GENE-40495; No. of pages: 10; 4C: Gene xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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Research paper

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MeiJing He, XinLei Yang, ShunLi Cui, GuoJun Mu, MingYu Hou, HuanYing Chen, LiFeng Liu ⁎

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North China Key Laboratory for Crop Germplasm Resources of Education Ministry, Laboratory of Crop Germplasm Resources of Hebei, Agricultural University of Hebei, Baoding 071001, People's Republic of China

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Article history: Received 4 September 2014 Received in revised form 23 March 2015 Accepted 5 May 2015 Available online xxxx

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Keywords: Peanut Annexin genes Cloning Expression analysis

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Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.)

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Annexin, Ca2+ or phospholipid binding proteins, with many family members are distributed throughout all tissues during plant growth and development. Annexins participate in a number of physiological processes, such as exocytosis, cell elongation, nodule formation in legumes, maturation and stress response. Six different full-length cDNAs and two partial-length cDNAs of peanut, (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8) encoding annexin proteins, were isolated and characterized using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins were 36.0 kDa with acidic pIs of 5.97–8.81. ANNAh1, ANNAh2, ANNAh3, ANNAh5, ANNAh6 and ANNAh7 shared sequence similarity from 35.76 to 66.35% at amino acid level. Phylogenetic analysis revealed their evolutionary relationships with corresponding orthologous sequences in soybean and deduced proteins in various plant species. Real-time quantitative assays indicated that these genes were differentially expressed in various organs. Transcript level analysis for six annexin genes under stress conditions showed that these genes were regulated by drought, salinity, heavy metal stress, low temperature and hormone. Additionally, the prediction of cis-regulatory element suggested that different cisresponsive elements including stress- and hormone-responsive-related elements could respond to various stress conditions. These results indicated that members of AnnAhs family may play important roles in the adaptation of peanut to various environmental stresses. © 2015 Published by Elsevier B.V.

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1. Introduction

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Annexin, always being considered as a kind of multifunctional soluble protein, is capable of Ca2+-dependent and Ca2+-independent binding to membranes (Clark et al., 2012). Annexins are ubiquitous in plants and can localize on all the plasma- and endo-membranes. Annexins are important to plant growth and development regulation and environmental adaptation. Annexins always take part in a series of relative metabolic process, such as wall synthesis, membrane construction, differentiation, exocytosis and stress responses (Seals and Randall, 1997; Proust et al., 1999; Lee et al., 2004; Cantero et al., 2006; Jami et al., 2008; Konopka-Postupolska et al., 2009; Talukdar et al., 2009). Phylogenetic tree analysis showed that plant annexins represent a unique subset originated from some ancient ancestor which now is

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Abbreviations: cDNA, complementary DNA; AnnAh(s), peanut annexin gene(s); ANNAh(s), peanut annexin protein(s); RT-PCR, reverse-transcription PCR; RACE-PCR, rapid-amplification of cDNA ends PCR; ABA, abscisic acid; SA, salicylic acid; gDNA, genomic DNA; ORF, open reading frame; UTR, untranslated region; GSPx, gene special primer for each AnnAh; TAIL-PCR, thermal asymmetric interlaced PCR; qRT-PCR, quantitative real-time polymerase chain reaction; EFL1B, translational elongation; UBI, polyubiquitin; YLS8, yellow leaf specific 8 gene; ACTIN7, cytoskeletal structural protein. ⁎ Corresponding author. E-mail address: [email protected] (L. Liu).

considered to be green alga (Clark et al., 2012). Plant annexins have a molecular weight in the range of 32–36 kDa. The typical structure of plant annexins generally consists of four repeats and each repeat has about 70 amino acids. In their animal counterparts, each repeat contains an endonexin sequence presenting as K-G-G-X-T-38-D/E, but in plant annexins the endonexin fold is considered to be selectively lacking in the second and third repeats (Hofmann, 2004; Gerke et al., 2005; Monastyrskaya et al., 2009). And consistently, at least 3 of the 4 repeats in animal annexins are highly conservative while just 1 or 2 in plant annexins (Hofmann, 2004; Gerke et al., 2005), and most of the structure differences are determined by their variable N-terminal region (Monastyrskaya et al., 2009). Compared with their animal counterpart, plant annexins tend to have a short N-terminal for 10 amino acids. The crystal structure of bell pepper (Capsicum annuum L.) annexin (ANNCa32) showed that the shorter N-terminal area can interact with its core domain, indicating that the regulation function is highly conserved (Monastyrskaya et al., 2009). Annexins exist in almost all eukaryotes, and always present in a form of multigene family. Since the first evidence given by Boustead in 1989, annexin-like proteins have been isolated from Arabidopsis thaliana, cotton (Gossypium spp.), maize (Zea mays L.), tomato (Lycopersicon esculentum L.), alfalfa (Medicago sativa l.,) etc. (Boustead et al., 1989; Blackbourn et al., 1992; Kovács et al., 1998; Lim et al., 1998; Hofmann et al., 2000; Dabitz

http://dx.doi.org/10.1016/j.gene.2015.05.004 0378-1119/© 2015 Published by Elsevier B.V.

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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Uniform and full peanut seeds of Jihua 2, the control of peanut regional test of Hebei Province with multi-resistance and wide adaptation, were surface sterilized by incubation with (1) 70% (v/v) ethanol for 1 min and (2) 0.1% (v/v) HgCl2 for 10 min and subsequently washed six times with sterile, deionized water. After peeling off the seed coat, the seeds were plated onto Murashige and Skoog medium with 3% (w/v) sucrose (Murashige and Skoog, 1962). The seeds were maintained in a growth chamber (light intensity of 275 mmol m−2 s−1, humidity of approximately 80%, and temperature of 27 ± 1 °C) under short-day conditions (14 h of light/10 h of darkness) for 2 weeks before transferring the seedlings to soil in separate pots. For organ specific expression, samples were collected from roots, stems, leaves and flowers in a 4-week stage. To investigate the effect of abiotic stress inducing compounds on the expression of different annexin genes (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, and AnnAh7), peanut was fed through separately with solutions

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2.2. Total RNA extraction and cDNA synthesis

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Total RNA of stress-treated and unstressed leaves, stems, roots and flowers was extracted with EASYspin Plant RNA mini kit (Aidlab) according to the manufacturer's instructions. About 4 μg of total RNA was taken to synthesize the first strand of cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara) with Oligo dT primer following manufacturer's protocol. For real-time PCR, the first strand cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara).

