Molecular Cloning and Expression Analysis of an ANS Gene

发布时间:2021-10-18 14:32:34

Plant Mol Biol Rep (2010) 28:112–121 DOI 10.1007/s11105-009-0133-0

Molecular Cloning and Expression Analysis of an ANS Gene Encoding Anthocyanidin Synthase from Purple-Fleshed Sweet Potato [Ipomoea batatas (L.) Lam]
Wei Zhou & Chengtao Huang & Yifu Gong & Qili Feng & Feng Gao

Published online: 12 August 2009 # Springer-Verlag 2009

Abstract Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of anthocyanin. This reaction is responsible for the formation of the colored anthocyanidins from the colorless leucoanthocyanidins. A full-length cDNA was isolated from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) cv. Yamakawamurasaki, designated IbANS, containing a 1,086-bp open reading frame encoding a 362-amino-acid polypeptide. Multiple alignments revealed that the deduced IbANS protein had high identity to ANS proteins of other plants such as Ipomoea nil (90.8% identities), Ipomoea purpurea (91.4% identities), and Brassica juncea (72.7% identities). Structural analysis showed that the IbANS protein might belong to the 2OG and Fe(II)-dependent oxygenase, containing three binding sites of 2OG (H236, D238, and H292) and three binding sites of Fe(II) (Y221, R302, and S304). Phylogenetic tree analysis revealed that IbANS shared the close relationships with I. nil and I. purpurea. Southern blotting showed that there were two copies of the IbANS gene in this genome. Real-time quantitative polymerase chain reaction revealed that expression of the IbANS gene was highest in storage roots and lowest in leaves. IbANS was expressed most abundantly during the formation of
W. Zhou and C. Huang contributed equally to this work. W. Zhou : C. Huang : Y. Gong : Q. Feng : F. Gao (*) Guangdong Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, People’s Republic of China e-mail: peak0041@vip.sina.com Y. Gong Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, People’s Republic of China

storage roots. In five cultivars of sweet potato, IbANS expression was strongly associated with anthocyanin accumulation, suggesting that ANS gene expression was associated with activation of anthocyanin biosynthesis. Keywords Purple-fleshed sweet potato . Ipomoea batatas . Anthocyanidin synthase . Cloning Abbreviations ORF open reading frame RACE rapid amplification of cDNA ends UTR untranslated region RT-PCR reverse transcription polymerase chain reaction

Introduction Purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) accumulates a large amount of anthocyanins in its storage roots (SR). Anthocyanin pigments and the related oligomeric proanthocyanidins are receiving more intensive interests because they play roles in pigmentation and act as signaling molecules (Bohm 1998) and have been reported to possess a variety of biomedicinal properties, including strong antioxidative activity (Suda et al. 1997; Kano et al. 2005), antimutagenicity (Yoshimoto et al. 1999), antihyperglycemic (Matsui et al. 2002), hepatoprotective, and antihypertensive effects (Suda et al. 2002). The anthocyanins biosynthesis pathway has been extensively characterized in terms of genetics, biochemistry, and molecular biology. Anthocyanidin synthase (ANS), a 2oxoglutarate (2OG) iron-dependent oxygenase, catalyzes the modification reaction of the central C-ring, and this reaction is responsible for the formation of the colored

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anthocyanidins from the colorless leucoanthocyanidins (Saito et al. 1999; Welford et al. 2005), which is the penultimate step in the biosynthesis of anthocyanin and is thought to be a essential biosynthesis step mediated solely by nonheme oxygenases (Heller and Forkmann 1994). Genes and cDNAs encoding ANS have been isolated from a number of plant species including Arabidopsis thaliana, Perilla frutescens, Spinacia oleracea, Phytolacca americana, and other plants (Wilmouth et al. 2002; Saito et al. 1999; Shimada et al. 2005; Wellmann et al. 2006; Shih et al. 2008). The research results imply that the expression rate of the ANS gene corresponds to the accumulation pattern of anthocyanin and the ANS gene might be the committed step of the anthocyanins biosynthesis pathway (Saito et al. 1999; Shimada et al. 2005; Gong et al. 1997). As we know, SR, derived from different purple-fleshed sweet potato cultivars, accumulate anthocyanin pattern that corresponds to the pigmentation phenotype. Therefore, it is a very important research item to know the gene structure and expression profile of ANS gene from purple-fleshed sweet potato, which produces colored SR under the soil. It is the basis of directional breeding of purple-fleshed sweet potato on the level of gene engineering. In this paper, the cloning and characterization of the ANS cDNA from the purple-fleshed sweet potato cultivar Yamakawamurasaki were reported. The expression profiles of IbANS in various tissues and cultivars and the copy number of the IbANS gene were investigated. It is suggested that ANS gene expression may activate anthocyanin biosynthesis in purple-fleshed sweet potato.

