Molecular Biology Reports

, Volume 36, Issue 1, pp 207–214

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Authors

  • Tianyun Wang
    • Laboratory for Cell BiologyZhengzhou University
    • Department of Biochemistry and Molecular BiologyXinxiang Medical University
    • Laboratory for Cell BiologyZhengzhou University
  • Xiang Ji
    • Laboratory for Cell BiologyZhengzhou University
  • Jie Li
    • Laboratory for Cell BiologyZhengzhou University
  • Yafeng Wang
    • Laboratory for Cell BiologyZhengzhou University
  • Yingcai Feng
    • Laboratory for Cell BiologyZhengzhou University
Article

DOI: 10.1007/s11033-007-9168-1

Cite this article as:
Wang, T., Xue, L., Ji, X. et al. Mol Biol Rep (2009) 36: 207. doi:10.1007/s11033-007-9168-1
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Abstract

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca2+-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.

Keywords

14-3-3 ProteinDunaliella salinaMolecular evolution

Abbreviations

ORF

Open reading frame

RACE

Rapid amplification of cDNA ends

RT-PCR

Reverse transcriptase-polymerase chain reaction

Introduction

14-3-3 proteins have been found in many eukaryotic organisms including higher plants, amphibians, insects, yeast and microalga Chlamydomonas since they were discovered in brain proteins by Moore and Perez [13]. It has been demonstrated that the amino acid sequences of the 14-3-3 proteins are highly conserved [2, 4], and they not only are mainly cytoplasmic molecules, which can interact with over 200 target proteins by phosphoserine- dependent and independent manners [5], but also exist in some subcellular organelles such as microsomes, endoplasmic reticulum, dictyosome and plasma membrane, etc [6]. The important consequences of these interactions include alterations of enzymatic activity, and inhibition or promotion of protein interactions enhancing post-translational modifications such as phosphorylation. Obviously, 14-3-3 proteins may be involved in many different cellular processes including mitogenesis, cellular trafficking and transport, cell division, cell cycle control, apoptosis and oncogenesis, etc [79].

So far, cDNAs encoding polypeptides with a high degree of similarity to the 14-3-3 proteins have been cloned and sequenced from diverse eukaryotic organisms including amphibians, insects, yeast and higher plants [1, 4, 6, 1013]. The names of 14-3-3 proteins from different organisms were different, e.g. GF14 for higher plants, BMH1 for yeast, KCIP-1 for sheep. It has known that 14-3-3 proteins of vertebrate are encoded by at least seven different genes, which have nine isomerides α, β, γ, δ, ε, η, ζ, σ and τ, and among them α and σ are the phosphorylation form of β and ζ, respectively [10, 14]; Meanwhile, 14-3-3 protein isomerides in Arabidopsis are a largest family of 14-3-3 proteins including 10 kinds: GF14Ψ, GF14χ, GF14φ, GF14υ, GF14μ, GF14ω, GF14κ, GF14ν, RCI2 and GF14 [15], of which five have been identified: GRF1χ–GF14χ, GRF2ω–GF14ω, GRF3ν–GF14ν, GRFφ–GF14φ, GRF5ν–GF14ν [16]. 14-3-3 proteins of higher plants have been classified into two isoforms, -like and non- like. The 14-3-3 protein cDNA fragment from Chlamydomonas reihardtii was first cloned by Liebich and Voigt [1], which is 1464 bp in length and encodes 259 amino acids; additionally, four 14-3-3 proteins of C. reihardtii were found to differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane [2, 17, 18].

However, studies on 14-3-3 proteins of D. salina, a halotolerant microalga, have not been reported so far. Here we identify a 1485 bp of complete cDNA sequence of the D. salina 14-3-3 gene, which contains a 774 bp of ORF encoding a polypeptide of 258 amino acids.

