A trehalose-6-phosphate synthase gene from Saccharina japonica(Laminariales, Phaeophyceae)

Yunyan Deng • Xiuliang Wang • Hui Guo •

Delin Duan

Received: 13 July 2013 / Accepted: 25 November 2013 / Published online: 30 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The full-length cDNA sequence of a trehalose-

6-phosphate synthase gene from Saccharina japonica

(designated as SjaTPS) (Accession: KC578568) was iso-

lated based on homologous cloning and RACE-PCR. It was

4,127 bp, with 320 bp 50-UTR, 21 bp 30-UTR, and open

reading frame (ORF) of 3,786 bp. The deduced 1,261

amino acids characterized with predicted molecular weight

of 137.84 kDa and theoretical isoelectric point of 7.12. The

SjaTPS had one N-terminal CBM20 (family 20 carbohy-

drate-binding module) domain, one TPS domain (treha-

lose-6-phosphate synthase) in the middle region and a

single TPP (trehalose-6-phosphate phosphatase) domain

near the C-terminus. Structural analysis suggested that the

SjaTPS putatively functioned as trehalose-6-phosphate

synthase, and might be related to laminaran metabolism in

S. japonica. Homology analysis indicated that the SjaTPS

shared 49–70 % similarities with the 13 known TPS

sequences of other algae; only 55 % amino acid similarities

were detected between SjaTPS and the previously reported

TPS sequence of S. japonica (Accession: DQ666325).

Phylogenetic analysis revealed close affinity between

SjaTPS and TPS of brown alga Ectocarpus siliculosus

(Accession: CBJ29609). Transcriptional analysis showed

that desiccation greatly enhanced SjaTPS expression and

the maximum appeared at 3 h, which was about 300-fold

compared to that of the start, implied that SjaTPS was

involved with drought adaption in kelp. In vitro expression

of SjaTPS showed that one distinct band existed at

*115 kDa, and western blot detection proved that it was

positive to the anti-His antibody with high specificity. Our

results increased the knowledge of trehalose-6-phosphate

synthase properties in S. japonica and also important for

better understanding the role trehalose plays in kelp abiotic

tolerance for adaption to the sublittoral habitats.

Keywords CBM20 domain � Drought adaption �Saccharina japonica � Trehalose-6-phosphate

synthase � TPS domain � TPP domain

Introduction

Saccharina japonica (Areschoug) Lane, Mayes, Druehl

and Saunders (Laminariales, Phaeophyta) is one of the

important commercial seaweeds, due to its application in

food, pharmaceutical and biotechnology. Currently in

China, it is mainly used as foodstuff and raw material for

alginate production. Naturally, S. japonica niches on the

substratum in sublittoral areas with complicated varied

conditions [1] and exhibits tolerance to various abiotic

factors such as light, dryness, salinity and temperature.

Trehalose is a non-reducing disaccharide sugar with the

two glucose units linked in a, a-1, 1 -glycosidic linkage. It

exists widely in organisms, including bacteria, yeast, fungi,

insects, invertebrates and plants [2]. In higher plants, tre-

halose functions as stress protection metabolite for struc-

ture stabilization in stress tolerance and as carbohydrate

storage [3, 4]. It also plays as signaling molecule in some

plant and yeast, and links trehalose metabolism to glucose

transport and glycolysis [4–9]. The biosynthesis of

Y. Deng � X. Wang � H. Guo � D. Duan (&)

Chinese Academy of Sciences, Institute of Oceanology,

Qingdao 266071, China

e-mail: dlduan@qdio.ac.cn

Y. Deng

e-mail: maimai0328@126.com

H. Guo

University of the Chinese Academy of Sciences,

Beijing 100049, China

123

Mol Biol Rep (2014) 41:529–536

DOI 10.1007/s11033-013-2888-5

trehalose has been best described in Escherichia coli and

Saccharomyces cerevisiae, and characterized with the tre-

halose-6-phosphate formation from glucose-6-phosphate

and UDP-glucose by trehalose-6-phosphate synthase (TPS)

followed by dephosphorylation to trehalose by the enzyme

trehalose-6-phosphate phosphatase (TPP) [10].