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2.3. Identification of AnnAhs by database search

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In order to retrieve annexins from different species (Glycine max, Medicago, L. japonicas and Arabidopsis thaliana, etc.), we performed a search of gene description with ‘annexin’ as the keyword on Plaza (http://bioinformatics.psb.ugent.be/plaza/organism/view). A series of degenerate primers (AxF/R) were designed based on the annexin amino acid sequences by CODEHOP (https://icodehop.cphi.washington.edu/icodehop– context/Welcome) and optimized through Oligo 7 (Supplementary Table 1).

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containing 250 mM sodium chloride (NaCl), 150 μM cadmium chloride (CdCl2), 10% PEG-6000, 100 μM ABA, and 100 μM SA. By adjusting the incubator at 15 °C to make the leaves experience low temperature stress. Leaves were collected at different times of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h after treatment and quickly-frozen in liquid N2 and stored at −80 °C for further use. Control seedlings were mock treated with water.

2.4. Isolation of cDNA clones from peanut

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et al., 2005; Konopka-Postupolska et al., 2009; Laohavisit et al., 2009). The whole gene family has been identified in Arabidopsis, tomato, mustard (Brassica juncea L.), and rice (Oryza sativa L.) (Clark et al., 2001; Jami et al., 2009, 2012b; Lu et al., 2012). The dynamic expression of annexin genes goes throughout the whole life circle of high plant. Medicago (Medicago truncatula) annexins have been observed during lateral root development (Carvalho-Niebel et al., 2002). Differential expression of individual annexins had also been observed in cotton fiber (Shin and Brown, 1999), seedlings of Arabidopsis (Clark et al., 2001), the pulvinus of mimosa (Mimosa pudica L.) (Hoshino et al., 2004), fruit ripening in bell pepper and strawberry (Fragaria × ananassa Duch.) (Wilkinson et al., 1995; Proust et al., 1996), tips of pollen tubes in Lilium longiflorum (Blackbourn et al., 1992), and in the leaf and stem cells of pea (Pisum sativum L.) (Clark et al., 1998). Annexin is widely distributed in plant. Although the structural and functional researches of plant annexins are still shallow, it is clear that they have a lot of significant functions, such as membrane binding, actin binding, peroxidase activity, ion channel, receptor of Ca2 +mediate signal transduction, etc. (Hu et al., 2000; Gorecka et al., 2005; Laohavisit and Davies, 2009). As well as being linked to growth and development, annexin expression and cellular position can change in response to abiotic and biotic stimuli, such as osmotic stress, salinity, drought, ABA, light, gravitropism and pathogen attack (Truman et al., 2007; Vandeputte et al., 2007; Jami et al., 2008; Mortimer et al., 2008; Divya et al., 2010; Huh et al., 2010). Peanut is an important vegetable oil and economic crop in China (Zhang et al., 2002) and its economic benefit is remarkable and growing acreage gradually expanded in recent years. However, the stresses such as drought, heavy metal and salinity limit the increase of peanut production and quality (Suthar and Patel, 1992; Rao and Wright, 1994; Rucker et al., 1995) as to stress affects membrane lipids and photosynthetic responses (Lauriano et al., 2000). In addition, nitrogen fixation by leguminous plants is reduced by water stress which would limit its fertility effect (Williams and Boote, 1995; Lauriano et al., 2000). Therefore, it has practical significance to explore peanut stress tolerant genes. In view of annexins in important roles of stress regulation, a functional study of annexins may offer an alternative approach to improve peanut stress tolerance. Information about the annexin gene family in Arabidopsis, tomato, mustard, rice and maize has been well characterized (Carroll et al., 1998; Lim et al., 1998; Clark et al., 2001; Cantero et al., 2006; Jami et al., 2012a; Lu et al., 2012; Zhou et al., 2013), but no such characterization of peanut annexin gene family has been reported yet in peanut. Consequently, the objectives of this study were to (1) clone annexin gene family from peanut, (2) study the expression of annexin genes in different organs of peanut, and (3) measure the transcript level for annexin genes under simulated stresses.

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The full-length cDNAs of AnnAhs were isolated using degenerate primers and gene specific primers for reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE)-PCR reactions. For the isolation of annexin cDNAs, total RNA from unstressed leaves was used in reverse transcription. For RT-PCR amplification, 2 μL of cDNA was used as a template along with a set of degenerate primers (Supplementary Table 1). Degenerate primer amplification with touchdown PCR amplifications was performed in a total volume of 25 μL using ExTaq DNA polymerase (Takara) at 94 °C for 1 min followed by 94 °C for 30 s (denaturation), 65 °C (− 0.5 °C/cycle) for 30 s (annealing), 72 °C for 1 min (elongation) for 20 cycles, then 94 °C for 30 s (denaturation), 55 °C for 30 s (annealing), 72 °C for 1 min (elongation) for 15 cycles with a final extension of 15 min at 72 °C. The resultant PCR products were purified with SanPrep Column DNA Gel Extraction kit (Sangon) and cloned in pMD19/T vector (Takara). Similarly, the 5′- and 3′-UTR regions of AnnAhs were amplified using SMART RACE cDNA amplification kit (Clontech) using the sequence information of the corresponding cDNA isolated by RT-PCR. The 5′ ready cDNA was synthesized by reverse transcribing 1 μg of total RNA using SMART™ II kit with an oligonucleotide, and a cDNA synthesis primer 5′-RACE CDS Primer A, supplied in the kit. First and nested PCR products were amplified using GSPx-1R primers (Supplementary Table 1) and Universal Primer Mix (UPM) for the first reaction and GSPx-2R primers (Supplementary Table 1) and Nested Universal Primer A (NUP) for the nested reaction. First strand cDNA for the 3′-RACE was performed using 1 μg of total RNA as a template using T7dT18 5′-ACGACTCACTATAGGGCTTTTdT18-3′, and first and nested PCR products were amplified using GSPx-1F primers (Supplementary Table 1) and anchor primer T7 5′-ACGACTCACTATAGGGCTTT TT-3′ for the first reaction and GSPx-2F primers (Supplementary Table 1) and anchor primer T7 for the nested reaction. PCR for 5′RACE was performed using Advantage® 2 PCR Kit (Clontech) under the following condition: 5 cycles (94 °C for 30 s, 72 °C for 3 min) and 5 cycles (94 °C for 30 s, 70 °C for 30 s, 72 °C for 3 min) followed by 25 cycles (94 °C for 30 s, 68 °C for 30 s, 72 °C for 3 min). PCR for 3′-