Materials and methods

Alga strain and culture

D. salina UTEX-LB-1644 was purchased from the Culture Collection of Algae at the University of Texas at Austin, USA and grown in medium modified by Ben-Amotz and Avron (1989) comprising 2 M NaCl, 50 mM NaHCO3, 5 mM KNO3, 0.2 mM KH2PO4, 6 μM EDTA, 2 μM FeCl3, 5 mM MgSO4, 0.2 mM CaCl2, 7 μM MnCl2, 1 μM ZnSO4, 1 μM \( {\text{Co(NO}}_{{\text{3}}} {\text{)}}_{{\text{2}}} \), 1 μM CuSO4, at 26°C and 4500 lux lit 12 h/day with continuously, gently shaking as described previously [19].

Cells of D. salina at logarithmic phase were transferred to a solid medium containing 0.8% agar, and cultured until single colonies appeared. Subsequently, one single colony was picked out and transferred to liquid medium mentioned above for further culture.

RNA isolation and cloning of the 14-3-3 cDNA

After incubation at 26°C for 7 days, cells of D. salina at logarithmic phase were collected by centrifugation at 4000 g for 5 min. RNA extraction was carried out using TRIzol Reagent according to the manufacturer’s instruction. The quality and concentration of RNA were examined by EB-stained agarose gel electrophoresis and spectrophotometry.

First of all, a group of 14-3-3 proteins and mRNA sequences from C. reinhardtii, Tobacco, Pisum sativum L., Arabidopsis, Homo sapiens and other organisms were multi-aligned on Vector NTI Suite 6 to design two degenerated homologous PCR primers P1 (5′-TCNGTNGCNTAYAARAAYGT-3′) and P2 (5′-YTGCATDAT NARNGTRCTRTC-3′), respectively, corresponding to their conserved amino acid sequences SVAYKN and DSTLIMQ. A 2.5 μg of total RNAs was reverse-transcribed into cDNA with a cDNA synthesis primer AP using an adaptor primer 5′-GACTCGAGTCGACATCG(T)16-3′. Subsequently PCR were carried out using the two degenerate primers mentioned above. The purified PCR products were ligated to pGEM-T Easy vector, followed by sequencing using T7/SP6 primers synthesized by Sangon Company, China.

3′- and 5′-RACE of the D. salina 14-3-3 gene

According to result of sequencing of the 14-3-3 cDNA fragment cloned above, three primers were designed and synthesized for 3’ RACE of the 14-3-3 gene. A 2.5 μg of total RNA was reverse-transcribed into cDNA as described above. The first PCR was carried out using the primer A 1 (5′-AAGATGGTGGAGAAGGAGCTGA-3′) and AP (5′-GACTCGAGTCGACATCG-3′) primers, the nested PCR was performed using A 2 primer (5′-ACAAGGACA GCACCCTTATCAT-3′) and AP primer mentioned above. According to the result of sequencing of the 3′-cDNA end, four primers were designed and synthesized for 5′-RACE of the 14-3-3 gene. A 5 μg of total RNAs extracted from D. salina was reversely transcribed using primer RT (5′-(P)CGCTGTGGGGATGAGGTGCTC-3′), followed by hybrid RNA was circulated according to the kit protocol. Primers A1 and S (A1: 5′-TCCAGCAT AGAGCAGAAGGA-3′; S: 5′-TGCGCCAATAACGTTTTGT-3′) were used for primary amplification using 5 μl circulated cDNA as template. After confirmation by electrophoresis on agarose gels, the PCR products were diluted 50-fold and used as templates for nested PCR using primers (A2: 5′-AAGATGGTGGAGAAGGAGCTGA-3′; S: 5′-TGCGCCAATAACGTTTTGT-3′). The PCR products were subjected to electrophoresis, extraction, ligation, E. coli transformation and sequencing as mentioned above.

Bioinformatics analysis of the full-length sequence of the 14-3-3 cDNA By comparing and aligning the sequences of 3′- and 5′-RACE products, a full-length sequence of 14-3-3 cDNA was obtained through RT-PCR using primer P 1 (5′-ATGGCTGAGGCGAGAGAGGAGTG-3′) and P 2 (5′-GGCATCGGCCTCCTCAACCTT-3′). After electrophoresis on agarose gels, DNA of the target bands was ligated to pGEM-T Easy vector and then transformed into competent cells of E. coli JM109. Three positive colonies harboring the amplified DNA were sequenced using T7/SP6 primers. Sequence alignments, ORF translation and molecular weight of the 14-3-3 protein were predicted with Vector NTI Suite 6. BLAST was carried out at the NCBI server (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), while structural analysis of the predicted 14-3-3 proteins was conducted on the website (http://cn.expasy.org). Phylogenetic analysis was performed using ClustalV method of MegAling program in DNA STAR package.

