiaceae

Castor bean

ABK59093

5

13

Sesamum indicum

Pedaliaceae

Sesame

AAX11454

5

14

Vernicia fordii

Euphorbiaceae

Tung tree

AAN87573

4

15

Vernonia galamensis

Asteraceae

Ironweed

AAF04094

5

16

Vitis labrusca

Vitaceae

Grape

AEI60129

4

polyunsaturated fatty acids, but the hydrogenation of vegetable oils leads to the formation of trans fats which have been linked with the heart disease and a type 2 diabetes [20]. Therefore, genetic engineering is used to improve the vegetable oil stability without hydrogenation [6, 21]. Since vegetable oils with desirable fatty acid compositions are needed for both nutritional and industrial purposes, there is considerable interest in modifying the oil composition by plant breeding or by using the new molecular tools of biotechnology such as mutational approaches [22, 23]. The importance of FAD2 in modifying the composition of seed oils draws attention of many investigators to manipulate FAD2 gene expression. Most of the studies conducted to reduce the expression of FAD2 gene for increasing the oleic acid content, for example sunflower high-oleic mutant Ol [7, 24], silencing of FAD2 in rapeseed [25], and developing and introducing sense suppression DNA construct into cotton to reduce FAD2 activity [7]. A homologues region based on the highly conserved sequence of a gene family can be used for isolation of a new gene or designing molecular markers in breeding. FAD2 gene family is interesting and important for the production of polyunsaturated fatty acids in plants. Therefore, it is of great importance to have a thorough understanding of the FAD2 genes and important application of bioinformatics studies is obvious. Bioinformatics approaches infer ancestral relationships among gene families and the information derived can be utilized for more survey. In the present study some characteristics of the FAD2-2 genes including structural, functional and

phylogenetics relationships were elucidated using bioinformatics analysis.

Materials and methods FAD2 sequences collection All protein sequences of FAD2 (FASTA format) belonging to oilseeds were collected from NCBI (http://www.ncbi. nih.gov) (Table 1). Bioinformatics analysis Protein sequences of FAD2 genes were analyzed using several online web services and software. Comparative and bioinformatics analysis of FAD2 protein sequences were performed online at two websites NCBI (http://www.ncbi. nih.gov) and EXPASY (http://expasy.org/tools). The MEME Suite web server (Multiple Em for Motif Elicitation; version 4.9, http://www.Meme.nbcr.net/meme/meme/.html) was used for searching and analysis of sequence motifs. Applied parameters of MEME were as following: minimum width for each motif, six; maximum width for each motif, fifty; maximum number of motifs to find three and occurrence of each motif, zero or one per sequence. A multiple sequence alignment of FAD2 loci was performed with Clustalw using default parameters. Phylogenetic tree was produced by MEGA software (Molecular Evolutionary Genetics Analysis; version 5.1) on the basis of aligned sequences of

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FAD2 proteins. The Neighbor-Joining (NJ) method was used to construct the phylogenetic tree. In order to investigate the protein structures of FAD2 in oilseeds, S. indicum FAD2 (Accession Number: AAX11454) was selected and analyzed by bioinformatics tools to predict the protein properties, such as molecular weight, theoretical pI, signal peptide, transmembrane and conserved domains. SignalP (http://www.cbs.dtu.dk/services/SignalP) [26] and TMHMM (http://www.cbs.dtu.dk/services/TMHMM) [27] were used to predict signal peptide and transmembrane helices respectively. Amino acid and atomic composition, extinction coefficients, half-life also were estimated.

Results and discussion There is at least one oleic acid desaturase isoform in all higher plants involving in linoleic acid synthesis by insertion of a double bond between carbons 12 and 13 of oleic acid. Desaturases are different in specificity as some of them have evolved the ability to introduce various functional groups, such as hydroxy, epoxy, acetylene groups and conjugated double bonds in flanking regions [28]. These enzymes have useful industrial applications. For example, castor oil is rich in hydroxy fatty acids and use as high-performance lubricant, tung oil is rich in conjugated fatty acids and use as drying agent, and ironweed oil is rich in epoxy fatty acids and use in the synthesis of industrial polymers. Arabidopsis harbors a single copy of the FAD2 gene which is constitutively and abundantly expressed [29], whereas crops including soybean, flax, sunflower, safflower and canola express at least one additional FAD2 gene(s), which is tightly regulated during seed development. FAD2 gene family is quite large and diverse in many plant species, whereas some oilseeds FAD2 genes were not included in this analysis including Gossypium hirsutum, Brassica juncea, B. oleracea, B. nigra and B. rapa. They were excluded from the analysis because of their destructive effects especially on inferring phylogenetic relationships among species and finding conserved domains. Linoleic acid is one of the two essential fatty acids which is not synthesized in mammals including the humans and therefore should be consumed through diets. FAD2 enzyme responsible for conversion of oleic acid to linoleic acid has a generally accepted predicted structure but no crystal structure exits for that. However, likely features have been revealed using comparative sequence analyses and structural information from known proteins. These features include highly conserved histidine-rich motifs that are located by three to five transmembrane anchors. Another important structural feature of FAD2 is the C-terminal endoplasmic reticulum signaling motif which

