Isolation, Expression and Characterization of rbcL Gene from Ulva prolifera J. Agardh (Ulvophyceae, Chlorophyta)

SHAO Zhanru, LI Wei, 2), GUO Hui, 3), and DUAN Delin, *

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Isolation, Expression and Characterization ofGene fromJ. Agardh (Ulvophyceae, Chlorophyta)

SHAO Zhanru1), LI Wei1), 2), GUO Hui1), 3), and DUAN Delin1), *

1),,266071,2),,07743,3),100049,

is a typical green alga in subtidal areas and can grow tremendously fast. A highly efficient Rubisco enzyme which is encoded by UpRbcL gene may contribute to the rapid growth. In this study, the full-lengthopen reading frame (ORF) was identified, which encoded a protein of 474 amino acids. Phylogenetic analysis ofsequences revealed that Chlorophyta had a closer genetic relationship with higher plants than with Rhodophyta and Phaeophyta. The two distinct residues (aa11 and aa91) were presumed to be unique for Rubisco catalytic activity. The predicted three-dimensional structure showed that one α/β-barrel existed in the C-terminal region, and the sites for Mg2+coordination and CO2fixation were also located in this region. Gene expression profile indicated thatwas expressed at a higher level under light exposure than in darkness. When the culture temperature reached 35℃, the expression level ofwas 2.5-fold lower than at 15℃, and the carboxylase activity exhibited 13.8-fold decrease.was heterologously expressed inand was purified by Ni2+affinity chromatography. The physiological and biochemical characterization of recombinant Rubisco will be explored in the future.

Ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit; sequence analysis; real-time PCR;expression;

1 Introduction

(Ulvophyceae, Chlorophyta) is one of the typical green algae which niches in subtidal areas along the seashore of China, and commonly attached to solid substratum. In recent years,bloom has been frequently occurring in Yellow Sea (Liu.,2009; Duan.,2012), and caused serious problems on coastal ecosystems (Sun., 2008). Green algal thalli exhibit rapid growth features during their floating state on the sea. It is believed thatpossesses particular physiological and ecological characteristics to maintain long period survival through high efficient photosynthesis (Lin., 2011; Zhang., 2012).

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Ru- bisco, EC 4.1.1.39) is a crucial enzyme in Calvin cycle of photosynthesis (Hartman and Harpel, 1994; Houtz and Portis, 2003); it is involved in the first step of carbon fixation in photosynthetic and chemoautotrophic organisms. Rubisco can simultaneously catalyze the carboxylation and oxygenation of ribulose bisphosphate (RuBP). RuBP carboxylation defines gross photosynthesis rate and determines productivity (Jensen and Bahr, 1977). Most reports to date have focused on unicellular algae, including(Dron., 1982),(Ginrich and Hallick, 1985), and, and few molecular studies concerning macroalgal Rubisco are available (Kai., 2002; Ying., 2011). Therefore, we studied the multicellular green algafor better understanding how Rubisco regulates its rapid growth and also the dynamics of real-time expression and enzymatic activity.

There are four forms of Rubisco (I, II, III and IV), and that of Chlorophyta is believed to be form I which comprises of eight large catalytic subunits (RbcLs) and eight small subunits (RbcSs) (Tabita., 2008). In terrestrial plants and green algae, RbcLs (50–55kD) are encoded by a singlegene present in the chloroplast genome, whereas the rbcSs (12–18kD) are encoded by nuclear genes (Dean., 1989; Spreitzer and Salvucci, 2002). So far, it is reported that RbcLs provide all the catalytically essential residues of Rubisco, while the function of RbcSs is not clear (Kaplan and Reinhold, 1999).

In this study, we isolated and characterized the gene encoding.RbcL protein. Variation ingene expression was monitored under different conditions, and recombinant protein was purified after it was expressed in.. Our research made contribution to decipher relationship between UpRbcL structure and function, and to explore physiological mechanisms supporting rapid growth of.during floating adaptation.