Analysis of 14-3-3 protein expression using real-time quantitative RT-PCR

Total RNA was isolated from synchronized D.salina cells which were harvested at 6, 9, 12, 15, 18 and 21 h after beginning of the light period, respectively. Real-time quantitative RT-PCR was performed using SYBR RT-PCR Kit (Takara) according to the manufacturer’s instructions. The primers for 14-3-3 protein gene were as follows: 5′-AGCCACTGTCGCAAGGTGAAGC-3′ (sense) and 5′-TTCTCATGGAAGTAACCCTCCTCTGTC-3′ (antisense). The resulting product of real-time quantitative RT-PCR was 367 bp long. The primers for 18S rRNA were 5′-TTGGGTAGTCGGGCTGGTC-3′ (sense) and 5′-CGCTGCGTTCTTCATCGTT-3′ (antisense). After heating at 95°C for 10 min, the PCR proceeded for 45 cycles of 10 s at 95°C, 30 s at 60°C and 40 s at 72°C, and a final extension at 72°C for 5 min. As targeted DNA was amplified, the fluorescence of the end of each cycle of RT-PCR was monitored by iCycler iQ (BIO-RAD), the targeted DNA amplified specifically was confirmed by electrophoresis and sequencing. The relative abundance of 18S rRNA was used as an internal standard.

Results and discussion

Amplification of 3′ and 5′ end of the 14-3-3 cDNA

Agarose gel analysis revealed that amplification with degenerate primers and resulted in a DNA band of about 530 bp. 3′-RACE result of first PCR with A 1 and S primers showed some faint bands and smearing, nested amplification of the 3′ end resulted in an ∼750 bp of bright band. BLAST-n analysis indicated that it had wide similar to known 14-3-3 protein genes from C.reinhardtii, Tobacco, Pisum sativum L., Arabidopsis, Tomato, Homo sapiens and others organisms, implying that it may be a partial fragment of the14-3-3 gene.

Electrophoresis analysis of the primary PCR product of 5′-RACE showed only smear, not specific band, however when nested amplification was carried out directly using this PCR product as the template and a specific band of about 260 bp appeared. BLAST of this sequence resulted in similar outcome to that from the 3′cDNA end BLAST. Based on the 54 bp overlap between the 5′ and the 3′ fragments, the full-length 14-3-3 cDNA of 1470 bp (poly (dA) not included) was deduced.

Characterization of the 14-3-3 gene

As anticipated, a 1470 bp of specific DNA band was amplified by PCR using full length primers P 1 and P 2. Sequencing result of this sequence completely coincided with the deduced full-length cDNA sequence (Fig. 1). The cDNA of 14-3-3 possessed a 774 bp ORF from 86 bp to 862 bp of the sequence, except for an 85 bp 5′ UTR and a 608 bp 3′ UTR. The 3′ UTR possessed typical low G + C content (43%) but not a typical polyadenylation signal TGTAA described firstly in algae [20]. A similar sequence (TGCAA), however, is present at position from 1102 to 1108 upstream from the poly (A)-tail, just like the 14-3-3 cDNAs of Chlamydomonas. The nucleotide sequence data reported here were deposited in GenBank database under accession no. AY 965896.
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Fig. 1

Nucleotide sequence of the gene encoding the 14-3-3 protein from the green unicellular alga D.salina and the deduced amino acid sequences (GenBank accession number AY965896). The nucleotide sequences used as degenerate oligonucleotide primers are underlined. The putative initiation codon (ATG) and the putative polyadenylation signal were underlined, and the stop codon (TAA) in italic was underlined

The ORF was obtained by ORF Finder on NCBI (http://www.ncbiikol.nlm.nih.gov/gorf/gorf.html) and the predicted 14-3-3 protein precursor was 258 amino acids in length, with a theoretical molecular weight of 29.33 kDa and a pI value of 4.46.