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Fig. 2 Sequence-specific MEME motifs for FAD2 proteins. Motif 1: c EWDWLRGALAT[YM]DRD[WY]GVILNKVFHITDTHV[AST]HHL [FV]STMPHYHAMEAT[KNR][AV], Motif 2: QGC[VIT][LMC]TGV WV[ILV]AHECGHHAFS[DK][YN]Q[WLG][LV]D[DN][TLIVM]VG [LF]HS[CASFVQ]L[LM]VP[YVN][FL][SL][WLG][KNE][YHIA][SQ] [HP][RP], Motif 3: I[KI]P[IV]LG[ENDQ]YY[GRH]FD[GE]T[PS][FIV] [YFVI]KA[ML][WY]RE[ATD][KR]EC[ILV]YV[ED][PKRSHA]D [EDQR][GSEQNA][DVTGEA][QKESP][NDSKT][KG][GV][VY]

allows them to selectively bind to and embed in the ER. Induction of mutations anywhere in this motif cause decoupling of the enzyme from the endoplasmic reticulum and its anchoring in the plasma membrane [30]. Three conserved histidine regions form the active site of the enzyme and are essential for catalysis. Based on the conserved motifs homologic primers can be designed for isolation of a new FAD2 gene. Using good alignment represented by 16 protein sequences from oilseeds, it is possible to identify three regions of strong conservation among them. The first histidine motif in all FAD2 polypeptide sequences is HECGHH. Consistent with all plant FAD2 enzymes, Ala (A) is the first amino acid immediately after the first histidine box. The second histidine motif is highly conserved as HRRHH in FAD2 sequences, except flax (Linum grandiflorum) that does not contain histidine box 2 and has four hydrophobic amino acids of proline instead. The conserved motif is represented in FAD2s by WKYSHRRHH that harbors second histidine box [31]. The HV[A/C/T]HH motif is repeated toward the carboxy terminus of each sequence in which three histidine residues representing third histidine box. Among delta-12 oleate desaturases EWDWLRGALAT and LFSTMPHYHAMEAT are two most strongly conserved motifs before and after third histidine box respectively (Figs. 2, 3, 4). The large number of highly conserved histidines among the blocks of homology may represent a type of iron binding site. The proteins may be helical in spacing places three histidine boxes. In the most selected FAD2 proteins, the ER retrieval sequence is YNNKL but in their orthologs the mentioned sequence is Y(K/N)NKF or YRNKI. Decreasing in motif conservation may indicate loss of the ER retrieval signal, resulting in dissociation of the enzyme from the endoplasmic reticulum [32]. To elucidate the phylogenetic relationship of FAD2 genes, 16 deduced polypeptide sequences were aligned and a neighbor-joining tree was constructed using MEGA5.1. As shown in the phylogenetic tree, most of the genes fell into three distinct groups. In first branch of the tree, FAD22 genes belong to three species of Asteraceae family including sunflower, safflower and ironweed were grouped together with a little distance from FAD2-2 genes of olive, rapeseed and pumpkin. In second group, FAD2 genes from peanut, castor bean and sesame were aligned with FAD2-2

Mol Biol Rep

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Mol Biol Rep Fig. 3 Motifs in FAD2 proteins produced by the MEME software. Motifs are shown as different-colored boxes

Fig. 4 The position of transmembrane anchors and histidine boxes in SeFAD2 protein sequence

Fig. 5 Phylogenetic comparison of sesame FAD2 gene and orthologous FAD2s from other plants. The phylogenetic tree was generated by ClustalW (MEGA 5.1). The Neighbor-Joining (NJ) method was used to construct the tree. The percentage of 1000 bootstrap replicates was given at each node. Based on the result of phylogenetic tree sixteen protein sequences of FAD2 were clustered in three main groups