2 Materials and Methods

2.1 Pre-culture and treatments of

Fresh.were collected from the green algal blooming area on the sea in Qingdao (36?03′N; 120?18′E) in August 2011. The algal filaments were maintained indoor at 25℃, with a continuous illumination of 45μmol m?2s?1under a light exposure of 12:12 L/D for 48h. Then.a cells were collected and subjected to different temperature conditions (10, 15, 20, 25, 30, 35℃) for 1h to test the influence of temperature on gene expression and carboxylase activity. For the light treatment, the alga was incubated at 25℃ under continuous illumination (45μmolm?2s?1) for 48h, and then in dark for 12h. After these treatments, the alga was collected and rinsed with 0.1% DEPC sterile seawater 3 times, frozen in liquid nitrogen and stored at ?80℃ for nucleic acid and crude protein extractions.

2.2 DNA, RNA Extraction and cDNA Synthesis

Genomic DNA was extracted with the Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. Total RNA extraction was carried out using CTAB (cetyltrimethylammonium bromide) method (Yao., 2009). Extracted RNA was desiccated and dissolved in 30μL RNase-free water, and any possible genomic DNA contamination was removed with the DNase I Kit (Fermentas, Burlington, USA). First strand cDNA was synthesized using the PrimeScript II cDNA Synthesis Kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions, and stored at ?20℃ for subsequentisolation.

2.3 Cloning of

One pair of specific primers, UpRbcL-F (5’-ATGG CTCCACAGACTG-3’) and UpRbcL-R (5’-TAATGTA TCAATAGCATCAAATTC-3’), was designed based on thesequence of.(DQ813497) to amplify full-length coding sequence ofcDNA. PCR was performed in a 20μL reaction volume containing 1μL of cDNA, 0.25μLTMDNA polymerase high fidelity (HiFi) (TransGen, Beijing, China), 2μL of 10×HiFi buffer, 1μL of each primer (10μmolL?1), 3.2μL of dNTP (10mmolL?1), and 11.55μL of RNase-free water. The amplification was carried out by predenaturing at 95℃ for 5min, followed by 30 cycles of denaturing at 95℃ for 30s, 47℃ for 30s, 72℃ 2min, and a final extension at 72℃ for 10min. PCR product was sequenced by Sangon (Shanghai, China).

The same pair of primers, UpRbcL-F and UpRbcL-R, was used to amplify thesequence from genomic DNA, and PCR product was treated through the procedure described above.

2.4 Analysis of UpRbcL Sequence

Sequence was analyzed for molecular weight (MW), isoelectric point (pI) with software ORF Finder (Sayers., 2012) and ProtParam (Gasteiger., 2005). The similarity of UpRbcL ORF with others was checked by BLAST analysis (Altschul., 1997) at NCBI database (http://www.ncbi.nlm.nih.gov/blast). Signal peptide of UpRbcL sequence was predicted by SignalP 4.0 Server (Petersen., 2011), and presence of trans-membrane domains was determined with the TMHMM version 2.0 program (Krogh., 2001). Hydrophobicity and hydro- philicity were analyzed by ProtScale program (Gasteiger., 2005), and the secondary structure was predicted by SOPMA (Geourjon and Deleage, 1995). SWISS- MODEL (Peitsch, 1995; Arnold., 2006; Kiefer., 2009) and Pymol Viewer programs were applied to construct the putative 3D-structure of UpRbcL by homology modeling with.

2.5 Phylogenetic Analysis

Phylogenetic tree was constructed using the neighbor- joining algorithm by MEGA 5.2 program (Tamura., 2011) and bootstrap values were calculated from 1000 replicates. UpRbcL amino acid sequence was aligned with RbcL sequences from Archaeplastida. Clustal X 2.0 software (Larkin., 2007; Thompson., 1994) was used to perform further alignments.

2.6 Transcriptional Profile of

Two specific primers UpRbcL-S (5’-ATGGCTCCACA GACTGAAAC-3’) and UpRbcL-A (5’-CCTGGTTGAG GAGTCATACGG-3’) were applied for amplifying a 139 bp amplicon. Primers UpLhcSR-S (5’-ACTGGTCCATT TGGTTTCTT-3’) and UpLhcSR-A (5’-CCGCTGATGTT ACCCTCCC-3’) were designed according to the coding sequence of light-harvesting chlorophyll a/b-binding protein (LhcSR) (HQ622096.1). Transcriptional levels were normalized by the expression of the gene encoding the small subunit rRNA amplified with the primers 28S-S (5’-AACACGGACCAAGGAGTCTAAC-3’) and 28S-A (5’-GAAACTTCGGAGGGAACCAG-3’).