Multi-alignment of 14-3-3 gene with that of other organisms was conducted (Fig. 2). According to NCBI protein-protein BLAST, the deduced 14-3-3 amino acid sequence showed 89% (231/258) identities and 94% (244/258) positives in local alignments to Chlamydomonas 14-3-3-like protein. It also shared very broad and high local identities and positives to the 14-3-3 gene from species of broad classes such as Pisum_sativum (80% identity and 89% positive), Mesembryanthemum_cr (77% identity and 87% positive), Lycopersicon_escule (82% identity and 90% positive), Solanum_tuberosum (80% identity and 88% positive), Nicotiana tabacum (80% identity and 88% positive), Arabidopsis thaliana (80% identity and 89% positive), Tomoto (78% identity and 88% positive). Vigna_angularis (80% identity and 89% positive), Xenopus_tropicalis (75% identity and 84% positive), Zea_mays (76% identity and 88% positive), Drosophila_melanoga (79% identity and 86% positive), Oryza_sativa_japon mspaeas (with 75% identity and 85% positive), Caenorhabditis_eleg (with 66% identity and 77% positive), Homo_sapiens (with 76% identity and 84% positive), Mus_musculus_house (with 64% identity and 76% positive), Rattus_norvegicus (with 64% identity and 76% positive), Bos_taurus_cattle (64% identity and 76% positive), Giardia_intestinali (62% identity and 78% positive).
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Fig. 2

Alignment of 14-3-3 protein using DNAMAN program. Multi-alignments were made by DNAMAN of the deduced 14-3-3 amino acid sequence with the 19 most homologous 14-3-3 proteins from Chlamydomonas_reinh (CAA55964), Pisum_sativum (CAB42546), Mesembryanthemum crystallinum (AAB40395), Lycopersicon_escule (AAL04426), Solanum_tuberosum (CAA72383), Nicotiana_tabacum (BAD12172), Vigna_angularis (BAB47119), Arabidopsis_thalian (AAA96253), tomato (T07387), Xenopus_tropicalis (AAC41251), Zea_mays (AAT06575), Drosophila_melanoga (NP_732311), Oryza_sativa__japon (AAX95656), Caenorhabditis_eleg (NP_509939), Homo_sapiens (P31946), Mus_musculus__house (Q9CQV8), Rattus_norvegicus_ (BAA04260), Bos_taurus__cattle_ (AAC02090 ), Giardia_intestinali (AAZ91664). The completely identical amino acids AKLAEQAERY, ERNLLSVAYKNV, SKVFYLKMKGDY, PTHPIRLGLALNFSVFYYEILNSP and EESYKDSTLIMQLLRDNLTLWT were dark shaded, underlined and boxed

There were five highly conserved domains at positions 12–21(I), 43–72(III), 120–137(β), 168–191(χ) and 197–237(δ), respectively. Two putative phosphorylation sites for cAMP-dependent protein kinase and Ca2+/calmodulin kinase were present in the second conserved domain (at positions 62 and 67). The Ser-62 was also a potential phosphorylation site for protein kinase C. Therefore, the D. salina 14-3-3 protein might act as an inhibitor of protein kinase C as reported for some members of the 14-3-3 protein family. A putative Ca2+ binding site (= EF hand like motif, EXDXXGXXSXXD) was found in the carboxy-terminal domain (amino acid positions 208-219) Ca2+ binding properties for the 14-3-3 proteins of Arabidopsis and Chlamydomonas.

