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Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. 227 Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. MENG-JUN LI, AI-QIN LI, HAN XIA, CHUAN-ZHI ZHAO, CHANG-SHENG LI, SHU-BO WAN, YU-PING BI and XING-JUN WANG* High-Tech Research Center, Shandong Academy of Agricultural Sciences, Key Laboratory for Genetic Improvement of Crop, Animal and Poultry of Shandong Province, Key Laboratory of Crop Genetic Improvement and Biotechnology, Huanghuaihai, Ministry of Agriculture, Ji’nan 250100, China *Corresponding author (Email, [email protected]) The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase (I, II, III), β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli. [Li M-J, Li A-Q, Xia H, Zhao C-Z, Li C-S, Wan S-B, Bi Y-P and Wang X-J 2009 Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.; J. Biosci. 34 227–238] 1. Introduction Oils are glycerol triesters of fatty acids and are mainly derived from plant sources. Peanut is widely grown and ranks fifth among the world oil crops (Moretzsohn et al. 2004). It is of great importance to study the fatty acid biosynthesis pathway for improving oil quality and increasing oil content of peanut through biotechnology-based approaches. Fatty acid biosynthesis is catalysed by two types of fatty acid synthase (FAS). Type I FAS, as found in vertebrates, yeast and some bacteria, contains all the active sites on one or two multidomain polypeptides. In type II FAS of many bacteria, plant plastids and mitochondria, the active centres reside in discrete gene products. E. coli serves as the paradigm for the type II FAS system. Acyl carrier protein (ACP) functions to sequester the growing acyl chain attached to the prosthetic phosphopantetheine group from solvent as it shuttles the intermediates between type II FAS enzymes (Zhang et al. 2003b). The condensation of malonyl-ACP with acyl-ACP to form β-ketoacyl-ACP is catalysed by small β-ketoacyl-ACP synthase (KAS) family enzymes. FabH (β-ketoacyl-ACP synthase III, KASIII) condenses acetylCoA with malonyl-ACP to form 4:0-ACP, FabB (KAS I) is responsible for the elongation of 4:0-ACP to 16:0-ACP, and FabF (KAS II) mediates the elongation of 16:0-ACP to 18:0-ACP. The formation of malonyl-ACP is catalysed Keywords. Arachis hypogaea L.; EST sequencing; gene cloning; type II FAS Abbreviations used: ACAT, acetyl CoA:ACP transacylase; ACP, acyl carrier protein; DAP, days after pegging; ENR, enoyl-ACP reductase; EST, expressed sequence tag; FAS, fatty acid synthase; HD, β-hydroxyacyl-ACP dehydrase; KAS, β-ketoacyl-ACP synthase; KR, β-ketoacyl-ACP reductase; MCAT, malonyl-CoA:ACP transacylase; NCBI, National Center for Biotechnology; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SNP, single-nucleotide polymorphism http://www.ias.ac.in/jbiosci J. Biosci. 34(2), June 2009, 227–238, © Indian Academy Sciences 227 J. Biosci.of34(2), June 2009 228 Meng-Jun Li et al by acetyl-CoA carboxylase and FabD (malonyl-CoA:ACP transacylase, MCAT). The reduction of β-ketoacyl-ACP to β-hydroxyacyl-ACP by FabG (β-ketoacyl-ACP reductase, KR) is the first reductive step. Two isozymes, FabA and FabZ (β-hydroxyacyl-ACP dehydratases, HD), catalyse the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP. FabA is a bifunctional enzyme involved in the introduction of a cis-double bond into the growing acyl chain (Heath and Rock 1996). The last reduction in each elongation cycle is catalysed by FabI (enoyl-ACP reductase, ENR). The isolation and cloning of cDNAs encoding plant plastid type II FAS genes have been reported in Arabidopsis thaliana (Post-Beittenmiller et al. 1989; Lamppa and Jacks 1991; Hlousek-Radojcic et al. 1992; Tai et al. 1994; Carlsson et al. 2002), Brassica napus (Kater et al. 1991; Simon and Slabas 1998), Cuphea sp (Klein et al. 1992; Voetz et al. 1994; Slabaugh et al. 1995), Spinacia oleracea (Scherer and