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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2.6. Sequence and phylogenetic analysis

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The amplification products were sequenced and analyzed with Blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgih) and predicted with ORF finder to test the alignment results and the integrity of coding sequences. The annotation of amino acid sequences, pI, subcellular localization and theoretical molecular mass of each of ANNAHs were analyzed using the Expasy proteomic server (http://www.ca.expasy.org). The conserved protein motifs of peanut annexin protein sequences and other plant sequences used in phylogenetic analysis were analyzed using the MEME 4.6.1 and MAST motif search software (http://meme. sdsc.edu/meme/cgi-bin/meme.cgi) with the default setting parameters excepting the number of different motifs settled as 10. The functional annotation of these motifs was analyzed by SMART databases (http:// smart.embl-heidelberg.de) and InterProScan (http://www.ebi.ac.uk/ interpro/scan.html). Multiple sequence alignment of the deduced ANNAHs was carried out by the Clustalw2 (http://www.ebi.ac.uk/ Tools/msa/clustalw2/). Phylogenetic analysis was performed to investigate the evolutionary relationships among deduced annexin sequences

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2.7. Relative transcript level analysis of AnnAhs

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Real-time PCR analyses were performed in a 96-well plate using Agilent MX3000P real-time PCR system (Agilent Technologies) with gene-specific forward and reverse primers of different annexin genes and reference genes (Supplementary Table 2). Control samples and treated samples by 10% PEG-6000 were analyzed using ELF1B as the endogenous reference gene for mRNA normalization. Roots, stems, leaves and flowers and samples treated by CdCl2 and 15 °C used UBI as the endogenous reference gene. Samples treated by NaCl used YLS8 as the endogenous reference gene for mRNA normalization. ACTIN7 was used as endogenous reference in analyzed samples treated by ABA and SA. For PCR, 2 μL of cDNA (after dilution) was used as a template in a reaction volume of 20 μL using SYBR® Premix Ex Taq™ II kit (Takara) with second fast cycling conditions of 95 °C 1 min; 95 °C 15 s, 60 °C 34 s (data collection), 40 cycles; then melting curve analysis at 95 °C 1 min, 55 °C 30 s, 95 °C 30 s. Real time quantitative PCR analyses were carried out with three independent total RNA samples and 2−△△ct was applied to analyze the expression of annexin genes (Livak and Schmittgen, 2001).

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Genomic DNA was extracted from 2-week-old peanut seedlings grown on MS basal medium (Murashige and Skoog, 1962) using CTAB procedure (Wang et al., 2002). 200 ng of DNA was used as a template for the PCR amplification of various genomic annexin clones (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7) with both forward and reverse primers GxF/R (Supplementary Table 1) that were used for the isolation of their corresponding cDNA sequences in a total volume of 25 μL using LA Taq DNA polymerase (Takara). The PCR cycling conditions were the same with the step for ORF amplification. The PCR products were gel-purified, cloned and sequenced. The exon–intron structures were generated using GSDS (http://gsds.cbi.pku.edu.cn/) by aligning the cDNA sequences with the corresponding genomic sequences. The 5′-upstream promoter sequence of AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, and AnnAh7 genes was isolated by TAIL-PCR using the Genome Walking kit (Takara) according to manufacturer's instructions. Genomic DNA (300 ng) was used in the primary PCR. It was performed using adaptor primers (AP1-AP4) and primary gene-specific primers (USxR-1) (Supplementary Table 1). The second PCR was carried out using adaptor primers (AP1–AP4) and nested secondary genespecific primers (USxR-2) (Supplementary Table 1). The third PCR was carried out using adaptor primers (AP1–AP4) and nested tertiary gene-specific primers (USxR-3) (Supplementary Table 1). Both primary and nested primers were designed using sequence information of the corresponding genomic annexin clones of Arachis hypogaea L. The amplified products were gel-purified, cloned and sequenced. The 2 kb of genomic sequences upstream of 5′-UTR of each annexin gene was obtained for putative cis-element analysis in the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) (Higo et al., 1999) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/).

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from peanut and diverse plant species. The unrooted phylogenetic tree was generated by neighbor-joining method using MEGA 5 from 1000 bootstrap replicates and the evolutionary distances were calculated by Poisson correction method corresponding to the number of amino acid substitutions per site. Protein sequences were aligned using Clustal with pairwise gap penalties of 10 for gap opening and 0.1 for gap extension, and multiple alignment penalties of 10 for gap opening and 0.2 for gap extension (Tamura et al., 2011).

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RACE using LA Taq DNA polymerase (Takara) was performed under the following condition: 94 °C for 1 min followed by 94 °C for 30 s (denaturation), 65 °C (−0.5 °C/cycle) for 30 s (annealing), 72 °C for 1 min (elongation) for 20 cycles, then 94 °C for 30 s (denaturation), 55 °C for 30 s (annealing), 72 °C for 1 min (elongation) for 15 cycles with a final extension of 15 min at 72 °C. The resultant 5′ and 3′-RACE-PCR products were purified, cloned and sequenced. The ORF of each AnnAh by gene primers AnnAhx F/R was amplified using Ex Taq DNA polymerase (Takara) at 94 °C for 1 min followed by 94 °C for 30 s (denaturation), 58 °C for 30 s (annealing), 72 °C for 1 min (elongation) for 35 cycles with a final extension of 15 min at 72 °C. The resultant ORF products were purified, cloned and sequenced.