Two- and three-dimensional structure predictions of D. salina 14-3-3 protein were conducted (Fig. 3). Based on the Hierarchical Neural Network [21]. 14-3-3 mature peptide is composed of 56.98 % alpha helix, 10.85 % extended strand and 32.17 % random coil. Alpha helices resided within the main body especially in the N-terminal region of 14-3-3 protein, while random coils penetrated through middle part of this sequence accompanied by a small amount of extended strands.
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Fig. 3

The two- and three-dimensional structures of the predicted D. salina 14-3-3 protein. (a) The two- dimensional structure of 14-3-3. 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 three-dimensional structure of 14-3-3. The α-helix was shown in red (helix-shaped), and the random coil also in white (line-shaped)

Molecular evolution analysis

To investigate the evolutionary relationship among different organism 14-3-3 genes, a phylogenetic tree was constructed based on the deduced amino acid sequences of D. salina 14-3-3 gene and other 14-3-3 genes. All the different organisms studied were grouped into four big clusters (Fig. 4). The first cluster comprised C. reinhardtii, Arabidopsis_thalian, Mesembryanthemum crystallinum, Giardia intestinali, which seemed to be made up of two taxonomically unrelated families, such as Mesembryanthemum crystallinum, Giardia_intestinali, The second cluster group contained Oryza_sativa_japon, Nicotiana_tabacum, Solanum_tuberosum, Vigna_angularis, Pisum_sativum, while the third cluster group comprised Lycopersicon_escule, Tomato, Zea_mays Caenorhabditis_eleg, Homo_sapiens, Mus_musculus_house, Rattus_norvegicus, Bos_taurus_cattle, Xenopus_tropicalis and Drosophila_melanoga constructed the fourth group.
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Fig. 4

Phylogenetic tree analysis. Pisum_sativum (CAB42546), Mesembryanthemum crystallinum (AAB40395), Lycopersicon_escule (AAL04426), Solanum_tuberosum (CAA72383), Nicotiana_tabacum (BAD12172), Vigna_angularis (BAB47119), Arabidopsis_thalian (AAA96253), tomato (T07387), Xenopus_tropicalis (AAC41251), Zea_mays (AAT06575), Drosophila melanoga (NP_732311), Oryza_sativa__japon (AAX95656), Caenorhabditis_eleg (NP_509939), Homo_sapiens (P31946), Mus_musculus__house (Q9CQV8), Rattus norvegicus_ (BAA04260), Bos_taurus__cattle_ (AAC02090 ), Giardia_intestinali (AAZ91664)

All the above four groups of the 14-3-3 genes were derived from a common ancestor in evolution, suggesting that these 14-3-3 genes from different organisms share a common evolutionary ancestor, 14-3-3 proteins of D. salina are more similar to the algal and plant sequences than to those of animals and yeasts, but it apparently diverged from this branch of the tree during early evolution.

Expression of the gene encoding 14-3-3 protein

The real time quantitative RT-PCR was used to analyze the expression profile- of the 14-3-3 protein gene from D. salina cells synchronized under a constant light-dark cycle of 12 h light/ day during the different light periods. As shown in Fig. 5, the level of total RNA was elevated considerably at ∼6 h after the light period (Fig. 5a). The relative proportion of the 14-3-3 protein mRNA was gradually increased from 6 h to 18 h after the light period with a highest level at 18 h (Fig. 5b). The findings were similar to the expression of the 14-3-3 protein cDNA of Chlamydomonas reinhardtii [22], suggesting that expression of the 14-3-3 protein of D.salina is cell cycle-dependent.
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Fig. 5

Expression of the 14-3-3 protein gene in the different growth periods. (a) Cell density and total RNAs isolated from cells of D. salina grown in the different periods after the light culture. (b) the relative proportion of the 14-3-3 protein mRNA was gradually increased from 6 h to 18 h after the light period with a highest level at 18 h

In summary, we have successfully cloned a new gene encoding 14-3-3 proteins from D. salina, which is involved in gene regulation. The cloned cDNA is 1485 bp in length containing a 774 bp ORF encoding a polypeptide of 258 amino acids, and the deduced amino acid sequence includes two putative phosphorylation sites within the N-terminal region. Bioinformatics analysis revealed that D. salina 14-3-3 had homology to that of other organisms, and might express constitutively.

Acknowledgements

This work was supported by the grants from National Natural Science Foundation of China (No.30470030), and carried out in Henan Key Laboratory for Molecular Medicine.

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