was isolated from leaves of 1-month-old seedlings using the modified cetyltrimethylammonium bromide method (Kim and Hamada 2005) for Southern blot analysis. The quality and concentration of RNA and DNA samples were examined by ethidium bromide-stained agarose gel electrophoresis and spectrophotometer analysis. Isolation of the IbANS Full-Length cDNA by RACE To isolate a conserved sequence of the IbANS gene, a pair of oligonucleotide primers ANSF1 (5′-CGCATCCCGAAG GAGTACATAA-3′) and ANSR1 (5′-AACCATAAT AATA CCAAACGGACT-3′) were designed and synthesized according to the conserved sequence of ANS shared by other species. RT-PCR was carried out to amplify the conserved fragment of the ANS gene from I. batatas using the OneStep RNA PCR Kit (Takara, Japan) as per recommended reaction. The amplified product was purified, ligated into the pMD19-T vector, and cloned into Escherichia coli strain DH5α followed by sequencing from both strands. BLAST analysis was conducted to confirm the conserved fragment homology to other plant ANS genes. This fragment was subsequently used for designing specific primers for cloning of the 5′ end and 3′ end of cDNA of IbANS by RACE. The SMART? RACE cDNA Amplification Kit (Clontech, USA) and 3′-RACE System for Rapid Amplification of cDNA Ends (Invitrogen, USA) were used to synthesize the 5′ end and 3′ end of cDNA of IbANS. The first-strand 5′-RACE-ready and 3′-RACE-ready cDNA samples from I. batatas were prepared according to the manufacturer's protocol and used as templates for 5′-RACE and 3′-RACE, respectively. The 5′ end of cDNA of IbANS was amplified using two 5′ end genespecific primers (GSP) and the universal primers provided by the kit. For the first PCR amplification, ANSR1 and universal primer A mix (provided by the manufacturer) were used as the primers and 5′-RACE-ready cDNA as the template. For the nested PCR amplification, ANSR2 (5′-CGCCTGGT CATTAGCATACTTC-3′) and nested universal primer A (provided by the manufacturer) were used as the nested PCR primers, while the dilution products of the first PCR amplification were used as templates. The first-round PCR for 3′-RACE was performed with ANSF1 and universal adapter primer (provided by the manufacturer).The PCR products were diluted and used as template for the nested PCR with ANSF2 (5′-AGGGGTTGTGAACAGGGAGAA GGTG-3′) and abridged universal anchor primer (provided by the manufacturer). All the reactions were carried out as per manufacturer's recommendation and the nested 5′-RACE and 3′-RACE products were purified, ligated into the pMD19-T vector, and cloned into E. coli strain DH5α, followed by sequencing for confirmation from both strands. To amplify the full-length cDNA of IbANS, two GSP, ANSFull-F1 and ANSFull-F2, were synthesized following

Materials and Methods Plant Materials and Treatments Four cultivars of purple-fleshed sweet potato (I. batatas (L.) Lam) cv. A5, A1, Yamakawamurasaki, and A3 and one of white-fleshed sweet potato cv. Yubeibai were obtained from Guangdong Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, People's Republic of China. The SR of purple-fleshed sweet potato cv. Yamakawamurasaki was used as material for isolating the full cDNA of IbANS. Isolation of RNA and DNA For real-time reverse transcription polymerase chain reaction (RT-PCR) analysis, 3′-rapid amplification of cDNA ends (RACE), and 5′-RACE, total RNA was extracted from different tissues using Trizol reagent (Invitrogen, USA) and treated with DNase I (Promega, USA) according to the protocol suggested by the manufacturer. Genomic DNA