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3. Results

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3.1. The partial sequence of AnnAhs

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A series of degenerate primers were designed based on high conserved region. After amplification and detection, several fragments with a length of 200 bp–700 bp were obtained and sequenced. BlastX analysis of these sequences showed that these fragments contained specific structures of annexin genes indicating that we obtained the partial sequence of multiple members of peanut annexin gene family (AnnAhs).

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3.2. Sequence analysis of AnnAhs

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The cDNAs of eight related genes encoding annexins in peanut were isolated and designated as AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8 (GenBank accession nos. KM267643, KM276779, KM276780, KM276781, KM276782, KM276783, KM276784 and KM276785. AnnAh4 and AnnAh8 obtained partial sequences.). The nomenclature of these annexin genes was based on the similarity of their sequences with their corresponding homologous sequences from its leguminosae relative, Glycine max or Medicago. The lengths of the six genes were verified by amplifying the gene special primer AnnxAh F/R. The AnnAh ORFs flanked by 45–132 bp and 141–237 bp as the 5′-and 3′-untranslated regions (5′-and 3′-UTRs), respectively. The amplification of genomic DNA of AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, and AnnAh7 with their specific primers of their corresponding coding regions or subsection specific primers resulted in fragments of 2000 bp – 5000 bp. Comparison of the genomic and cDNA sequences revealed the intron/exon boundaries, which carried the consensus nucleotide GT/AG sequence at 5′- and 3′-splice site junctions mostly and also indicates that the splicing method of peanut annexin genes is accorded with the classic GT-AG rule (Breathnach and Chambon, 1981). Gene structure analysis showed that the coding region of each of the AnnAh consisted of exons separated by variable lengths of introns (Fig. 1). AnnAh1 and AnnAh2 had five exons each, and AnnAh3, AnnAh5 and AnnAh6 have six exons each, but AnnAh7 has the least

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Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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Fig. 1. Gene structures of peanut annexins and representative leguminous genomes (Arachis hypogaea L, Glycine max and Medicago truncatula). As shown in the legend, the intron phases in between exon–intron junctions are given as 0 and 1; exons are represented by black filled round corner rectangles, introns by black lines. Gene structures were generated from online tool Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/).

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Phylogenetic tree was analyzed in MEGA5 from the subject protein sequences of 41 annexins from various plant species. Nine species were formed five orthologous groups according to the result of phylogenetic tree performed in Fig. 2. Six annexins (ANNAh1, ANNAh3, ANNAh5, ANNAh6 and ANNAh7) from peanut had close phylogenetic relationships with leguminous crops while they had distant relationship with Arabidopsis and monocots. ANNAh2 had close phylogenetic relationships with Arabidopsis. In each group, all the annexins from monocot origin had high homology. These results were accordance to the traditional botanical classification. Comparative analysis of gene structures in leguminous annexin genomes in identical group revealed that each group exon–intron organization had a similar distribution (Fig. 1). Analysis of annexin genes (AnnAh6 and AnnAh7 had a small difference) for intron phases revealed that their first exons are flanked by an intron in phase 1 (intron located between the first and second nucleotide of one codon), while the rest of their exons are present in phase 0 (intron between two codons, which refers to the third nucleotide in one codon and the first nucleotide in another codon) in the exon–intron junctions (Fig. 1).

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At the nucleotide level, the coding regions of these AnnAhs shared 46.23–66.56% identity among each other, whereas their corresponding

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exons (four exons). Except AnnAh7, intron phases in other AnnAhs were similar (the first intron phase is 1 and the others are 0) and the sizes of 1 to 4 exons were found to be highly congruent. The difference in nucleotide sequences could be due to deletions or small insertions within each of these annexins (Jami et al., 2009).

deduced protein sequences shared 17.93–65.72%. The lowest identity was found in AnnAh7 with other AnnAhs, while AnnAh1 and AnnAh2 shared the highest identity of 65.72% (Fig. 3). All deduced proteins had a predicted molecular mass of approximately 36 kDa (35.85 to 36.29 kDa), but their predicted isoelectric points were very divergent, ranging from 5.97 to 8.81 (Table 1). These results were in accordance to annexin proteins reported (Jami et al., 2009, 2012a; Lu et al., 2012). Localization predicted by PSORT showed that most ANNAhs were distributed in the cytoplasm. Some proteins were located in the chloroplast, endoplasmic reticulum and microbody, but only ANNAh3 has a distribution in the nucleus. These results indicated that ANNAhs may involve some common pathway and particular regulation way. The authentic localizations of ANNAhs should be identified further. In the primary structure of plant annexins, the N-terminal region is short in length and the type-II Ca2+-binding residues are absent in repeats 2 and 3 (Clark and Roux, 1995; Moss and Morgan, 2004). The definition of core repeat regions for all six of the peanut annexins was compared to the annexin consensus sequences as analyzed by Barton et al. (1991). Based on SMART and InterProScan databases, we founded 2–4 annexin repeats with different calcium binding residues. These deduced ANNAhs contained a type II Ca2+-binding site (G-X-G-T-(38)-D/ E) in the first or fourth repeat (Fig. 3), suggesting that the speculated ANNAhs possess the classical characteristics of the annexin family proteins. The deduced amino acid sequences of peanut annexins contain several conserved residues/motifs, but annexin domains were only partly conserved in peanut annexins, as clearly shown by the analysis of their deduced amino acid sequences in Fig. 3. The residues in color background are represented in Fig. 3 referring to the annexin consensus sequences mentioned in Lu et al. (2012) and also found in repeat regions of other plant annexins. Except for ANNAh7, other five peanut annexins contain conserved His residue at the N-terminal region.