Generally, TPS is regarded as the crucial enzyme for

catalyzing the first step in trehalose synthesis. In Arabi-

dopsis, disruption of TPS1 led to embryo lethal phenotype

[8]. So far, TPS genes have been isolated from E. coli [11,

12], S. cerevisiae [13], Arabidopsis thaliana [8, 14, 15],

Selaginella lepidophylla [16], Gossypium hirsutum [17]

and Metarhizium anisopliae [18]. These TPS sequences

contained at least a TPS domain and most members also

included a C-terminal TPP domain. While in algae, only

Wang et al. [19] and Xuan et al. [20] preliminarily reported

TPS genes from 10 seaweed species, one of which was

from S. japonica (Accession: DQ666325) (SjTPS). All

these TPS genes encoded 908 amino acids and had only

two functional domains: one N-terminal TPS domain and

one C-terminal TPP domain. It is intrigued that they were

highly conserved both in nucleotide ([94 %) and in amino

acid sequence ([96 %). Recently, TPS orthologs were also

detected in genomes of the algal species Ectocarpus silicu-

losus (Accession: CBJ29609) (EsTPS) [21], Thalassiosira

pseudonana (Accession: XP_002288483) (TpTPS) [22] and

Phaeodactylum tricornutum (Accession: XP_002180425)

(PtTPS) [23]. However, compared with sequences of Wang

et al. [19], they only shared 42, 36, 34 % average amino acid

similarities, respectively.

With curiosity, we aimed to examine structure of TPS

gene of S. japonica and attempted to explore its physio-

logical functions, especially in desiccated condition. Based

on homologous cloning and the rapid amplification of

cDNA ends (RACE), the full-length cDNA of another TPS

gene of S. japonica (designated as SjaTPS) was isolated.

The transcription analysis of SjaTPS in desiccation and

in vitro prokaryotic expression were also conducted. The

new SjaTPS gene enriched our knowledge about TPS

properties in S. japonica, and helped us to better under-

stand the role trehalose plays in kelp abiotic tolerance for

adaption to the sublittoral habitats.

Materials and methods

Sample collection and treatment

Saccharina japonica sporophytes were collected from

cultivated rafts in Rongcheng, Shandong, China, in March,

2011. Healthy individuals were rinsed with sterilized sea-

water for several times to remove epiphytes. The washed

materials were put immediately into liquid nitrogen and

stored at -80 �C for the following RNA extraction.

For desiccation treatments, the washed materials were

precultured at 8 �C for 12 h, and then were under dryness

at 0 �C at about 40 % humidity for 0, 1, 2, 3, 4, 5, 6 and

7 h, respectively. All the treated materials were collected

for the following RNA extraction and transcriptional

analysis.

RNA extraction and cDNA synthesis

Total RNA extraction was conducted according to Yao

et al. [24], and was treated with RNase-free DNase I (Ta-

KaRa, Dalian, China) to remove residual genomic DNA.

The first strand cDNA was prepared using the PrimeScript

II cDNA synthesis kit (Takara, Tokyo, Japan) following the

manufacturer’s instructions and stored at -20 �C.

SjaTPS cDNA cloning

One pair of specific primers, P1 (50-GTGAGGCCTCCGC

CAGCTTACGAGC-30) and P2 (50-GATCTGTTCGAAGT

AAGCGCTCATCGTCCC-30), were designed based on the

TPS sequence from brown alga E. siliculosus (Accession:

CBJ29609) for amplifying SjaTPS cDNA fragment about

3,800 bp. PCR was performed in a 25 ll reaction volume,

which contained 12.5 ll 29 TransTaqTM High Fidelity

(HiFi) PCR SuperMix (TransGen Biotech, Beijing, China),

1 ll of S. japonica cDNA (30 ng/ll), 1 ll of each primer

(10 lM) and 9.5 ll of RNase-free water. The amplification

protocol was conducted at 94 �C for 5 s, followed by 35

cycles of 95 �C for 30 s and 55 �C for 30 s, 72 �C 2 min,

and a final extension at 72 �C for 10 min. The PCR pro-

ducts were subcloned into pMD-19T vector (Takara,

Tokyo, Japan) for sequencing (BGI, Qingdao, China).