RT-qPCR was performed with the SYBRTMII (Takara, Tokyo Japan) on the TP800 Thermal Cycler Dice? (Takara, Tokyo, Japan). The PCR mixture (25μL) contained 12.5μL Premix, 1μL of each primer (10μmolL?1), 2μL of diluted cDNA and 8.5μL of RNase-free water. Thermal cycling protocol was: 95℃ for 30s, followed by 40 cycles of 95℃ for 5s and 58℃ for 30s. Specificity of primers was checked by observing relevant dissociation curve. Three independent biological replicates were carried out for each sample, and relative quantitative values were calculated with 2?△△Ctmethod (Schmittgen., 2000).

2.7 Measurement of Carboxylase Activity

Extraction of total protein from.was conducted according to the method described by Rousvoal(2011). Protein content was determined according to the Bradford method (Bradford, 1976) using theProtein Quantitative Kit (TransGen, Beijing, China).

To determine Rubisco carboxylase activity, we referred to the method described by Lan and Mott (1991), and applied the Plant Rubisco Carboxylation Activity Detection Kit (GenMed Scientifics, China). The reaction was conducted at 25℃ for 5min in a Microplate Reader (BioTek, Vermont, USA). Five μg of algal protein extract was added into the reaction mixture (250μL), and the reaction was initiated by adding the substrate RuBP. Production of NADH was determined by measuring ultraviolet (UV) absorbance at 340nm, and the extinction coefficient of 6.22mmolL?1was used to determine the initial carboxylase activity (Unit: mmolL?1CO2mg?1min?1). Boiled algal protein extract was used as negative control.

2.8 Heterologous Expression of

Specific primers UpRbcL-F (5’-ATGGCTCCACAGA CTG-3’) and UpRbcL-R (5’-TAATGTATCAATAGCA TCAAATTC-3’) were used for amplifying. The thermal cycling applied was: 94℃ for 5 min, followed by 30 cycles of 94℃ for 30s, 48℃ for 30s, 72℃ for 2min, and a final extension step of 72℃ for 10min. The positive PCR product was subcloned into the expression vector-E2 (TransGen, Beijing, China). The recombinant plasmid was transformed into.BL21 (DE3) and transformants was spread on antibiotic selection media containing ampicillin (0.1mgmL?1final concentration). Plates were incubated at 37℃ overnight, and positive clones were screened by colony PCR detection.

To obtain production of the recombinant protein, pre- culture of 2mL of LB medium containing ampicillin was incubated overnight at 37℃, and was then used to start a fresh 200mL LB plus ampicillin culture, until the OD600valuereached to 0.5. Expression ofwas induced by adding 0.5mmolL?1IPTG into the medium at 120 rpm for hours at 37℃.

In order to optimize the duration of induction and the production of recombinant protein, 500μL of culture were collected every 1h and then centrifuged at 4000rmin?1for 15min. Fifty μL of deionized water and an equal volume of 2×sodium dodecyl-sulfate polyacrylamide gel electrophoresis loading buffer (Tris-HCl 25mmolL?1, DTT 100mmolL?1, SDS 2%, Glycerol 20%, Bromophenol Blue 0.016%) were added to the pellet. After boiling for 10min, mixtures were loaded on a 12% SDS-PAGE (ATTO, Tokyo, Japan). Electrophoresis was carried out at a constant voltage of 80 V for 20min, and then 160V for 60min. Bacterial protein extracts produced from.containing the empty vector were used as negative control.

2.9 Western Blot Analysis

After migration on SDS-PAGE, proteins from the gels were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (0.45μm) at 30V for 2h with an AE-6675 HorizBlot system (ATTO, Tokyo, Japan). After rinsing with TBST buffer (150mmolL?1NaCl, 20mmolL?1Tris-HCl pH 8.0, 0.05% Tween-20) 3 times, membrane was blocked to avoid non-specific binding by placing it in a freshly prepared 5% solution of non-fat milk (5g dry milk dissolved in up to 100mL TBST buffer), and shaking at 30rmin?1for 2h at room temperature. The membrane was then rinsed twice with TBST buffer for 5min. To detect the target protein, a 1:250 dilution of primary rabbit anti-His antibody (Top- Science, Beijing, China) was incubated with the membrane under gentle agitation for 2h at room temperature. After additional rinsing with TBST, membranes were exposed to a 1:2500 dilution of goat-anti-rabbit IgG antibody conjugated with horseradish peroxidase (Tiandz, Beijing, China) for 1h. DAB sensitive chromogenic reaction (Tiandz, Beijing, China) was used to detect binding of antibodies, and reaction was terminated by transfer of the membrane into deionized water.