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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Fig. 2. Phylogenetic tree of peanut annexins and other plants. Unrooted neighbor joining (NJ) phylogenetic tree was constructed with MEGA5 software using 41 full-length amino acid sequences from four dicot species and monocot species. The tree was classified into five orthologous groups represented by figure adjacent to the tree branches. The distribution of ten conserved motifs represented in the colored boxes was identified in annexin protein sequences using the MEME 4.6.1 software. The order of the motif corresponds to their position in the individual protein sequences. The motifs are not drawn to scale (color figure manually) and showed on Table 2.

Many putative post-translational modification sites were detected in peanut annexins, including phosphorylation, N-glycosylation, and Nmyristoylation sites (Table 1). Conserved motifs were identified from the 41 full-length annexins that were used in phylogenetic studies using the MEME 4.6.1/MAST motif search software (Bailey and Elkan, 1994; Bailey and Gribskov, 1998). A total of 10 motifs containing 15 to 50 residues were identified and each of these motifs was annotated by InterProScan and SMART databases (Fig. 2, Table 2). The diversity of motif patterns based on combinations of motifs was found to be in agreement with the phylogenetic analysis and these motif patterns were conserved and specific to each of the group. Within the same group, annexins containing the motif pattern structures might have conserved during evolution, while between the groups the different

motif patterns indicated the functional divergence across groups (Jami 389 et al., 2012a). 390 3.5. Organ-specific expression of AnnAhs in peanut

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The expression patterns of the six peanut annexin genes in four organs (stems, leaves, roots and flowers) were investigated using real time quantitative RT-PCR (Fig. 4). RT-PCR using gene-specific primers for partial sequences that specifically amplify each of AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6 and AnnAh7 genes indicated that AnnAhs expressed in examined organs but at different levels under non-stress condition. All the six genes showed expression constitutively in all the organs examined. AnnAh1, AnnAh2 and AnnAh7 expressed at higher

392

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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E

Fig. 3. Amino acid sequence alignment of peanut annexins by Clustalw2. Peanut annexins share a core domain made up of four internal repeats named the endonexin fold, with length of approximately 70 amino acids and containing a highly conserved consensus sequence. The putative annexin repeats (I to IV) are shown below the sequences with color lines. Identical (*), highly conserved (:), and moderately conserved (.) regions are designated. The sequences marked are as follows: yellow, endonexin (type II Ca2+ binding) sequences; green, His 40 residue; atrovirens, S3 clusters; red, IRI actin-binding motif; and black box, putative GTP-binding motif (GXXXXGKT, DXXG) (color figure manually). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

404

3.6. Regulation of AnnAhs in response to various abiotic stresses

405

408 409

AnnAhs (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6 and AnnAh7) RNA transcription levels were observed through real-time PCR strategy to detect the correlation between AnnAhs expression and abiotic stress and hormones from various time points at the seedling stage (Fig. 5). Most peanut annexin genes were induced by single or several stresses.

t1:1 t1:2

Table 1 Characteristics of peanut annexin were predicted by bioinformation.

R

R

O

C

Protein name

AAa

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11

ANNAh1 ANNAh2 ANNAh3 ANNAh4 ANNAh5 ANNAh6 ANNAh7 ANNAh8

316 317 321 211 315 315 318 187

t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20

MW/Pib

U

t1:3

N

406 407

Relative to untreated control, AnnAh1, AnnAh3, AnnAh5 and AnnAh6 induced by cold and AnnAh3 was in high expression particularly. Treatment with NaCl (250 mM) caused an increase in transcript level in all AnnAhs in different time period with different folds. The levels of induction for AnnAh1, AnnAh5 and AnnAh6 increased from 2 h to 12 h and declined thereafter. For AnnAh3, the transcript level increased the maximum fold on 10 h than other genes and decreased in the following hours slightly, whereas mild induction was observed in AnnAh2 and AnnAh7 at 2 h to 24 h. In the case of 150 μM CdCl2 treatment, slight induction was observed in AnnAh5 and AnnAh6, particularly AnnAh5 had the lowest transcript level. AnnAh1, AnnAh2 AnnAh3 and AnnAh7 had the same trend that the levels got peaked at 10 h and the level of

E

402 403

level in stem than in other organs. The transcripts of AnnAh3, AnnAh5 and AnnAh6 showed more expression in roots. Based on the results, we can clearly differentiate the quantification in transcript level of six annexin family members in different organs of peanut.

36.08/7.10 36.06/5.97 36.24/6.11 – 35.85/8.81 36.13/8.58 36.29/7.70 –

Anx repeatsc

PKC

Caesin

N-Myr

Amidation

cAMP-cGMP

Tyr

N-Glyc

Prediction by PSORT

4 4 4 1 4 3 2 1

6 6 5 – 5 4 7 5

8 9 9 3 6 8 3 5

1 2 2 2 3 1 2 2

– – – – – 1 2 –

3 2 – 1 – 1 1 –

– 3 1 – 1 2 – –

2 2 1 – 1 2 – –

Cyto, Mit, ER Cyto, Chlo Nucl, ER Cyto, Mic, Mito Cyto, Mic, Mit, ER ER, Chlo Cyto, Mic, Chlo Cyto, Mito, ER

The post-translational modifications such as PKC protein kinase C, phosphorylation sites caesin casein kinase II, N-Myr N-myristoylation, amidation amidation site, cAMPcGMP cAMP- and cGMP-kinase, Tyr tyrosine kinase, N-Glyc N-glycosylation were predicted at ScanProsite (http://ca.expasy.org/tools/scanprosite/); the post-translational modifications such as PKC protein kinase C, phosphorylation sites caesin casein kinase II, N-Myr N-myristoylation, amidation amidation site, cAMPcGMP cAMP- and cGMP-kinase,Tyr tyrosine kinase, N-Glyc N-Glycosylation were predicted at ScanProsite (http://ca.expasy.org/tools/scanprosite/). Location on Cyto cytoplasm, Mit mitochondrial matrix space, ER endoplasmic reticulum (membrane), Mic microbody, Nucl Nucleus, Chlo chloroplast were predicted at PSORT (http:// psort.hgc.jp/form.html). a Number of amino acids. b Molecular weight (KD) and isoelectric point. c Anx represents annexin repeat.