Rapid amplification of SjaTPS cDNA ends

SmartTM RACE cDNA Amplification Kit (Clontech) was

used for the RACE according to the manufacturer instruc-

tion. Nested-PCR amplification was carried out to clone the

30 end using primers P3 (50-TTGTGATGCCAACGCACCT

CCTTCCC-30) and P4 (50-GGGACGATGAGCGCTTAC

TTCGAACAGA-30), while primers P5 (50-AACGGACCCT

TCGAGCTACCCGGTG-30) and P6 (50-CTGGGGCACGG

GGAAGTGATTTACG-30) were designed to generate the 50

end. The products were migrated on 1 % agarose gel and the

objective bands were selected and purified with agarose gel

DNA fragment recovery kit (TaKaRa, ToKyo, Japan), then

was subcloned into pMD-19T vector (TaKaRa, Tokyo,

Japan) and sequenced (BGI, Qingdao, China).

530 Mol Biol Rep (2014) 41:529–536

123

Analysis of SjaTPS deduced amino acid sequence

The cDNA sequence and deduced amino acid sequence of

SjaTPS were analyzed by using the ORF Finder and BLAST

algorithm [25]. The yielded amino acid sequence was pre-

dicted for theoretical molecular weight and isoelectric point

using the ProtParam softwares [26]. The signal peptide was

predicted with SignalP 4.0 Server [27]. TMHMM Server

version 2.0 program [28] was used to analyze transmem-

brane topological structure. SOPMA program [29] was

applied to predict secondary structure of SjaTPS.

Phylogenetic analysis of SjaTPS

For phylogenetic analysis, together with the known 13 TPS

sequences from other algae species, which including Pha-

eophyta: E. siliculosus (Accession: CBJ29609), S. japonica

(Accession: DQ666325), Sargassum henslowianum (Acces-

sion: GQ352536), Undaria pinnatifida (Accession: GQ35

2535); Bacillariophyta: T. pseudonana (Accession: XP_0022

88483), P. tricornutum (Accession: XP_002180425); Rho-

dophyta: Porphyra yezoensis (Accession: AY729671),

P. haitanensis (Accession: DQ666326), Chondrus ocellatus

(Accession: DQ666328), Gracilaria lemaneiformis (Acces-

sion: DQ666327); and Chlorophyta: Monostroma angicava

(Accession: DQ666324), Ulva prolifera (Accession:

DQ666330), U. pertusa (Accession: DQ666329) were adop-

ted for the analysis. Multiple sequence alignments and cluster

analysis were carried out by DNAMAN software (Version 6,

Lynnon Corporation).

Quantitative PCR analysis of SjaTPS mRNA expression

qPCR was performed with the SYBR� Premix Ex TaqTM

(TakaRa, Tokyo, Japan) on the Takara TP800 Thermal

Cycler DiceTM (TakaRa, Tokyo, Japan). Two specific

primers, qSjaTPS-F (50- CGAGCGGGAACAGGACTA-30)and qSjaTPS-R (50- CTCGCACTGCCGTGTTTAT-30)were used to amplify the 186 bp product. Another pair of

primer, b-actin-F and b-actin-R was applied b-actin frag-

ment of 184 bp as the internal control [30]. Specificity of

each pair of primers was examined by the dissociation

curve. All the reactions were performed in biological

triplicates, and the results were expressed relative to the

expression levels of b-actin in each sample by using the

2-DDCt method [31].

In vitro expression of SjaTPS in E. coli

One pair of specific primer, SjaTPS-F (50-ATGGTGAGG

CCTCCGCCAGCTTACGA-30) and SjaTPS-R (50- CGGG

CTGGAGGCTGTCCGTCTCGTCG-30) was used to

amplify the SjaTPS cDNA fragment of 3,000 bp, which

contained the initiator ATG codon and the three conserved

domains. The objective clone confirmed by sequencing

(BGI, Qingdao, China) was subcloned into the pEASY-E2

expression vector with a His-tag (TransGen Biotech, Bei-

jing, China). The valid recombinant plasmid was then

transformed into E. coli expression strain Transetta (DE3)

(TransGen Biotech, Beijing, China). Positive transformants

were incubated in Lysogeny broth (LB) medium (con-

taining 0.1 mg/ml ampicillin) at 37 �C with shaking at

170 rpm. When the culture medium reached OD600 of

0.5–0.7, the cells were incubated for additional 5 h with the

induction of isopropyl-b-D-thiogalactopyranoside (IPTG)

at the final concentration of 1.5 mM. Cultures were then

collected, lysed by Sodium dodecyl-sulfate (SDS) poly-

acrylamide gel electrophoresis loading buffer (Tiandz,

Beijing, China) and separated on the 7.5 % SDS-PAGE

(ATTO, Tokyo, Japan). The expression of the empty vector

and positive recombinant without induction were used as

two negative tests.