2.10 Purification of Recombinant UpRbcL

After induction with 0.5mmolL?1IPTG for 5h, LB plus ampicillin culture (200mL) was centrifuged at 4000rmin?1for 15min. Pellet was re-suspended in the buffer which contained 300mmolL?1NaCl, 50mmolL?1NaH2PO4, 10mmolL?1imidazole and 10mmolL?1Tris base (pH 8.0). The suspension was sonicated intermittently and centrifuged at 15000for 20min at 4℃. The supernatant was filtered and loaded on theTMNi-NTA Resin (TransGen, Beijing, China). Elution was conducted by applying successively imidazole solutions (40, 80, 120, 160, 200mmolL?1), and fractions were loaded on 12% SDS-PAGE. All the purification steps were carried out at 4℃. Concentrations of UpRbcL recombinant protein were quantified according to the method described above.

3 Results

3.1 Sequence Analysis of UpRbcL

Full-length open-reading frame of(1425bp), encoding a protein of 474 amino acids, was submitted to GenBank (JQ867403.1). Comparison between sequences obtained from cDNA and genomic DNA showed that there was no intron in. BLAST analysis confirmed thatencoded a protein belonging to Rubisco large superfamily, with a theoretical molecular mass of 52.34kD and an isoelectric point (pI) of 5.82. UpRbcL was considered as a comparatively stable enzyme with an instability index of 38.71.

Maximal values of the original shearing site (C score), signal peptide (S score), and synthesized shearing site (Y score) were 0.115, 0.105 and 0.123, respectively, meaning that there was no signal peptide in UpRbcL amino acid sequence. With the transmembrane topological struc- ture analysis, all parts of UpRbcL were identified to be outside the membrane.

In relation to the secondary structure prediction, proportions of α-helix, extended strand, β-turn and random coil of UpRbcL were 42.83%, 15.82%, 9.92%, and 31.43%, respectively, indicating that α-helix and random coil were its major components. The tertiary structure of UpRbcL was constructed with SWISS-MODEL and Pymol Viewer prediction programs based on the structure template of(Taylor, 2001). It was obvious that one α/β-barrel existed in the C-terminal end, and 7 sites (K174, R294, H297, H326, K333, G402 and G403) in this region were responsible for the RuBP activation and catalization (Fig.1). The two adjacent acidic residues D202 and E203 were assumed for Mg2+coordination, and the role of residue K200 was for CO2fixation (Fig.1).

Fig.1 Predicted 3D structure of UpRbcL. (a) Global tertiary structure of UpRbcL. The white rectangle indicated the α/β-barrel in the C-terminal end. (b) Zoom in on the white rectangle region which contained the barrel and serial important amino acid residues: D202 and E203 were reported to coordinate Mg2+; K200 provided a site for CO2 fixation; amino acids important for catalysis of carboxylation reaction (K174, R294, H297, H326, K333, G402 and G403) were presented in red color.

3.2 Phylogenetic Analysis of UpRbcLs

Alignment of amino acid sequences showed that UpRbcL was more closely related to that of other green algae and land plants than to that of,and(Fig.2). The long C-terminal in..and.indicated that brown and red alga sequences might have originated from the same common ancestor. Thr 11 was unique in RbcLs sequences from, while Ala or Val were located at site 11 in other species of Archaeplastida. A deletion of amino acid was observed at site 91. It was deduced that both sites might have function on the catalytic characterization of Rubisco from other species.

A phylogenetic tree was constructed based on 14sequences from algae and land plants (Fig.3). It shows that UpRbcL was in the subgroup of green algae and shared more than 99% identities with those from.and..alsoexhibited 75.7% identities withand 77.1% with. In reference to the phylogeny of RbcLs,was more closely related to terrestrial plants than to glaucophytes and Rhodophyta.