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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M. He et al. / Gene xxx (2015) xxx–xxx t2:1 t2:2

7

Table 2 Conserved motifs were identified by MEME 4.6.1/MAST motif search software.

t2:3

Number

Length(a)

Conserved motif

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13

1 2 3 4 5 6 7 8 9 10

50 41 41 41 25 29 21 21 15 46

GTDEWSLTRVIVTRAEIDMQKIKEEYQKRNSVPLDRAVAGDTSGDYKDML VPPPVPSPTQDCEQLRKAFQGWGTNEKMIISILGHRNAAQR WVLVEIACTRTPNQLFFVRQAYHSRFKCSLEEDVAAHTSGD HKQYNDDDFIRIFSTRSKPQLNATFNHYNNMYGHSINKDLK ETYNEDLIKRLHSELSGDFERAVML DEFMSLLRTVIWCITCPEKYFAKVLRNSM WTLDPAERDAVLANEATKMWT FRKLLVPLVSSYRYEGDEVNM MLAQCEAKILHEKIN TDKRMVTRAILGSDDVGMDEIRSVFKSSYGKNLADFIQENLPQGDY

Note: number 1–10 in line with number showed in Fig. 2. Conserved motifs were not listed on order.

422

439

AnnAh3 at 24 h was far above level of control. The transcript level of AnnAh3 increased from 2 h to 12 h relative to untreated control. Under drought stress with 10% PEG, the expression of AnnAhs in leaves showed different expression trend. The level of induction for AnnAh1 reached the highest level of transcript at 12 h. For AnnAh2, AnnAh3 and AnnAh7, the transcript increased consistently and reached the highest level of transcript at 2 h, whereas mild induction was observed in AnnAh6,all time points. Hormones play important roles in the growth, development and environmental adaptation of plants. Previous studies have shown that annexins can respond to different exogenous hormones. In our study, we examined the effects of SA and ABA on the expression of peanut annexin genes in leaves by real-time qRT-PCR (Fig. 5). AnnAh1, AnnAh3 and AnnAh6 were induced by ABA or SA obviously. Other AnnAhs showed slight inductions or no-induction on 2-24 h. These results showed that the transcript levels of annexin genes in peanut are differentially regulated by various stresses, implying certain AnnAh induced by distinct signaling pathways and revealing specific function.

440

3.7. Analysis of cis-element in the upstream region of AnnAhs

441 442

Cis-elements and the interaction with their corresponding transregulatory factors are important for gene expression involved stress responses. The 5′-upstream promoter regions of peanut annexin genes were isolated and characterized for identifying the potential cisregulatory elements using online software on PlantCARE and PLACE database. The result showed that at least 20 putative cis-elements can be identified on the upstream of transcription start site (+1). There were classical TATA-box, CAAT-box and a series of light responsive elements G-box (CACGTG), B-box4, SP1 etc., but some other important regulating elements such as high level transcription regulatory factors 5′UTR pyrimidine enrichment region (GTTTCTTTTCT), abscisic acid responsiveness element ABRE(TACGTG), salicylic acid responsiveness TCAelement (CCATCTTTTT), anaerobic induction cis-regulatory element ARE (TGGTTT), MYB binding site MBS (CAACTG) for drought response,

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Fig. 4. Expression profiling of AnnAhs in four organs. UBI was as the endogenous reference gene. The x-axis represents different organs. The y-axis represents the relative expression levels of AnnAhs. Results are normalized to annexin levels in roots (value of 1). Error bars represent standard error of the mean for three replicates.

455

4. Discussion and conclusions

463

With more completions of the genome sequencing and functional genomics research in different species, the numbers of annexin genes identified recently increased. Genome sequencing had revealed ten annexin genes in rice (Cantero et al., 2006), ten in tomato (Lu et al., 2012), and eight in Arabidopsis (Jami et al., 2012a), twelve in maize (Zhou et al., 2013) and at least five in Indian mustard (Jami et al., 2009). In the present study, we have cloned and characterized six fulllength and two partial-length genes encoding annexins in peanut (AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6, AnnAh7, AnnAh4 and AnnAh8), which were homologous to other higher plant annexins. Based on the results of phylogenetic analysis (Fig. 2), peanut has a close genetic relationship with soybean and alfalfa, which were are both leguminous. Currently, since tetraploid peanut genomes have not been sequenced, we resorted to using their ancestral diploid genome sequenced A and B (http://www.peanutbase.org/). In our study, sequence alignments had been conducted using all the annexins that had been noted online in soybean (in Plaza), alfalfa and AnnAhs cloned with the published sequencing results of peanut genome A and genome B. The results showed that the annexins localizations of peanut, soybean and alfalfa on the peanut genome were highly consistent, and so did the different members of the same subtribe with soybean (data not shown). Moreover, according to the published sequencing results of peanut genome A and genome B, AnnAh genomic sequences we obtained by amplifying DNA just had tiny differences on intron sequence. These results indicated that we obtained the majority of the members of annexin gene family. Increased annexin gene number may be due to gene duplication events, which can be seen from amino acid sequence similarities and their genome locations (Clark et al., 2001; Mortimer et al., 2008). Duplication events have also been found in the O. sativa and Arabidopsis annexin genes (Jami et al., 2012a). In our study, AnnAh1 had sequence identities of 32%–100% at nucleotide level distributed in different chromosomes, which may be attributed to the same group just like soybean had two or more annexin genes in the same group. These genes had high homology at amino acid level. Along with the publication of peanut sequencing result, we will explore more information with AnnAh family and understand their mechanism in depth. The expression of annexin was dynamic in the whole life and is expressed in most of tissues of higher plant, such as embryo, roots, stems, leaves, inflorescence and fruit (Kovács et al., 1998; Hofmann et al., 2000; Clark et al., 2001; Cantero et al., 2006). In this study, the results of tissue specific expression for AnnAhs showed that AnnAhs

464

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low temperature responsive element LTR (CCGAAA), ethyleneresponsive element ERE (ATTTCAAA), GA responsive element P-box (GCCTTTTGAGT), cis-acting element involved in heat stress responsiveness HSE (AAAAAATTTC) and defense and stress responsiveness element TC-rich repeats (ATTTTCTTCA), can only be found in certain genes, which suggested potential nonredundant functions in vivo. Taken together, these data suggested the very complex regulatory patterns of the annexin family genes.