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the assembling result of 5′-RACE and 3′-RACE. The RTPCR amplification was carried out following the protocol of One-Step RNA PCR Kit (TaKaRa, Japan). ANSFull-F1 (5′ACGCGGG GGAATGAAAATCAA-3′) and adapter primer ( A P ; 5 ′ - G G CC A C G C G TC G A C T A G TA C TT T T T TTTTTTTTTTTT-3′) with a poly(T) tail were used as RTPCR primers and 1 ?g total RNA as template. The one-roundamplified products were diluted and used as template of the nested PCR in which ANSFull-F2 (5′-GGGAATGAAAAT CAAGAGATAATATA-3′) and AP were used as the nested primers. The amplified products were purified, ligated into the pMD19-T vector, and cloned into E. coli strain DH5α, followed by sequencing for confirmation from both strands. Bioinformatics Analysis The nucleotide sequence, deduced amino acid sequence, and open reading frame (ORF) were analyzed online (http://www. ncbi.nlm.nih.gov). Structural analysis of deduced protein was carried out at the website of Expasy Molecular Biology Server (http://cn.expasy.org/tools/). Homology-based structural modeling was performed by Swiss-Model (Schwede et al. 2003). WebLab ViewerLite was used for threedimensional model display. The software vector NT1 TM Suit 9.0 was used for sequences multialignment. A phylogenetic tree was constructed using neighbor-joining method (Saitou and Nei 1987) and the MEGA version 3.1 software (Kumar et al. 2001). The reliability of the tree was measured by bootstrap analysis with 1,000 replicates (Felsenstein 1992). Southern Blot Analysis Aliquots of DNA (20 ?g/sample) were digested respectively with EcoRI, BglII, and BamHI (Takara, Japan), which did not cut within the full-length genomic region. Fully digested samples were fractionated on 0.8% agarose gel electrophoresis, denatured, and transferred onto a positively charged Hybond-N+ nylon membrane (Amersham Pharmacia, England). Using a pair of special premier RTANSF3 (5′-CCTCACCTTCATCCTCCACAACA-3′) and ANSR1 (5′-AACCATAATAATACCAAACGGACT-3′), a 498-bp fragment was amplified using 3′-RACE-ready to use cDNA as template. DNA probe was random premier-labeled with Digoxigenin-11-dUTP (DIG DNA Labeling and Detection Kit, Roche) using the PCR product as template. DNA gel blot hybridization (DIG Easy Hyb, Roche) was performed at 42°C for 16 h. The hybridized signals were visualized by exposure to Fuji X-ray film at room temperature for 1 h. Expression Profile of IbANS by Real-Time Quantitative PCR Total RNA, isolated from different tissues including leaves, stems, fibrous roots (FR), thick roots (TR), and