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genes of soybean, grape and tung tree. In this branch, soybean and peanut are from the same family of Leguminosae. Based on the blast results, castor bean FAD2 has 89 and 85 % identity with this protein of tung tree and flax respectively. In third branch, FAD2-2 genes belong to linum species with 86 % similarity were separately grouped (Fig. 5). In order to investigate the protein structures of FAD2 in oilseeds, S. indicum FAD2 (Accession Number: AAX11454) was selected and analyzed by bioinformatics tools. Existence of the complete sequence information for sesame FAD2 in the databases was the reason of its selection for more analyze. The SeFAD2 protein (length: 383 aa) has a molecular mass of 44265.1 Da and the value of its isoelectric point (pI) is 8.56. SeFAD2 atomic composition is C2068H3066N522O536S14. Its amino acid composition was showed in Table 2. The characteristics of an enzyme derived from the sequence of amino acids determine the structure of the active site and hence the

Mol Biol Rep Table 2 Amino acid composition of SeFAD2 protein Amino acid

No. of amino acids

Percentage of amino acids (%)

Ala (A)

22

5.7

Arg (R)

17

4.4

Asn (N)

11

2.9

Asp (D)

19

5

Cys (C)

9

2.3

Gln (Q)

8

2.1

Glu (E) Gly (G)

12 22

3.1 5.7

His (H)

19

5

Ile (I)

19

5

Leu (L)

39

10.2

Lys (K)

19

5

Met (M)

5

1.3

Phe (F)

20

5.2

Pro (P)

26

6.8

Ser (S)

23

6

Thr (T)

20

5.2

Trp (W)

12

3.1

Tyr (Y)

28

7.3

Val (V)

33

8.6

Fig. 6 No N-terminal signal peptide in FAD2 protein was discriminated by SignalP server

specificity of the enzyme. Electrostatic bonds may occur between oppositely charged groups. Such electrostatic bonds can occur with groups that are completely positively or negatively charged or with groups that are partially charged. According to the amino acid composition, SeFAD2 contains 31 negatively and 36 positively charged amino acids. The computed instability index (II) was 37.32, therefore this protein is classified as a stable protein (http:// www.expasy.org/cgi-bin/ProtParam/ProtParam) [33].

Three algorithms of Psort (http://psort.hgc.jp/cgi-bin/ runpsort.pl) [34], TargetP (http://www.cbs.dtu.dk/services/ TargetP/) [35] and SignalP (http://www.cbs.dtu.dk/ser vices/SignalP/) were employed for the prediction of protein sorting signals and cellular localization sites in N-terminal region of SeFAD2. Based on the results, no N-terminal signal peptide for probable localization of SeFAD2 in chloroplast, mitochondria, Golgi and secretary pathway was recognized (Fig. 6). In this protein ER retention motif in the C-terminus (KDEL) is absent and instead it contains a C-terminal aromatic amino acid-containing sequence (YKNKF) which is necessary for maintaining localization in the ER indicating that the gene encodes a microsomal enzyme. The low degree of motif conservation among FAD2s might be due to the loss of the endoplasmic reticulum retrieval signal and result dissociating the enzyme from the ER and its anchoring in the plasma membrane [32]. NKF amino acid sequence has been identified as the peroxisomal targeting signal in the C-terminus of the protein [36]. FAD2 isoforms commonly contain five transmembrane domains, three histidine boxes and the ER retrieval motif representing the characteristics of fatty acid desaturase [31]. There are highly conserved stretches of the hydrophobic residues form biological transmembrane traverse three to five times. The hydrophobic residues are necessary for positioning the three histidine-rich motifs in the N-terminus region constituting active site of the enzyme. The conserved His-containing regions are crucial for proper enzymatic function, since substitution of a histidine with a different amino acid disrupts the desaturase function [37]. The hydropathy plot of SeFAD2 amino acid sequence generated by the method of Kyte and Doolittle [38] showed SeFAD2 protein has five prominent hydrophobic peaks at a position of 55–77 aa, 82–104 aa, 119–136 aa, 176–198 aa and 223–272 aa (Fig. 7a). Predicted transmembrane helices using TMHMM software confirmed existence of five transmembrane domains in that (Fig. 7b). The number of transmembrane domains among FAD2 proteins is different. For example FAD2 from L. grandiflorum has three transmembrane domains whereas other FAD2s span the membrane four or five times (Table 1; Fig. 7). These segments may be involved in substrate specificity [39]. Seed-specific SeFAD2 expression is controlled by cisregulatory elements in its promoter and enhancers in the 50 UTR intron. For further analysis of known cis-acting elements in SeFAD2 intron and 50 UTR, a web search of publicly available databases (http://www.dna.affric.go.jp/ htdocs/PLACE and http://www.intra.psb.ugent.be:8080/ PlantCARE) was done (Table 3). Intron region of SeFAD2 promoter is responsible for the expression of gene