Knauf 1987; Schmid and Ohlrogge 1990; Tai and Jaworski 1993), Hordeum vulgare (Hansen and von Wettstein-Knowles 1991; Hansen and Kauppinen 1991), Coriandrum sativum (Mekhedov et al. 2001), and Allium ampeloprasum (Chen and Post-Beittenmiller 1996). The crystal structure determination of E. coli type II FAS enzymes has been completed (White et al. 2005), but only the crystal structure of B. napus KR and ENR has been determined in higher plants (Rafferty et al. 1995; Fisher et al. 2000). Most of the work with plant plastid type II FAS is focused on ACP (Hlousek-Radojcic et al. 1992; Kopka et al. 1993; Suh et al. 1999; Bonaventure and Ohlrogge 2002) and KAS (Clough et al. 1992; Olsen et al. 1999; Abbadi et al. 2000; Dehesh et al. 2001; Pidkowich et al. 2007). Plant ACPs are encoded by a multigene family which expresses in a constitutive manner (HlousekRadojcic et al. 1992) or in a tissue-specific manner (Bonaventure and Ohlrogge 2002). The altered expression levels of KASII and KASIII lead to a change in oil content and qualities in A. thaliana (Abbadi et al. 2000; Dehesh et al. 2001; Pidkowich et al. 2007). In contrast to the well-studied E. coli type II FAS system, plant type II FAS enzymes located in plastids are largely uncharacterised except in A. thaliana. Plant mitochondrial fatty acid synthesis is also catalysed by the type II FAS system (Olsen et al. 2004), in which only ACP from Pisum sativum (Wada et al. 1997) and A. thaliana (Shintani and Ohlrogge 1994), and KAS from A. thaliana (Yasuno et al. 2004) have been characterised. The crystal structure of mitochondrial KAS has been determined (Olsen et al. 2004). Mitochondrial ACP functions as an essential cofactor in lipoic acid synthesis (Shintani and Ohlrogge 1994; Wada et al. 1997). Mitochondrial KAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis (Yasuno et al. 2004). The role of the mitochondrial fatty acid synthetic pathway J. Biosci. 34(2), June 2009 is unclear because mitochondrial membrane fatty acids are believed to originate outside mitochondria (Wada et al. 1997). In this study, we report the cloning of A. hypogaea type II FAS genes in plastids and two ACPs in mitochondria by expressed sequence tag (EST) sequencing of a fulllength cDNA library and homology-based cloning from immature peanut seeds. The primary structures of plant type II FAS enzymes were analysed by sequence alignments, and compared with that of E. coli. Furthermore, homologues of each peanut type II gene in developing seeds were investigated by cloning and sequencing. The goal of our study was to provide a basis for elucidating the molecular mechanism of fatty acid synthesis in peanut seed development. 2. 2.1 Materials and methods RNA isolation and cDNA library construction Peanut (A. hypogaea cultivar luhua-14) was grown in the farm and gynophores were labelled. The immature peanut seeds from 20 to 60 days after pegging (DAP) were collected, frozen in liquid N2 immediately and stored in a freezer at –80ºC. Total RNA was extracted from the seeds by the RNAgent kit (Promega, Madison, WI, USA). Messenger RNA was isolated and purified from total RNA (Promega). Directional cDNA synthesis (using adapters of EcoRI, XhoI restriction sites) and library construction followed the protocol of Stratagenes pBluescript II cDNA library construction kit. 2.2 Analysis of ESTs Each sequence obtained was edited using the Lasergene SeqMan II Module (DNAStar) (http:/www.DNAStar.com). Comparison of peanut ESTs with non-redundant protein sequence databases (tBLASTx) at the National Center for Biotechnology (NCBI) was performed to determine the open reading frame (ORF) of the cDNA and probably gene function. 