SR (derived from five cultivars of sweet potato: A5, A1, Yamakawamurasaki, A3, and Yubeibai), were subjected to produce cDNA by oligo(dT)18 primer using the First-Strand cDNA Synthesis Kit (TakaRa, Japan) according to the protocol suggested by the manufacturer. Gene expression analysis was measured by real-time PCR, using the SYBR green method on a 7300 real-time cycler (AB Applied Biosystems, USA). Each PCR reaction (20 ?L) contained the following components: 250 nM primer (each), cDNA (appropriate dilution), 1× SYBR Green ROX mix (AB Applied Biosystems, USA).The real-time quantitative PCR thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 95°C for 15 s, 60°C for 1 min for 40 cycles, followed by a melt cycle 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. The premiers RTANSF1111 (5′GACACGCCCAAACCTGATGAA-3′) and RTANSR1255 (5′-ATACCAAACGGACTCCACAAAGC-3′) were used for real-time PCR to amplify a 145-bp fragment of IbANS from different types of cDNA. A metallothionein-like protein gene (G14 gene), which exhibited constitutive expression pattern in all tissues of sweet potato and had no significant variation of gene expression level among tissues and stages analyzed (Chen et al. 2003, 2006), was chosen for normalization of gene expression. The premiers G14F (5′GTGCGGAAACTGCGACTGC-3′) and G14R (5′-CGTCC ATCTTGCTTCCCTTCCT-3′) were designed to amplify a 62-bp G14 gene fragment from different cDNA samples. The products of real-time quantitative PCR were confirmed by determining the melt curves for the products at the end of each run by analysis of the products using agarose gel electrophoresis and by sequencing. Quantification of the gene expression was done with the comparative Ct method (Bogs et al. 2006; Muller et al. 2002). Each data represents the average of three independent experiments. Anthocyanin Quantification Extraction and quantitation of anthocyanins was performed following the protocols of Mano et al. (2007). One milliliter of acidic methanol (1% HCl, w/v) was added to 0.3 g of fresh plant material. Samples were incubated for 18 h at room temperature under moderate shaking. After centrifugation (21,500×g at room temperature for 3 min), 0.4 ml of the supernatant was added to 0.6 ml of acidic methanol. Absorbance of the extracts at wavelengths of 530 and 657 nm (UV-2450 Spectrophotometer, SHIMADZU) was determined photometrically. Quantification of anthocyanins was performed using the following equation: QAnthocyanins = (A530 ? 0.25A657)× M?1 where QAnthocyanins is the concentration of anthocyanins, A530 and A657 are the absorptions at the indicated wavelengths, and M is the fresh weight (in grams) of the plant material used for extraction. Quantitative values of anthocyanin were calculated based on the

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absorbance data. Each data represents the average of three independent experiments. Error bars indicate the SD of the average of the anthocyanin content.

Results Isolation of the Full-Length cDNA of IbANS Gene Based on the alignment of the ANS sequences from other plants, a pair of primers ANSF1 and ANSR1 was designed to amplify a core fragment of the ANS gene. Agarose gel analysis showed a specific band of about 1160 bp in length, and the sequence analysis showed that it was highly homologous to ANSs from plant species. Thus, this fragment was then used to design the GSP for amplifying the 5′ and 3′ ends of cDNA by 5′-RACE and 3′-RACE. Nested amplification for the 5′ and 3′ ends of the IbANS gene resulted in a bright band of anticipated length. Through sequencing and BLAST-n analysis of the two fragments, 411 bp 5′ end and 429 bp 3′ end of cDNA sequences were obtained by 5′RACE and 3′-RACE, respectively. By assembling these two end sequences and the core fragment, a full-length cDNA sequence of IbANS was generated primarily. To confirm the integrity of the sequence, nested PCR was used to amplify the full-length cDNA of IbANS. In conclusion, an integrity cDNA sequence of IbANS was obtained, which was 1,352 bp in length (GenBank accession number FJ478179). Sequence Analysis of IbANS The full-length cDNA of IbANS possesses a 1,086-bp ORF, which encodes a polypeptide of 362 amino acids, with a calculated molecular mass of 40.45 kDa and a predicted isoelectric point of 5.27. In addition, a 45-bp 5′-UTR and a 221-bp 3′-UTR including a poly(A) tail exist in the isolated cDNA (Fig. 1). Homology search results in GenBank (NCBI) showed that IbANS shared high identities to other plant species of ANSs DNA and cDNA sequences, with the highest identity with Ipomoea nil (80.8% identities, accession number AB073919). The amino acid sequences multialignment (Fig. 2) showed that IbANS had high identities with I. nil (90.8% identities, 93.3% positives, AB073919), Ipomoea purpurea (91.4% identities, 93.8% positives, EU032613), Brassica juncea (72.7% identities, 81.3% positives, EU927147), Pyrus communis (76.3% identities, 83.2% positives, DQ230994), G. elegans (75.0% identities, 83.2% positives, AY256380), and A. thaliana (32.5% identities, 48.1% positives, AY093302). These high identities suggested that IbANS was structurally a member of the ANS family. Conserved domain search (Marchler-Bauer and Bryant 2004) revealed that IbANS belonged to the 2OG–Fe(II)-dependent oxygenase super