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Mol Biol Rep Fig. 7 a Hydropathy plot of Sesamum indicum FAD2. b Predicted transmebrane domains in Sesamum indicum FAD2. c Predicted transmebrane domains in FAD2-2 from Linum usitatissimumand d from Linum grandiflorum by TMHMM server (CBS; Denmark). Red color boxes represent the putative transmembrane domains, blue and pink color lines indicate the inner and outer membranes of the cell respectively

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Mol Biol Rep Table 3 Cis-regulatory elements in SeFAD2 promoter

Cis- element

Sequence

Function

DOF core

AAAG

Involved in carbon metabolism

DPBP core

ACACNNG

ABA-responsive and embryo-specification element

ABRE/E-box

CANNTG

Light-responsive element

Myb-core

cg/tgtta

Water stress-responsive element

G-box-like element/ACGT motif

ACGTGKC

ABA-responsive element

GTGA-box

GTGA

Pollen-specific expression

GT-1

GRWAAW

GCN4

TGTGTCA

Light-responsive, salicylic acid (SA)-inducible element Involved in endosperm expression

RY motif

CATGCATG

ABA-responsive element

G-box

CACGTG

ABA and light-responsive element

GATA motifs

GATA

Light regulated, and tissue-specific expression

T-box

TTWTWTTWTT

Stress-responsive element

MYB core

CNGTTR

Dehydration stress-responsive element

HSE

AAAAAATTTC

Heat stress-responsive element

B-box

CAAACAC

ABA-responsive element, involved in seed-specific expression

AAAC-motif

CAATCAAAACCT

Light-responsive element

LTR

CCGAAA

Low temperature-responsive element

GTCAT motif ARE

GTCAT TGGTTT

Element required for endosperm expression Element essential for the anaerobic induction

TC-rich repeats

ATTCTCTAAC

Defense and stress-responsive element

CAAT-box

CAAT

Common cis-acting element in promoter and enhancer regions

TATA-box

TTTTA/ taTATAAAg

Core promoter element around -30 of transcription start

Box 4

ATTAAT

Light-responsive element

TGA-element

AACGAC

Auxin-responsive element

CAT-box TGACG-motif

GCCACT TGACG

Element related to meristem expression MeJA-responsive element

Circadian

CAANNNNATC

Circadian control element

CATT-motif

GCATTC

Part of a light-responsive element

CGTCA-motif

CGTCA

MeJA-responsive element

ERE

TTCGACC

Elicitor-responsive element

specifically in seeds. It has been reported that deletion of this region results in a switch from seed-specific expression to constitutive gene expression [40]. In addition to this control mechanism for seed-specific expression, interaction between cis-elements in the 50 UTR regions and trans-acting factors is another well-known regulation mechanism. For example, the interaction between the prolamin box (Pbox, AAAG, or CTTT) and endosperm-specific transcription factors, such as bZIP, have a positive function in the coordinate activation of zein gene expression during the development of the endosperm [41]. DOF proteins, a family of plant transcription factors, are endosperm-specific proteins that bind to prolamin box involve in carbon metabolism [42]. The SeFAD2 promoter exhibited several potential cis-elements are known to be essential to seed or

endosperm-specific expression including DOF cores, 2xRY, GCN4, ACGT, AACA and ABRE motifs and B, E, G and T-boxes. Positive cis-elements for SeFAD2 gene expression in developing seeds include CACA element, CCAAT, E, G and P boxes, ABRE motif, G-box-like and RY repeat elements. It was shown that mutations in these elements result in a profound reduction of promoter activity in seeds [40]. Several stress-related elements including ERE, TTGACC, T-box; TTWTWTTWTT, GT-1 consensus and GRWAAW were localized to the sequences of the SeFAD2 intron. Suitable promoters with strong expression of the gene in a specific tissue (such as seeds) are critical factors for development of new cultivars through biotechnological methods [43]. Based on the SeFAD2 promoter cis-acting elements, this promoter is

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Mol Biol Rep

probably useful for modification of seed characterization in order to make transgenic plants applicable to agriculture. Based on the studies, environmental stresses such as cold, heat, drought and salt induce changes in fatty acid composition and the cell membrane fluidity. The main role of fatty acid desaturase is to modulate the fluidity of membranes which helps to improve tolerance to physiological stresses. In the other hand, when photosynthetic organisms are exposed to stress, the fatty acids of membrane lipids are desaturated. FAD2 enzymes are involved in fatty acid desaturation and maintenance of the lipids balance in cell membrane and enhance the tolerance of organism to stresses [44]. Application of various bioinformatics tools in biological research promotes better understanding of biological system in fullness. Comparative genetics of the plant genomes could provide reference for illustrating the evolution among gene family members and also for evaluating the potential use in improving stress resistance and quality of crops [45].

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Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds.

Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fat...
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