2.3 Gene cloning The full-length cDNAs of ACP, HD, ENR were identified from our EST contigs. The 3′-end MCAT was cloned by 3′-rapid amplification of cDNA ends (3′-RACE). The KR, KASI, KASII and KASIII genes were isolated by homologybased cloning. Primers were designed based on the homology of KAS sequences from the NCBI. Fragments of KAS genes were amplified by polymerase chain reaction (PCR) using One-Shot LA PCRTM Mix (TaKaRa, Dalian, China). The Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. 5′-RACE and 3′-RACE (5′/3′ RACE Kit, 2nd Generation, Roche) primers were constructed based on known sequences of KAS genes. PCR products were cloned into the pMD18-T vector (TaKaRa). 2.4 Homologue analysis of type II FAS genes Total RNA was isolated at six different seed development stages (stage 1, 15 DAP; stage 2, 20 DAP; stage 3, 25 DAP; stage 4, 35 DAP; stage 5, 45 DAP; stage 6, 70 DAP) using RNAiso Reagent (TaKaRa) as described by the manufacturer. Five microgram of RNA was reverse transcribed using a PrimerScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa) with oligo (dT) as the primer according to the protocol provided by the supplier. The resulting cDNA was mixed and diluted 10-fold and 1 μl was used as a template for PCR amplification using 2×pfu PCR MasterMix (Tiangen Biotech, Beijing, China) with the specific primers of each type II FAS gene. The PCR products were cloned into the pMD18-T vector (TaKaRa). Primers applied in homologue analysis are available in supplementary materials. Plasmid DNA was prepared using the EZNA Plasmid Minipreps DNA Purification System (Omega Bio-Tek, USA). Purified plasmid DNA was sequenced to obtain the 5′- and 3′-end with BigDyeR Terminator v3.1 Cycle Sequencing Kit (ABI) on an ABI 3730XL DNA Analyzer. Nucleotide sequence assembly and homology searches were performed using the tBLASTx tool online. Sequence reassembly and coding region prediction were performed using the Lasergene SeqMan II Module (DNAStar) (http: //www.DNAStar.com). Multiple sequence alignments were analysed using the ClustalW1.83 software (http: //www.ch.embnet.org/software/ClustalW.html). Sequences were shaded using the BoxShade program (http:// www.ch.embnet.org/software/BOX_form.html). 3. 3.1 Results Cloning and sequence analysis of AhACP Forty-three ACP cDNA clones were identified from ESTs derived from the full-length cDNA library; these could be divided into three groups: AhACP1 (36 clones), AhACP2 (6 clones) and AhACP3 (1clone) based on their amino acid similarity. Twenty-seven ACPs from 6 plant species revealed primary structure conservation among plant ACPs (figure 1). Plant ACPs can clearly be divided into two types, which are located in plastids (figure 1A) and mitochondria (figure 1B). The central region encompassing the phosphopantetheine attachment site consists of a serine residue within a DSL motif recognised by members of the phosphopantetheinyl transferase family (Mofid et al. 2002). The prosthetic 229 group forms a thioester bond with fatty acids resulting in activation of the carboxyl carbon of the acyl group (Suh et al. 1999). Helix II was the most conserved in plant ACP 4 α helices. Helix II plays a dominant role in the interaction with type II FAS partner enzymes in plastids and has been termed the ‘recognition helix’ of ACP (Zhang et al. 2003a). Three other α helices, helix I, helix III and helix IV, were the main distinguishing features of plant ACPs in plastids and mitochondria (figure 1). A CT-rich sequence and the motif CTCCGCC and its derivative are conserved in the 5′-leader region of 18 plastid ACP cDNA (Bonaventure and Ohlrogge 2002). Site-directed mutagenesis of the CT-rich sequence, TTCTCTCTCCT, resulted in a three-fold reduction in transcription of the AtpC::uidA gene fusion (Bolle et al. 1996). The motif CTCCGTC and two CT-rich sequences were identified in the 5′-leader region of AhACP1 but not in AhACP2 and AhACP3 (table 1). Lack of these two motifs may lead to a difference in expression between AhACP1 and AhACP2, AhACP3 in developing peanut seeds. 