family, which was characterized by the presence of a 2OG– Fe(II)-Oxy conserved domain. SOPMA (Geourjon and Deléage 1995) prediction results indicated that the secondary structure of IbANS was mainly composed of α-helices (32.6%) and random coils (46.13%), while extended strands (17.13%) and β-turns (4.14%) contributed a little (Fig. 3a). The region between V66 and G207 is dominated by six α-helices, which are connected by random coils and extended strands. The homologybased three-dimensional structural modeling of IbANS was analyzed using Swiss-Modeling and displayed using WebLab ViewerLite (Fig. 3b). The protein has a large space in the binding sites of 2OG (H236, D238, and H292) (Matsuda et al. 1991; Shimada et al. 2005) and Fe (II) (Y221, R302, and S304 ) (Wilmouth et al. 2002), which is composed of extended strands connected by random coils. Phylogenetic Relationships of ANSs To evaluate the molecular evolutionary relationships of IbANS with ANSs from other species, a phylogenetic tree was constructed by the neighbor-joining method. As shown in Fig. 4, a phylogenetic tree analysis for the ANS genes classified them into many subfamilies and all the different plants studied were derived from a common ancestor in evolution. IbANS and ANSs from I. purpurea (EU032613) and I. nil (AB073919) clustered into the same subgroup, which got the farthest phylogenetic distance from other groups of ANSs from other species, including A. thaliana (AY093302) and G. elegans (AY256380). This dendrogram suggested that the IbANS protein might have a similar function to anthocyanidin synthase of Ipomoea plants such as I. purpurea and I. nil. Southern Blot Analysis To investigate whether IbANS belonged to a multigene family, 20 ?g per sample genomic DNA of I. batatas was digested with restriction endonucleases EcoRI, BglII, and BamHI, respectively. Southern blotting was performed using the DIGlabeling probe described above. EcoRI and BglII digestions both resulted in only a single band, but BamHI digestion yielded two bands (Fig. 5). Because all the three restriction enzymes (EcoRI, BglII, and BamHI) have no cutting site in the IbANS gene genomic DNA, it is suggested that the I. batatas genome may contain two ANS genes at least. Anthocyanin Accumulation and Expression Profile of IbANS in Different Tissues and Different Sweet Potato Cultivars Real-time quantitative PCR was subjected to examine the expression profile of IbANS. To investigate the expression

116 Fig. 1 The full-length cDNA and deduced amino acid sequences of I. batatas anthocyanidin synthase (IbANS). The deduced amino and the corresponding cDNA sequences are indicated in capital letter. The start codon (ATG), the stop codon (TGA), and the poly(A) tail are underlined

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patterns of IbANS during SR formation, we classified the adventitious roots into three categories: FR (maximum diameter <2 mm), TR (maximum diameter 2–5 mm), SR (maximum diameter >5 mm; Fig. 6a). Total RNAs were isolated from FR, TR, SR, leaf , and stems of sweet potato cv. Yamakawamurasaki. Results indicated that IbANS expressed constitutively in all examined tissues but most highly in SR. The expression levels of leaf and stem were

lowest, followed by FR and TR (Fig. 6). As shown in Fig. 6c, the pattern of different tissues that accumulate anthocyanin was consistent with the expression patterns of the IbANS gene during SR formation, which the SR accumulated the highest levels of anthocyanin, followed by TR, FR, stems, and leaf. The relationship between anthocyanin accumulation and IbANS gene expression was examined in four purple-

Plant Mol Biol Rep (2010) 28:112–121 Fig. 2 Multialignment of the amino acid sequences of anthocyanidin synthases (ANSs). Identical amino acids are indicated in white foreground and black background; conserved amino acids are indicated in black foreground and light gray background; block of similar amino acids are indicated in white foreground and gray background; nonsimilar amino acids are indicated with black foreground and white background. The aligned ANSs are from A. thaliana (accession number AY093302), G. elegans (accession number AY256380), I. batatas (accession number FJ47817), I. nil (accession number AB073919), I. purpurea (accession number EU032613), B. juncea (accession number EU927147), and P. communis (accession number DQ230994). The iron-binding residues (H236, D238, and H292) are indicated with an black triangle. Those involved in binding 2OG (Y221, R302, and R304) are shown with black dots