Western blot analysis

After 7.5 % SDS-PAGE electrophoresis, the sample (5 h

after 1.5 mM IPTG induction) was electroblotted onto

nitrocellulose membrane using the Idea electrophoresis

system (Idea scientific, MN). The membrane was blocked

with blocking solution (19 PBS, 0.1 % Tween-20 and 5 %

nonfat dry milk powder) and incubated with primary anti-His

mouse monoclonal antibody (1:1,000; TransGen Biotech,

Beijing, China) under gentle agitation for 2 h at room tem-

perature. After rinsing with PBST buffer, the membrane was

exposed to horseradish peroxidase conjugated secondary

antibody (IgG goat anti-mouse; 1:2,000; TransGen Biotech,

Beijing, China) for 1 h at room temperature. DAB sensitive

chromogenic reaction (Tiandz, Beijing, China) was used to

detect the specific blot, and the membrane was transferred

into deionized water for the termination of reaction.

Results

Characterization of deduced SjaTPS protein sequence

The products of 50 RACE with primers P5 and P6, and 30

RACE with primers P3 and P4 were 479 bp and 137 bp in

length, respectively. The complete sequence was obtained

by overlapping the two fragments with the 3,747 bp frag-

ment amplified by primers P1 and P2. The full-length of

SjaTPS cDNA was 4,127 bp, characterized with a 50 ter-

minal untranslated region (UTR) of 320 bp, a 30 UTR of

21 bp, and an open reading frame (ORF) of 3,786 bp. The

complete sequence of SjaTPS was deposited in GeneBank

with accession number KC578568.

Mol Biol Rep (2014) 41:529–536 531

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The ORF of SjaTPS (3,786 bp) encoded a 1261 amino

acids polypeptide with predicted molecular weight of

137.84 kDa and theoretical isoelectric point of 7.12.

Maximal values of the original shearing site (C score),

signal peptide (S score), and synthesized shearing site (Y

score) were 0.208, 0.115 and 0.143, respectively, which

indicated that there was no signal peptide in the amino acid

sequence. The transmembrane topological structure ana-

lysis inferred SjaTPS was outside the membrane. The

secondary structure prediction showed that the ratios of a-

helix, extended strand, b-turn and random coil were 41.32,

14.83, 5.47 and 38.38 %, respectively.

Structural and phylogenetic analysis of SjaTPS

BLAST analysis revealed that the deduced amino acid

sequence of SjaTPS included an N-terminal CBM20 (family

20 carbohydrate-binding module) domain, a TPS domain in

the middle region and a TPP domain near the C-terminus

(Fig. 1). In the CBM20 domain, the putative carbohydrate-

binding site 1 and site 2 were composed of 4 (Lys36, Arg70,

Trp82, Glu83) and 9 (Leu18, Gly19, His20, Gly21, Glu22,

Val23, Thr46, Pro52, Trp54) amino acid residues, respectively

(Fig. 2); 13 amino acid residues (Phe181, Ser202, Leu203,

Tyr258, Gly322, His324, His346, Lys460, Lys465, Leu544,

Asn564, Leu565, Glu568) were presumed to be associated

with the UDP and glucose-6-phosphate binding and catalysis

in the TPS domain (Fig. 2); while in the TPP domain, 3 con-

served motifs characteristic of the L-2-haloacid dehalogenase

(HAD) superfamily were deduced (Fig. 2).

Homology analysis showed that the amino acid similari-

ties between SjaTPS and the known 13 TPS sequences of

algal species ranged from 49 to 70 %. SjaTPS was clustered

into one clade with those of brown alga E. siliculosus and

diatoms T. pseudonana, P. tricornutum; while the remaining

10 sequences were grouped in another clade (Fig. 3). Con-

trary to average 57 % similarities with other 13 orthologs,

SjaTPS had 70 % similarities with EsTPS; In addition, only

55 % similarities were revealed between our SjaTPS and the

previous reported SjTPS (Accession: DQ666325).

Transcriptional analysis of SjaTPS

For both SjaTPS and b-actin genes, there was only one

peak at the corresponding melting temperature in the dis-

sociation curve analysis, which indicated that the qPCR

was specifically amplified. The mRNA accumulations were

dramatically up-regulated with increasing of desiccation

times from 1 to 3 h. The transcriptions reached the maxi-

mum at 3 h, which was about 300-fold compared to that of

start (Fig. 4). After that, the transcript level gradually

decreased.