3.2 Cloning and sequence analysis of AhMCAT In Sreptomyces coelicolor, MCAT is a key enzyme in both fatty acid and polyketide synthesis (Keatinge-Clay et al. 2003). A MCAT cDNA clone was obtained from the peanut cDNA library. AhMCAT contained a 1158 bp ORF, encoding a protein of 385 amino acids with a predicted molecular weight of 40.9 kD and a pI of 7.793. The deduced AhMCAT sequence shared 32.7% sequence identity with E. coli FabD (Serre et al. 1995). In EcFabD, the active site, Ser92, is hydrogen-bonded to His201. Gln250 serves as an H-bond acceptor during interaction with His201. Arg117 might play a role in binding the free carboxyl group. Gln11 can serve as an H-bond donor (Serre et al. 1995). The residues of AhMCAT, Gln89, Ser174, Arg199, His287 and Gln336, equivalent to EcFabD Gln11, Ser92, Arg117, His201 and Gln250, were strictly conserved in higher plants (figure 2). The GLSLGEY motif containing the catalytic residue (serine174 in AhMCAT) was completely conserved in higher plants, compared with the E. coli GHSLGEY motif (figure 2). The G(H/L)SLG pentapeptide belongs to the GXSXG motif (where x is any residue) prevalent in α/β hydrolases (Keatinge-Clay et al. 2003), which is conserved in all bacterial species (Simon and Slabas 1998). 3.3 Cloning and sequence analysis of AhKAS In higher plants, five types of KAS have been reported; namely, KASI, KASII, KASIII, KASIV and mitochondrial KAS, but there were no KAS ESTs in our peanut full-length J. Biosci. 34(2), June 2009 Meng-Jun Li et al 230 Figure 1. Multiple sequence alignment of selected acyl carrier protein (ACP) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm and shaded using BoxShade. Identical and conserved residues are shaded black and grey, respectively. The 4 α helices are indicated. Plant ACP Ser, corresponding to E. coli prosthetic group attachment site (Ser36), is marked with a triangle. GenBank accession numbers are as follows : Ah1, EE127470; Ah2, EG373603; Ah3, EU823319; At1, NP_187153; At2, NP_175860; At3, NP_564663; At4, NP_194235; At5, NP_198072; At6, NM_130026; At7, NP_176708; At8, NP_199574; Cl1, CAA54714; Cl2, CAA54715; Cl3, CAA54716; Cl4, CAA64542; So1, CAA36288; So2, P07854; Os1, NP_001050125; Os2, NP_001051948; Os3, NP_ 001055387; Os4, NP_001059204; Os5, NP_001062441; Os6, NP_001066930; Os7, NP_001067983; Hv1, AAA32920; Hv2, AAA32921 Hv3, AAA32922; Ec, AAB27925. Abbreviations: Ah, A. hypogaea; At, A. thaliana; Cl, Cuphea lanceolata; So, S. oleracea; Os, Oryza sativa; Hv, Hordeum vulgare; Ec, E. coli. Table 1. Proximal upstream sequence of acyl carrier protein (ACP) genes in peanut ACP isoform Sequence* AhACP1 gcattctcattaccacaaacactcttctcgtgctCTCCGTCcaaatctcagatctctctctctgtgaaa atg AhACP2 gagctaaagagagaagaactgagaagtgagaaccgagaatagagaagaagcaaagaagggttttaggtttttgtgtagatcgattttgca atg AhACP3 gacactcactcattcattcttcaaagaagaagaa atg * The motif CTCCGTC is indicated in upper case. The CT-rich sequences are underlined. The ATG start codon is separated by one space at the right end of the sequences. cDNA library. We cloned three members of the KAS family by a homology-based approach using KAS sequences of Glycine, Medicago and peanut ESTs in GenBank. The peanut AhKASI, AhKASII and AhKASIII contained a 1413 J. Biosci. 34(2), June 2009 bp, 1647 bp and 1206 bp ORF, encoding for a protein of 470, 548 and 401 amino acids with a predicted molecular mass of 50.0, 58.7, 42.5 kDa and a pI of 8.192, 8.090 and 6.940, respectively. AhKASI shared 51.5% sequence identity with Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. 