E

437 438

T

435 436

C

433 434

E

431 432

R

429 430

R

427 428

N C O

425 426

U

423 424

F

t2:14

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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Relative expression

2.5 CK

2

Cold NaCl

1.5

CdCl2 1

Drought SA

0.5

ABA

10

Cold 6

NaCl CdCl2

4

Drought SA

2

ABA

0 10h

12h

24h

2h

AnnAh3

6h

8h

6 5

CK

Cold

4

NaCl 3

CdCl2

2

Drought SA

1

4

3 2

12h

24h

CK

Cold NaCl CdCl2 Drought

E

ABA

5

1

10h

AnnAh5

6

Relative expression

7

4h

O

8h

R O

6h

P

4h

D

2h

Relative expression

CK

8

0

SA ABA

0

0 4h

6h

8h

10h

12h

24h

C

AnnAh6

9

T

2h

8

E

7

R

6 5

3

1 0

Drought SA

4h

6h

8h

ABA

2h

4h

6h

8h

10h

12h

24h

AnnAh7

20

18 16 14

CK

12

Cold

10

NaCl

8

CdCl2

6

Drought

4

SA

2

ABA

0 10h

12h

24h

2h

4h

6h

8h

10h

12h

24h

N

2h

Cold

CdCl2

O

2

CK

NaCl

R

4

C

Relative expression

AnnAh2

Relative expression

Relative expression

12

AnnAh1

3

F

8

U

Fig. 5. AnnAhs levels under different stresses. Results are shown comparing annexin transcript levels under stress conditions as compared to untreated controls. Peanut seedlings grown for v-2 on MS medium were treated with solutions containing 250 mM sodium chloride (NaCl), 150 μM cadmium chloride (CdCl2), 10% PEG6000, 100 μM abscisic acid (ABA), 100 μM salicylic acid (SA) and keep in 15 °C to make the leaves experience low temperature stress. Samples collected 0 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h. Samples treated by NaCl used YLS8 as the endogenous reference gene for mRNA normalization. Control samples and treated samples by 10% PEG6000 were analyzed using ELF1B as the endogenous reference gene for mRNA normalization. Samples treated by CdCl2 and 15 °C used UBI as the endogenous reference gene. ACTIN7 was used as endogenous reference in analyzed samples treated by ABA and SA. Data is presented as the fold change in transcript levels as normalized to Control untreated (value of 1). Data at 0 h is omitted for visual simplicity. Error bars are the standard deviations of three replicates.

505 506 507 508 509 510 511 512 513 514

(AnnAh1, AnnAh2, AnnAh3, AnnAh5, AnnAh6 and AnnAh7) were constitutive expression in peanut with different levels, and the transcript abundance of AnnAh6 was highest in root, and AnnAh1, AnnAh2, AnnAh7 were in stems and AnnAh3 and AnnAh5 were in flowers. The transcript levels of peanut annexin genes vary in different tissues, suggesting specific functions at different developmental stages in different peanut tissues (Cantero et al., 2006; Vandeputte et al., 2007; Vellosillo et al., 2007). Analysis of ANNAh amino acid sequences revealed that they belong to annexin family and have precious annexin conservative area. As calcium residues in the endonexin sequence are important in binding

membranes of the lipid in the convex side, the variable presence or absence of calcium residues in their corresponding annexin repeats might result in different protein conformations and different specificities for binding phospholipids. Function prediction showed that annexin proteins have typical KGXGT-38-D/E Ca2+ binding sites and His40 residues and conserved tryptophan required for membrane binding (Mortimer et al., 2008). Recent studies suggested this His residue was significant for maintaining the secondary structure (Konopka-Postupolska et al., 2009). Six peanut annexins including conserved salt bridges that are hypothesized to involve in the channel function which is also exist in

Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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Conflict of interest

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of other important eukaryotes cis-regulatory elements on the upstream of transcription start site in addition to some classical structure like TATA-motif and CAAT-motif. There were low temperature responsive element LTR (CCGAAA), which is reported to take a significant role in the regulation of barley gene blt4.9 under low temperature stress (Dunn et al., 1998); MYB binding site MBS (CAACTG) for drought response, which is necessary to the promoter of drought resistance gene in most of the researches (Yamaguchi-Shinozaki and Shinozaki, 1994); anaerobic induction cis-regulatory element ARE (TGGTTT) (Riou et al., 2002); GA responsive element P-box (GCCTTTTGAGT) and some light responsive elements such as G-box (CACGTG) and B-box4 (Mena et al., 2002; Zou et al., 2008). It has been reported that the combination of G-box and OsABI5 structures can raise the plant resistance to high salt (Millar and Kay, 1996). The existence of those elements may indicate that peanut responds to the environment stresses like low temperature, drought, anaerobic through regulating the expression of AnnAhs by those structures. It is difficult to deduce the expression properties of a certain gene by only depending on cis element analysis exactly. For example, both putative SA- and ABA-responsive elements can be found in most peanut annexin genes promoter region. However, in the current work, only AnnAh1, AnnAh3 and AnnAh6 responded to ABA and SA (Fig. 5). There was also LTR element existing in some AnnAhs, but just AnnAh3 had a obvious expression under cold treatment, which is inconformity with the results (Lu et al., 2012). Development and environment also control the expression of annexins (Mortimer et al., 2008) Under certain conditions, gene expression was regulated through specific signal molecules or trans-regulatory factors interacting with specific cis elements (Lu et al., 2012). In summary, eight annexin family members in peanut have been identified and characterized in our study. Their sequence characteristics, genomic structures and expression patterns were analyzed. These dates would provide fundamental knowledge and useful information for further confirming the functions of AnnAhs family and molecular breeding in peanut.