3.3 Transcriptional Profile of

Real-time PCR detection ofandtranscripts was conducted on algal samples harvested after incubation at various temperatures and illuminations. It was shown that both genes were highly expressed after exposure to continuous light illumination for 48h, while their abundance decreased significantly after being kept in dark for 12h (1.5-fold and 7.3-fold change, respectively) (<0.05) (Fig.4a). When the alga was incubated at a range of temperatures, from 10℃ to 35℃, transcript abundance ofwas high at 10℃, 15℃, 20℃ and 25℃, with a maximum at 15℃, and declined significantly (2.5-fold) at 35℃ (<0.05) (Fig.4b).

3.4 Measurement of Endogenous Rubisco Activity

After extracting crude protein from.(0.1g each) treated at 15℃, 25℃, 35℃, respectivelywe conducted SDS-PAGE electrophoresis. The bands at 55kD show the potential presence of Rubisco, as it is the major protein within crude extract (Fig.5a). In addition, carboxylase activity of Rubisco was determined using equal amounts of total protein for the three conditions tested. It was observed that the activity reached maximum at 15℃, half at 25℃, and almost completely abolished at 35℃ (Fig.5b).

3.5 Heterologous Expression and Purification of Recombinant UpRbcL

After optimizing the protein induction time, heterologous protein was significantly expressed in one hour after inducing with 0.5mmolL?1IPTG, and it became stably expressed within 2 to 5h. After being separated on 12% SDS-PAGE, 55kD protein band was distinctly identified from the recombinant bacterial extract while no other band correspondingly appeared after induction of bacteria containing empty plasmid (Fig.6a). The optimal imidazole concentration for the elution of purified UpRbcL was 80mmolL?1(Fig.6b), and western blot indicated that the 55kD band was positive to the RbcL anti-His antibody (Fig.6c).

Fig.2 Alignments of Ulva RbcL with those of land plants and algae. The green rectangle indicates a unique characterization at site 11; the red rectangle indicates a Pro deletion distinctively in the Rubisco activase contact region in U. porlifera, respectively; the blue rectangle shows highlights a long C-terminal extension in brown and red algal sequences.

Fig.3 Phylogenetic tree of RbcL subunits constructed with selected sequences from Archaeplastida. The tree is obtained by the neighbor-joining algorithm using MEGA 5.2 program. Bootstrap values are calculated from 1000 replicates, and the branch lengths are proportional to estimated evolutionary change. The scale bar corresponds to 0.05 estimated amino-acid substitution per site. Green letters show the species having Green-like Rubisco, and red letters indicates Red-like Rubisco. The sequences include Ulva prolifera (JQ867403.1), U. linza (DQ813496.2), U. pertusa (JQ867404.1), Chlorella pyrenoidosa (EU038283.1), Chlamydomonas reinhardtii (ACJ50136.1), Volvox carteri (ACI31253.1), Zea mays (NP_043033.1), Oryza sativa (NP_039391.1), Arabidopsis thaliana (NP_051067.1), Nicotiana tabacum (NP_ 054507.1), Spinacia oleracea (NP_054944.1), Cyanophora biloba (KF631394.1), Cyanophora paradoxa (NP_043240.1), Pyropia yezoensis (DQ227866.1).

Fig.4Influence of illumination and temperature on UpRbcL and UpLhcSR expression level. (a) Relative transcript abundance of UpRbcL and UpLhcSR exposed to different illuminations. (b) Relative abundance of UpRbcL transcript at different temperatures. All data are expressed as mean±SD (n=3).

4 Discussion

Recently, green tides unceasingly occur on large scale in the due seasons each year (Leliaert, 2008). In China, green tide occurs almost every year in Yellow Sea since 2007 (Liu, 2009; Duan, 2012). More and more investigations have been carried out to identify the reason behind this naturally occurring phenomenon. Nevertheless, previous works were concentrated on the morphological features and the origination survey (Zhao., 2011; Duan., 2012). In present study, we isolated and characterized, and carried out the over-ex- pression and purification of this large subunit protein from..

There was no intron in the sequence of, which was previously observed in.(Ying., 2011). From the evolutionary tree of form I Rubisco, taxa are commonly divided into two groups. One clade included sequences from Chlorophyta and land plants (Green-like Rubisco group), while the other was Rhodophyta (Red- like Rubisco group). Our results showed that although belonging to Archaeplastida, green algae have closer evo- lutionary relationship with land plants than with Glaucophyte algae.