231 Figure 2. Multiple sequence alignment of selected malonyl-CoA:ACP transacylase (MCAT) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. The five conserved residues of A. hypogaea MCAT corresponding to E.coli FabD Gln11, Ser92, Arg117, His201 and Gln250 are shaded black and position numbers are indicated. The motif G(L/H)SLG is boxed. GenBank accession numbers are as follows: Ah, EU823322; Gm, ABB85235; At1, AAM14913; At2, AAM64515; Bn, CAB45522; Pf, AAG43518; Ca, ACF17665; Os, ABF95452, Ec, 1MLA. Abbreviations: Ah, A. hypogaea; Gm, Glycine max; At, A. thaliana; Bn, B. napus; Pf, Perilla frutescens; Ca, Capsicum annuum; Os, O. sativa; Ec, E. coli. AhKASII and only 6.5% with AhKASIII. EcFabF shared 43.8% and 45.3% identity with AhKASI and AhKASII, whereas EcFabB shared 33.7% and 31.8% identity, which is consistent with results in Arabidopsis (von WettsteinKnowles et al. 2000). EcFabF was more closely related to peanut plastid KASI and KASII than EcFabB. The active site triads, Cys22–His36–His397 of AhKASI and Cys299–His439–His475 of AhKAS II, were revealed by sequence alignments. The Cys–His–His active site triad was strictly conserved in KASI and KASII of higher plants (figure 3). The greatest distinction between the active-site architecture of KASI, KASII and KASIII is the presence of two histidines in KASI and KASII and a histidine plus an asparagine in KASIII (von Wettstein-Knowles et al. 2000). AhKASIII shared 41% sequence identity with EcFabH and also had a Cys–His–Asn active site triad (Cys177, His327, Asn357), corresponding to EcFabH Cys112, His244, Asn274 (Qiu et al. 1999). The Cys–His–Asn active site triad and the motif GNTSAAS were strictly conserved in higher plants with the exception of Elaeis oleifera KASIII (Cys→ Tyr) (figure 4). Deletion of the tetrapeptide of GNTS led to a change in secondary structure and complete loss of AtKASIII condensing activity. The motif GNTSAAS was proposed to be responsible for the binding of acyl-ACPs (Abbadi et al. 2000). The Arg332 of AhKASIII, corresponding to Arg249 of EcFabH, a critical residue in the interaction between EcFabH and ACP (Zhang et al. 2001), was also strictly conserved in higher plants (figure4). 3.4 Cloning and sequence analysis of AhKR No KR clone was identified in our sequenced ESTs. By searching public databases, we found two AhKR ESTs (GenBank accession no. EG029580, ES715710) and the two sequences were used for primer design. The cDNAs of AhKR were cloned, which contained a 972 bp ORF encoding a protein of 323 amino acids with a predicted molecular mass of 33.8 kDa and a pI of 9.004. The deduced amino acid sequence of AhKR was 69.7% and 49.6% identical to B. napus KR (Fisher et al. 2000) and E. coli FabG (Price et al. 2001). On comparing BnKR with EcFabG, three active site residues – Ser217, Tyr230 and Lys234 of AhKR – corresponding to Ser138, Tyr151 and Lys155 of EcFabG, were found. The Ser–Tyr–Lys catalytic triad was completely conserved in higher plants (figure 5). The tyrosine and lysine residues are involved in actual catalysis, whereas serine participates in substrate binding and alignment (Price et al. 2001). Lys208 (Arg in BnKR) and Arg251 of AhKR, corresponding to Arg129 and Arg172 of EcFabG, made a significant contribution to ACP docking and were strictly conserved in higher plants (Zhang et al. 2003b). The catalytic YX3K motif conserved in FabG was also highly conserved in higher plants. 3.5 Cloning and sequence analysis of AhHD One full-length cDNA of HD was identified in the peanut cDNA library, which contained a 663 bp ORF encoding a protein of 220 amino acids with a predicted molecular mass of 24.0 kDa and a pI of 9.069. The deduced amino acid sequence shared 43.1% and 13.4% identity with EcFabZ and EcFabA, respectively. The two genes, FabA and FabZ, encoding β-hydroxyacylACP dehydratases have been well studied in E. coli. The catalytically important active site residues are His70 and Asp84′ in EcFabA (Leesong et al. 1996) and His54 and J. Biosci. 