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fleshed cultivars and one white-fleshed cultivar (Fig. 7). Among various sweet potato cultivars, cv. A5 showed the highest levels of gene expression, followed by cv. A1, Yamakawamurasaki, A3, and the white-fleshed cultivar

Yubeibai. Among various sweet potato cultivars, cv. A5 accumulated the highest levels of anthocyanin, followed by cv. A1, Yamakawamurasaki, and A3, whereas the white-fleshed cultivar Yubeibai accumulated the lowest anthocyanin (Fig. 7c).

118 Fig. 3 The predicated secondary and tertiary structure of IbANS established by homology-based modeling. a The secondary structure of IbANS. α-helix and extended strand were denoted as vertical long bars and vertical short bars, respectively, with the horizontal line presenting the random coil running through the whole molecule. b The tertiary structure of IbANS. The iron-binding residues (H236, D238, and H292) and those involved in binding 2OG (Y221, R302, and R304) are indicated, respectively

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150 C-terminus Glu 353

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350

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Y221 H236 H292 R302 S304 D238

N-terminus Arg 10

The IbANS expression profile in different sweet potato cultivars corresponded to the pattern of anthocyanin accumulation.

Discussion In this paper, the full-length cDNA of IbANS was isolated, which is 1,352 bp in length and encodes a 362-amino-acid polypeptide. Nucleotide BLAST-n revealed that the cloned cDNA sequence of IbANS was most identical to ANS genes from other species and the deduced IbANS protein also
Fig. 4 Phylogenetic tree of IbANS and other plant ANSs generated by? the neighbor-joining method. The numbers at each node represent the bootstrap values (with 1,000 replicates). The ANSs used in phylogenetic tree analysis were from plants including A. thaliana (accession number AY093302), G. elegans (accession number AY256380), I. batatas (accession number FJ478179), I. nil (accession number AB073919), I. purpurea (accession number EU032613), B. juncea (accession number EU927147), P. communis (accession number DQ230994), V. vinifera (accession number EF192468), G. hybrid cultivar Tacora (accession number AY997840), B. oleracea (accession number AY228485), Fragaria x ananassa (accession number AY695818), G. hirsutum (accession number EF187442), M. domestica (accession number AF117269), S. scutellarioides (accession number EF522157), T. fournieri (accession number AB044091), and A. andraeanum (accession number EF079869)

showed high identity to ANS proteins from other plant species via multialignments. Bioinformatics analysis showed that the IbANS protein might belong to the 2OG and Fe(II)-dependent oxygenase super family, which was thought to catalyze the formation of the plant hormone ethylene by oxidative desaturation of 1-aminocyclopropane-1-carboxylate and the hydroxylation (ACC) and desaturation steps in the synthesis of other plant hormones, pigments, and metabolites such as

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BamHI

Eco RI

23.1 kb 9.4 kb 6.5 kb 4.3 kb 2.3 kb 2.0 kb 1.0 kb

Flavonoid biosynthesis in dicot plants is controlled by at least two separate gene subsets. Chalcone synthase is involved in early biosynthetic genes, which are responsible for upstream steps in the flavonoid biosynthetic pathway. Anthocyanidin synthase and dihydroflavonol 4-reductase, in contrast, are responsible for the downstream steps that produce anthocyanins and proanthocyanidins and are thus called late biosynthetic genes (Koes et al. 2005; Quattrocchio