E

538 539

T

536 537

C

534 535

E

532 533

R

531

R

529 530

N C O

527 528

animal annexins (Laohavisit and Davies, 2011). The S3 cluster was found in cotton annexin ANNGh1 and also present in ANNAt1 from Arabidopsis (Hofmann et al., 2003; Konopka-Postupolska et al., 2009). The Cys residues in the S3 cluster in certain annexins may serve as target sites for modification by ROS (Konopka-Postupolska et al., 2009). All six peanut annexins (except for ANNAh7) have these two Cys residues. In addition, ANNAhs have a series of metabolism related structures such as protein phosphorylation binding sites and nitrosylation binding site. Protein phosphorylation and dephosphorylation were significant in stress signal recognition, conduction and processes of photosynthesis, cell growth, gene expression and even cancer (Stone and Walker, 1995; Schenk and Snaar-Jagalska, 1999). Studies have been proved that the processes of stimulus transformation from extracellular into intracellular were relevant to protein phosphorylation (Schenk and SnaarJagalska, 1999). Protein kinase also widely participated in the response to drought, high salt, ABA induction, light induced stress, and so on. The multiple comparison of amino acid sequences showed that annexin of peanut have close genetic relationships to annexin of leguminous crops such as soybean and alfalfa, with a high similarity to annexin gene family in Arabidopsis, suggesting that the fundamental plant annexin properties can be evolutionarily conserved (Lu et al., 2012). The high homology among these species displayed in phylogenetically tree indicated that AnnAhs may have the similar function of response to abiotic stress such as water shortage, osmotic stress and anaerobic environment as it has been reported in mustard (Jami et al., 2009) and Arabidopsis (Clark et al., 2001; Cantero et al., 2006). In identical group, leguminous annexin genomes including A. hypogaea, G. max and M. truncatula had similar organization (Fig. 1). The positions of introns in each group and their phases (AnnAh7 and GM13G26040 had a small difference) with symmetric exons are well conserved suggesting that these annexin genes from the leguminous plants even land plants might have a common ancestor (Jami et al., 2012a). The conserved intron phases in the gene structure may have provided stability during evolution similar to that observed in vertebrate annexins (Fernandez and Morgan, 2003). Annexin, always be considered as a kind of polygenes and multifunctional protein. Different members of annexin genes has different change trend in different stress treatment. 8 members of annexin in Arabidopsis showed different expression pattern and most of genes were induced by salinity, drought, ABA and high or low temperature (Cantero et al., 2006). Together, one study found the expression patterns of AnnBj genes in mustard were regulated by various stress conditions such as exposure to signaling molecules, salinity and oxidative stress and wounding (Jami et al., 2009). Recent research showed that the expressions of 9 tomato annexin genes were adjusted by development course and environment stimulation and most of these genes were induced by salinity, drought, wounding and hot and cold stress (Lu et al., 2012). Expressions of 12 annexin genes of maize based on maize genomic sequence were revealed that these genes were response to heavy metal stress, such as Ni, Zn and Cd (Zhou et al., 2013). In our study, the correlation between AnnAhs expression and abiotic stress (PEG, NaCl, Cold and CdCl2) and hormones (ABA and SA) at the seedling stage were observed through Real-Time PCR strategy (Fig. 5). Most peanut annexin genes were induced by single or several stresses. AnnAh1, AnnAh3, AnnAh5 and AnnAh6 induced by cold and AnnAh3 was in high expression particularly; under drought stress with 10% PEG, the expression of AnnAhs in leaves showed different expression trends. AnnAh1 increased after treated 2 h, and reached the highest level of transcript at 12 h. For AnnAh2, AnnAh3 and AnnAh7 reached the highest level of transcript at 2 h. In our study, we examined the effects of SA and ABA on the expression of peanut annexin genes in leaves by real-time qRT-PCR (Fig. 5). AnnAh1, AnnAh3 and AnnAh6 were induced by ABA or SA obviously. Other AnnAhs showed slight inductions or no-induction on 2-24 h. These results indicated that peanut AnnAhs involved in stress response. The promoter sequences of peanut AnnAhs were obtained through TAIL-PCR. The online function analysis showed that there were a series

U

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9

The authors declare no conflict of interest.

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Authors' contributions

628

LFL designed the study and wrote the manuscript. MJH carried out the experiments, analyzed data and wrote the manuscript; XLY and SLC carried out the field experiments for screening material; GJM, MYH and HCY assisted in writing the manuscript.

629

Acknowledgments

633

We thank Dr. Charles Y. Chen from Auburn University for review and comment. This work was financially sponsored by National Natural Science Foundation of China (No. 31471523), Grant of 948 Project (2013-Z65) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No.2012302110002).

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Appendix A. Supplementary data

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osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell Online 16, 1378–1391. Lim, E.-K., Roberts, M.R., Bowles, D.J., 1998. Biochemical characterization of tomato annexin p35 independence of calcium binding and phosphatase activities. J. Biol. Chem. 273, 34920–34925. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408. Lu, Y., Ouyang, B., Zhang, J., Wang, T., Lu, C., Han, Q., Zhao, S., Ye, Z., Li, H., 2012. Genomic organization, phylogenetic comparison and expression profiles of annexin gene family in tomato (Solanum lycopersicum). Gene 499, 14–24. Mena, M., Cejudo, F.J., Isabel-Lamoneda, I., Carbonero, P., 2002. A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone. Plant Physiol. 130, 111–119. Millar, A.J., Kay, S.A., 1996. 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Please cite this article as: He, M., et al., Molecular cloning and characterization of annexin genes in peanut (Arachis hypogaea L.), Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.004

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