34(2), June 2009 Meng-Jun Li et al 232 Figure 3. Multiple sequence alignment of selected β-ketoacyl-ACP synthase I (KASI) (A) and KASII (B) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. (A) The three conserved residues of A. hypogaea KASI corresponding to E.coli FabB Cys163, His298 and His333 are shaded black and position numbers are indicated. GenBank accession numbers are as follows: Ah1, EU823325; Gm11, AAF61730; Gm12, AAF61731; At11, AAC49118; At12, AAM65396; Pf1, AAC04691; Rc1, AAA33873; Jc1, ABJ90468; Os1, BAD35225; Hv1, AAA32968; Ec1, AAC67304. (B) The three conserved residues of A. hypogaea KASII corresponding to E. coli FabF Cys164, His304 and His341 are shaded black and position numbers are indicated. GenBank accession numbers are as follows: Ah2, EU823327; Gm21, AAW88762; Gm22, AAW88763; Gm23, AAF61737; At21, AAK69603; At22, AAL91174; Pf2, AAC04692; Rc2, AAA33872; Jc2, ABJ90469; Os2, BAC79989; Hv21, CAA84022; Hv22, CAA84023; Ec2, CAA84431. Abbreviations: Ah, A. hypogaea; Gm, G. max; At, A. thaliana; Pf, P. frutescens; Rc, Ricinus communis; Jc, Jatropha curcas; Os, O. sativa; Hv, H. vulgare; Ec, E. coli. Glu68′ in EcFabZ (Kimber et al. 2004). In AhHD, the His/Glu catalytic dyad, His121 and Glu135′, and the motif LPHRFPFLLVDRV were completely conserved in plant HD and EcFabZ (figure 6), which suggested that plant HD may function, like EcFabZ, in the metabolism of both saturated and unsaturated long chain acyl-ACPs (Kimber et al. 2004). 3.6 Cloning and sequence analysis of AhENR One ENR homologue was isolated from the peanut cDNA library. It contained a 1170 bp ORF encoding a protein of 389 amino acids with a predicted molecular mass of 41.4 kDa and a pI of 8.571. The predicted protein sequence J. Biosci. 34(2), June 2009 showed 29.0% identity with EcFabI and 84.3% identity with BnFabI (Rafferty et al. 1995). AhFabI had 15.5% sequence identity with AhKR, similar to that of BnFabI and BnKR (Fisher et al. 2000). The Tyr–Tyr–Lys active site triad is characteristic of FabI homologues. The first tyrosine hydroxyl is directly involved in catalysis (Rafi et al. 2006). The second tyrosine might donate a proton to the enolate anion and lysine might act to stabilise the negatively charged transition state (Rafferty et al. 1995). In AhENR, the catalytic triad Tyr260–Tyr270– Lys278 and the motif YGGGMSSAK, corresponding to the YX6K motif in EcFabI, were completely conserved in higher plants (figure 7). Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. 233 Figure 4. Multiple sequence alignment of selected β-ketoacyl-ACP synthase III (KASIII) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. The four conserved residues of A. hypogaea KASIII corresponding to E. coli FabH Cys112, His244, Arg249 and Asn274 are shaded black and position numbers are indicated. The motif GNTSAAS is boxed. GenBank accession numbers are as follows: Ah, EU823328; Gm, AAF70509; At1, AAA61348; At2, CAA72385; Ca1, ACF17661; Ca2, ACF17662; Cw1, AAA97533; Cw2, AAA97534; Ch1, AAF61398; Ch2, AAF61399; Pf1, AAC04693; Pf2, AAC04694; Aa, AAB61310; Ps, CAC08184; Rc, ABR12417; Jc, ABJ90470; So, CAA80452; Ha, ABP93352; Eg, ABE73469; Eo, ABE73470; Ec, AAA23749. Abbreviations: Ah, A. hypogaea; Gm, G. max; At, A. thaliana; Ca, C. annuum; Cw, C. wrightii; Ch, C. hookeriana; Pf, P. frutescens; Aa, A. ampeloprasum; Ps, Pisum sativum; Rc, R. communis; Jc, J. curcas; So, S. oleracea; Ha, Helianthus annuus; Eg, Elaeis guineensis; Eo, E. oleifera; Ec, E. coli. Figure 5. Multiple sequence alignment of selected β-ketoacyl-ACP reductase (KR) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. The five conserved residues of A. hypogaea KR corresponding to E. coli FabG Arg129, Ser138, Tyr151, Lys155 and Arg172 are shaded black and position numbers are indicated. The motif YX3K is boxed. GenBank accession numbers are as follows: Ah, EU823329; At1, AAG40337; At2, CAA45794; Bn1, CAC41362; Bn2, CAC41363; Bn3, CAC41364; Bn4, CAC41365; Bn5, CAC41370; Ca, ACF17653; Cl, CAA45866; Os1, ABA97197; Os2, BAD22913; Ec, ACF17653. Abbreviations: Ah, A. hypogaea; At, A. thaliana; Bn, B. napus; Ca, C. annuum; Cl, C. lanceolata; Os, O. sativa; Ec, E. coli. J. Biosci. 