SDS-PAGE assay of the recombinant SjaTPS protein

After 5 h induction with 1.5 mM IPTG, the recombinant

proteins were separated on the SDS-PAGE and one distinct

band about 115 kDa was identified (Fig. 5a).

Fig. 1 Schematic structure of SjaTPS. The CBM20, TPS and TPP domains are highlighted in dark gray background. The predicted molecular

weight of deduced amino acids polypeptide is shown on the right

Fig. 2 The deduced amino acid sequences of SjaTPS (Genbank

Accession: KC578568). The amino acid residues are numbered on the

left. Three conserved domains are shown in dark gray background. In

the CBM20 domain (11–86 aa), residues composed of carbohydrate-

binding site 1 and 2 are marked with underlines and triangles,

respectively; In the TPS domain (175–653 aa), the active sites are

highlighted with arrows; In the TPP domain (698–964 aa), 3

conserved motifs typical of the HAD superfamily are boxed

532 Mol Biol Rep (2014) 41:529–536

123

No obvious expression of 115 kDa was observed in the

control tests (positive recombinant without IPTG induction

and empty vector with IPTG induction). Western blot

indicated that the 115 kDa band was positive to the anti-

His antibody with high specificity (Fig. 5b).

Discussion

Structural characterization of SjaTPS

Similar to the EsTPS [21], the SjaTPS contained one

N-terminal CBM20 domain, a middle TPS domain and a

TPP domain near the C-terminus (Fig. 1). About 70 %

amino acid similarities were detected between the two

sequences, which coincided with their phylogenetic affinity

in the dendrogram result (Fig. 3). It was reported that the

TPS and TPP were encoded by multi-gene family [32]. 11

TPSs, 10 TPPs and 9 TPSs, 9 TPPs have been identified in

Arabidopsis and Oyrza genomes, respectively [33, 34].

Referred to our reported transcriptome data of S. japonica

(Accession: GSE33853) [30], several unigenes identified

with partial sequence of SjaTPS were found, suggested that

the SjaTPS was probably another TPS gene of S. japonica.

Wang et al. [19] previously isolated TPS sequences from

10 seaweed species, one of which was the SjTPS [19, 20].

All the 10 sequences only included one N-terminal TPS

domain and one C-terminal TPP domain. Homology

comparison indicated that they shared over 96 % amino

acid similarities [19]. Our new generated SjaTPS appar-

ently exhibited distinct structural characters, and shared

less amino acid similarity (only about 55 %). Much more

works on additional typical seaweeds might shed more

light on the algal TPS properties in the future.

Fig. 3 A dendrogram analysis of 14 TPS genes from algal species

using the DNAMAN program. The new sequence submitted in this

study is in bold. Es: Ectocarpus siliculosus (Accession: CBJ29609);

Tp: Thalassiosira pseudonana (Accession: XP_002288483); Pt:

Phaeodactylum tricornutum (Accession: XP_002180425); Py: Por-

phyra yezoensis (Accession: AY729671); Ph: Porphyra haitanensis

(Accession: DQ666326); Co: Chondrus ocellatus (Accession:

DQ666328); Gl: Gracilaria lemaneiformis (Accession: DQ666327);

Ma: Monostroma angicava (Accession: DQ666324); Upr: Ulva

prolifera (Accession: DQ666330); Upe: Ulva pertusa (Accession:

DQ666329); Sj: Saccharina japonica (Accession: DQ666325); Sh:

Sargassum henslowianum (Accession: GQ352536); Unp: Undaria

pinnatifida (Accession: GQ352535)

Fig. 4 Relative mRNA expression of SjaTPS gene at different

desiccation times. Values are mean ± standard deviation, n = 3

Fig. 5 SDS-PAGE (7.5 %) and western blot analysis of SjaTPS

recombinant protein. a SDS-PAGE analysis of the induced recombi-

nant SjaTPS. M, protein marker; lane 1, expression of recombinant

protein after 1.5 mM IPTG induction for 5 h; lane 2, negative control

(positive recombinant without IPTG induction); lane 3, negative

control (empty vector with IPTG induction). b Western blot analysis

of SjaTPS recombinant protein. M, protein marker; lane 1, western

blot result based on the induced recombinant

Mol Biol Rep (2014) 41:529–536 533

123

Functional deducing of SjaTPS

The CBM20 domain has been classically characterized in

fungi and known as starch-binding domain [35–37]. It was

found in many starch degrading enzymes which played reg-

ulatory role in starch metabolism in plants (such as a-amylase)