Being crucial to the photosynthesis, UpRbcL was supposed to be highly efficientand the major contributing factor in sustaining the rapid growth of.. In this study, we found that several crucial residues (K174, R294, H297, H326, K333, G402 and G403) in α/β-barrel were responsible for the catalysis of Rubisco, and these residues were consistent with early reports (Lorimer and Miziorko, 1980; Kellogg.,1997; Cleland., 1998). Previously, genetic screening has identified some residues influencing the specificity and stability of Rubisco: V331/ T342/ G344/ R339/ I393 in loop 6, D473 in C-terminus and A222/ V262/ L290 between large and small subunits (Spreitzer and Salvucci, 2002; Satagopan and Spreitzer, 2004; Karkehabadi, 2007; Andersson and Backlund, 2008). These residues were all found in UpRbcL sequence, which showed high conservation of the active sites under positive selection. However, two amino acid residues (aa11 and aa91) were unique in UpRbcL compared to other RbcL sequences. According to the previous publications (Li., 2005; Portis., 2008), we suspected that the deletion of Pro at site 91 might affect the activation of Rubisco due to the formation of an extra α-helix between the sites 89 and 93. Moreover, the substitution of hydrophobic amino acid A11 (in,,.) or V11 (in land plants) into hydrophilic T11 was presumed to affect the secondary structure of loop 1, and eventually influence the catalytic efficiencies and thermal stability. Site-specific mutagenesis is required to verify the function of these sites in the future.

When the green tide outbreaks,can float on the sea surface and rapidly grow within a few weeks..may possess unique physiological and biochemical properties that allow it to adapt to persistent illumination on the sea. We applied excessive light exposure, up to 48h, to monitor the transcription ofand. We observed that both genes were expressed at a higher level when alga was exposed to extensive irradiance. This indicated thatandcan be transcribed under continuous illumination, and might be coupled into complicated physiological process for utilizing excessive solar energy for rapid growth.

Comparative analysis on transcript abundance and enzymatic activity was carried out. The maximumexpression level and the initial activity of Rubisco both emerged in the range of 15 to 20℃, which was consistent with the ecological features of.(Wang, 2007). After 1h at 35℃,mRNA expression and carboxylase activity were highly decreased; indicating limited heat-resisting ability of UpRubisco. Referred to the studies on rice and spinach (Schneider, 1992; Li., 2002), we assumed that these thermal properties might due to the thermal instabilities of Rubisco activase.

For the prokaryotic expression, the content of recombination enzyme reached maximum whenwas induced with 0.5mmolL?1IPTG for 2h, and the recombinant UpRbcL could be completely eluted with 80mmolL?1imidazole. So far, no attempt to reconstitute active homologous enzyme has succeeded for form I Rubisco to our knowledge (Whitney, 2011), owing to the indispensability of chaperone and small subunit (Tarnawski, 2008; Liu, 2010). Although no activity of UpRubisco could be measured in the present study, we purified UpRbcL and paved the way for the successful overexpression of the whole enzyme UpRubisco.

In conclusion, this study isolated and characterized Rubisco large subunit gene of. Features detected in its secondary and tertiary structures, such as conservation of important catalytic residues, were consistent with its high photosynthetic efficiencies. Moreover, high transcription levels ofandunder continuous illumination should be part of the mechanisms favoring rapid growth of.. These molecular observations provided clues supporting the fast development of the green algal bloom, and pave the way for further investigation of key physiological processes in..

Acknowledgements

This research was financially supported by the Scientific and Technical Supporting Programs of China (2008 BAC49B01) and State Ocean Administration Project (200805069). The authors appreciated Drs. Thierry Tonon and Zimin Hu for revision and suggestions of the manuscript.

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(Edited by Qiu Yantao)

DOI 10.1007/s11802-015-2661-6

ISSN 1672-5182, 2015 14 (6): 1087-1095

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

(May 9, 2014; revised July 7, 2014; accepted September 10, 2015)

* Corresponding author. Tel: 0086-532-82898554 E-mail: dlduan@ms.qdio.ac.cn