34(2), June 2009 Meng-Jun Li et al 234 Figure 6. Multiple sequence alignment of selected β-hydroxyacyl-ACP dehydrase (HD) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. The catalytic dyad of A. hypogaea HD corresponding to E. coli FabZ His54, Glu68 and E. coli FabA His70, Asp84 is shaded black and position numbers are indicated. The conserved domain in HD is boxed. GenBank accession numbers are as follows: Ah, EU823332; At1, AAD23619; At2, AAM64548; At3, AAO24548; Bn, AAK60545; Ca, ACF17652; Os, AAT58880; Pm1, ABA25920; Pm2, ABA25921; Ec1, AAC36917; Ec2, 1MKB_A. Abbreviations: Ah, A. hypogaea; At, A. thaliana; Bn, B. napus; Ca, C. annuum; Os, O. sativa; Pm, Picea mariana; Ec, E. coli. Figure 7. Multiple sequence alignment of selected enoyl-ACP reductase (ENR) homologues. Protein sequences were aligned using the CLUSTALW alignment algorithm. The three conserved residues of A. hypogaea ENR corresponding to E. coli FabI Tyr146, Tyr156 and Lys163 are shaded black and position numbers are indicated. GenBank accession numbers are as follows: Ah, EU823333; At1, AAF37208; At2, AAM45010; At3, CAA74175; Bn1, AAB20114; Bn2, CAA64729; Bn3, CAC41366; Bn4, CAC41367; Bn5, CAC41368; Bn6, CAC41369; Oe, AAL93621; Ca1, ACF17650; Ca2, ACF17651; Nt1, CAA74176; Nt2, CAA74177; Os1, BAD03622; Os2, BAD26009; Os3, CAA05816; Ec, P29132. Abbreviations: Ah, A. hypogaea; At, A. thaliana; Bn, B. napus; Oe, Olea europaea; Ca, C. annuum; Nt, Nicotiana tabacum; Os, O. sativa; Ec, E. coli. 3.7 Homologue of type II FAS genes and subcellular target prediction Homologues of type II FAS genes were cloned by RT-PCR using total RNA from a peanut immature seed mixture by J. Biosci. 34(2), June 2009 gene-specific primers. More than six clones of each gene were picked randomly and sequenced. Homologue numbers of each type II FAS gene expressing in peanut seed development varied from 5 in ENR to only 1 in KASII, KASIII and HD. The number of single-nucleotide polymorphisms (SNPs) was Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. Table 2. Homologues of each type II FAS gene in peanut Gene Accession number ACP1 EG374024* 0 EE127470 4 EE124662 2 ACP1-2 423 EU823318 1 ACP1-2 423 EE127527 3 ACP2-1 393 EG373603 3 ACP2-2 393 EU823319 2 ACP3-1 375 EU823320 1 ACP3-2 375 EU823321 4 ACP3-3 375 EU823322 1 MCAT1 1158 EU823323 4 MCAT2 1158 EU823324 3 MCAT3 1161 EU823325 4 KASI-1 1413 EU823326 2 KASI-2 1413 KASII EU823327 10 KASII KASIII EU823328 7 KASIII KR EU823329 4 EU823330 3 ACP2 ACP3 MCAT KASI 235 Clone numbers Protein ORF (bp) SNP InDel ACP1-1 423 4 0 ACP1-2 423 4 0 3 0 7 1 20 0 1647 0 0 1206 0 0 KR1 972 3 0 KR2 972 EU823331 1 KR3 972 HD EU823332 7 HD-1 663 0 0 ENR EU823333 1 ENR1 1170 18 0 EU823334 3 ENR2 1170 EU823335 2 ENR3 1170 EU823336 1 ENR3 1170 EU823337 1 ENR3 1170 * EG374024 was found in the peanut full-length cDNA library but we did not find it on PCR-based homologue searching. quite different in each gene. The most were identified in KASI and ENR, while no SNP was identified in KASII, KASIII, HD. Indel was only found in MCAT (table 2). The percentage of transition was more than that of transversion in the identified SNPs. The results indicated that most type II FAS genes had more than two homologues expressing in developing peanut seeds. To clarify the possible subcellular compartment of peanut type II FAS genes, amino acid sequences were used for targeting prediction by the TargetP1.1 Server. Results clearly indicated that ACP1, MCAT, KASI, KASII, KASIII, KR, HD and ENR all targeted chloroplast, while ACP2 and ACP3 were confidently predicted to target mitochondria (table 3). 