BglII

a

b

0.5 kb
Fig. 5 DNA gel blot hybridization of the IbANS gene. Genomic DNA was digested with restriction enzymes (EcoRI, BglII, and BamHI) and detected with the digoxigenin-labeled ANS probe

a

b
gibberellins, anthocyanidins, and flavones (Lukacin and Britsch 1997; Zhang et al. 1997), and was characterized by the presence of a 2OG–Fe(II)–Oxy conserved domain (Turnbull et al. 2004). Similar to other members of the super family, IbANS contains three active sites of His236, Asp238, and His292, which are believed to be the ironbinding resides, and the other three active 2OG binding sites of Tyr221, Arg302, and Ser304 (Wilmouth et al. 2002; Welford et al. 2005). The sequence similarities between ANSs and the other flavonoid 2OG-dependent dioxygenases imply close structural relationships and functions. IbANS has the most close relationship with the same genus plants of I. nil and I. purpurea and the furthest evolution distance with A. thaliana in taxonomic system. The studies of the ANSs phylogenetic tree in detail can help to visualize how evolution produced what we see today and to track down genes from the past to the present. Southern blotting showed that ANS was present as a multiple copy (at least two copies) in the sweet potato genome. This is different to A. thaliana and grape being a single copy of ANS, which is responsible for both anthocyanin and proanthocyanidin synthesis (Boss et al. 1996; Pelletier et al. 1997). The fact that different ANS protein isomers can be located in separate subcellular compartment and responded to produce colored cyanidin in different developmental and environmental signals with varying substrate specificities in sweet potato needs to be further studied.

b c c

c
ab a

b c c

Fig. 6 Anthocyanin accumulation and gene expression of IbANS in different tissues of I. batatas. a The classified adventitious roots and different tissues including FR (maximum diameter <2 mm), TR (2– 5 mm), SR (>5 mm), stems (ST), and leaf (LF). b Real-time quantitative PCR analysis of IbANS gene. c Anthocyanin contents in different tissues of purple-fleshed sweet potato cv. Yamakawamurasaki were determined photometrically. Bars with the same lowercase letter are not significantly different (P >0.05). Each data was presented as the means ± SD from three replicates

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b
20 relative IbANS expression 16 12

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8 4

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5 1 3 i as am Y ub ur ei ba ak A A A i

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10 9 8 7 6 5 4 3 2 1 0

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et al. 1993, 1998). The fact that the mRNA expression profiles of IbANS correspond to the regulation of anthocyanin accumulation imply that ANS may be a key enzyme gene responding for anthocyanin production. Expression profiles revealed that, in spinach, ANS was not expressed in most tissues and organs except seeds in which ANS may contribute to proanthocyanidin synthesis and the lack of anthocyanins in the seeds of Caryophyllales (Shimada et al. 2005). In the SR of purple-fleshed sweet potato, A5 accumulates 18-fold of anthocyanidin content more than A1, while the accumulation of anthocyanidin is not detected in the SR of the white-fleshed cultivar Yubeibai. More evidences are needed to confirm the direct relationship of ANS with anthocyanidin accumulation in SR, for example, the difference of the ANS gene active sites, the content of protein enzyme, and the catalytical ability of enzyme (Shimada et al. 2005) in different sweet potato cultivars. So, we constructed the recombinant expression vector pPROEXTM HTa-IbANS and have obtained the recombinant protein in E. coli DH5α. Next, we will prepare the antibody and pay attention to the protein level of IbANS and construct RNA interference expression vector for Agrobacterium tumefaciens-mediated transformation in sweet potato to investigate the relationships between IbANS gene expression and anthocyanidin accumulation in SR. In conclusion, the cloning of IbANS enables us to investigate its role in depth in the biosynthesis of anthocyanin by overexpressing or silencing it in sweet potato in the near future. Furthermore, it is a basis work for molecular directional breeding of sweet potato.
Acknowledgements This work was funded by the China National Natural Science Foundation (30671320) and Guangdong Natural Science Foundation (05005945).

Y

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References
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Fig. 7 Anthocyanin accumulation and gene expression of IbANS in different cultivars of sweet potato. a Cross-sectional appearance of flesh of the mature tuberous roots of the five sweet potato cultivars. b Expression of the IbANS gene in the tuberous roots of the sweet potato cultivars detected with real-time quantitative PCR. c Anthocyanin contents in the mature tuberous roots of each cultivar were determined photometrically. Bars with the same lowercase letter are not significantly different (P >0.05). Each data was presented as the means ± SD from three replicates

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