4. Discussion Fatty acid biosynthesis in higher plants is carried out by type II FAS, which has been most extensively studied in E. coli. The crystal structural determination of E. coli FAS enzymes has been completed (White et al. 2005). Here, we report the cloning of type II FAS genes from A. hypogaea for the first time including MCAT, KASI, KASII, KASIII, KR, HD, ENR and ACP. FabF has acetyl CoA:ACP transacylase (ACAT) activity and seems able to initiate fatty acid synthesis, but it may not play this role when FabH is functional (Lai and Cronan 2003). The ACAT activities of E. coli, spinach and Streptomyces glaucescens FabH are approximately 0.5%, 1% and 12% of the KAS activities (Tsay et al. 1992; Olsen et al. 1999; Han et al. 1998). In avocado, KAS III and ACAT activities have been separated from each other and the native molecular mass of KAS III is 69 kDa and that of ACAT is 18.5 kDa (Gulliver and Slabas 1994), which indicates that there is a separate ACAT enzyme in avocado. However, in higher plants, it is still uncertain whether ACAT is a separate enzyme or a partial reaction of a condensing enzyme. No ACAT cDNA has been reported in E. coli and higher plants. J. Biosci. 34(2), June 2009 Meng-Jun Li et al 236 Table 3. Subcellular compartment of peanut type II FAS enzymes predicted by the TargetP1.1 Server Protein Accession Len cTP mTP Localisation RC TPlen ACP1 EE127470 140 0.938 ACP2 EE127527 130 0.109 0.016 Chloroplast 1 55 0.878 Mitochondria 2 40 ACP3 EU823319 124 0.082 0.923 Mitochondria 1 36 MCAT EU823322 385 0.968 0.080 Chloroplast 1 60 KI EU823325 470 0.910 0.104 Chloroplast 1 48 KII EU823327 548 0.624 0.007 Chloroplast 3 39 KIII EU823328 401 0.527 0.071 Chloroplast 5 72 KR EU823329 323 0.618 0.022 Chloroplast 3 73 HD EU823332 220 0.917 0.050 Chloroplast 1 41 ENR EU823333 389 0.937 0.143 Chloroplast 2 70 *Len, sequence length; cTP, chloroplast transit peptide; mTP, mitochondrial targeting peptide; RC, reliability class, from 1 to 5, where 1 indicates the strongest prediction; TPlen, presequence length Sequence comparisons revealed that the primary structure of plant plastid type II FAS enzymes was strictly conserved, especially the catalytic residues, which suggested that these enzymes may have similar functions in higher plants as those in E. coli. The helix II of plant ACPs was highly conserved, but plastid ACP and mitochondrial ACP can be distinguished by three other α helices. E. coli FabB and FabF shared 35.2%, 49.9% identity with A. thaliana mitochondrial KAS, and 33.7%, 46.0% with plastid KASI, KASII, respectively. These observations suggested that two type II FAS systems in higher plants originated from E. coli type II FAS. Plant type II FAS in mitochondria was more closely related to E. coli type II FAS than that in plastids. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 5 to 1. The number of SNPs was also quite different in each gene. The result was consistent with that in peanut acyl-ACP thioesterase (GenBank accession no, EF117305-EF117309). We cloned genomic DNAs of peanut ACP1, ACP2 and ACP3 by PCR using gene-specific primers (data not shown). More cDNAs of ACP1 and ACP3 could be deduced from genomic DNA clones. The cDNAs not identified in developing seeds may be silent or may be expressed in other tissues. More than two types of the 5′-terminal of KAS cDNA were cloned by 5′-RACE. Based on these results, we have reason to propose that more than two homologues of most type II FAS genes exist in the peanut genome although RT-PCR has its disadvantages in gene cloning. Gene redundancy was widespread in the peanut fatty acid synthesis pathway, which made this process more sophisticated. The findings obtained in this study provide the basis for future investigation of peanut FAS genes in terms of regulation, expression pattern analysis, evolution and gene engineering study. J. Biosci. 34(2), June 2009 Acknowledgements This work was supported by grants from the National High Technology Research and Development Program of China (2006AA10A114), Shandong Academy of Agricultural Sciences Foundation (2006YCX030), (2007YCX001) and Postdoctoral Foundation of Shandong Province (200701004). 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