[37]. It was known that CBM20 s employed 2 carbohydrate-

binding sites to provide bivalent interaction with 2 or 3 con-

served solvent accessible aromatic residues [35, 36]. Site 1

was shallower and more solvent exposed than site 2 which

undergone significant structural changes upon binding [36]. In

the SjaTPS, carbohydrate-binding site 1 composed of 4 amino

acid residues and site 2 contained 9 residues were deduced

(Fig. 2), which suggested the SjaTPS-CBM20 domain could

promote the recognition and binding of laminaran. In plant and

yeast, many reports showed that TPS played roles in sugar

metabolism and glycolysis regulation [4–9]. Functional ana-

lysis of unigenes relevant to the SjaTPS suggested that they

were closely related to starch and sucrose metabolism [30].

The presence of CBM20 domain implied that SjaTPS might

implicate in laminaran metabolism in S. japonica, future

investigations are needed to test the hypothesis.

Trehalose is synthesized from UDP glucose and glucose-

6-phosphate in two reactions catalyzed by TPS and TPP. In

E. coli, the two enzymes were separate entities; in yeast, the

two activities resided in a large complex together with a

regulatory subunit [38]. All the known plant TPS protein had

the two conserved regions (TPS and TPP domains), which

implied that fusion of TPS genes might occurred in plant

[38]. To our data, BLAST analysis revealed that one TPS

domain coexisted with a putative TPP domain in the SjaTPS

(Fig. 1). TPS domain represents the catalytic region of the

TPS enzyme. In the SjaTPS-TPS domain, the 13 residues

were presumed to involve with binding and catalysis of UDP

and glucose-6-phosphate (Fig. 2), which suggested that the

SjaTPS functioned as TPS; on the other hand, similar to TPS

group I in A. thaliana [33, 39], the deduced SjaTPS-TPP

domain lacked the two consensus sequences of phospha-

tases. However, it had 3 conserved motifs characteristic for

the L-2-haloacid dehalogenase (HAD) superfamily (Fig. 2),

which embraced a broad range of phosphatases and hydro-

lases [30–42]. The presence of motifs formed the active sites

of HAD enzyme prompted the speculation that the SjaTPS

might have TPP activity. Nevertheless, no such activity were

found in the group II of A. thaliana which also had the 3

conserved motifs of HAD [33], further work are remained to

explore the function of SjaTPS-TPP domain.

Expression levels of SjaTPS with desiccation

treatments

The transcriptional analysis of SjaTPS was conducted under

different desiccation times. Up-regulated transcriptions

appeared in 1–7 h treatments (Fig. 4), which implied that

SjaTPS was positive gene associated with desiccation.

Desiccation significantly elevated SjaTPS transcription in

3 h, and the maximum production occurred at 3 h was over

300-fold compared to that of start. It seemed that SjaTPS was

most probably involved in the kelp drought adaption. Nat-

urally, S. japonica niches on the substratum in sublittoral

areas and the exposure duration varies with tidal oscillations

and seasonal changes. Here the SjaTPS transcription mark-

edly increased by desiccation indirectly reflected the well

adaption of S. japonica to the sublittoral environment.

Ecologically, seaweeds experience various natural stres-

ses such as temperature, ultraviolet radiation and desiccation

duration. Adverse stresses influenced the algal physiological

processes and resulted in adapted molecular pathways

through regulation of stress responsive genes [43, 44]. Tre-

halose was reported to accumulate in many organisms for

drought tolerance, and functioned as osmoprotectant for

membranes preserved during desiccation stress [3, 45].

Transgenic plants overexpressing microbial trehalose bio-

synthesis genes exhibited increased levels of trehalose and

tolerances to drought, salt and cold [34]. To our SjaTPS, it

was hypothesized as stress responsive gene in S. japonica

which might play roles in adverse stress resistance. However,

intensive works are necessary to confirm the hypothesis.

Prokaryotic expression of recombinant SjaTPS protein

Primers (SjaTPS-F and SjaTPS-R) were used to generate

the SjaTPS cDNA fragment of 3,033 bp which included the

initiator ATG codon and the 3 function domains. The

fragment encoded 1011 amino acids with predicted

molecular weight of 111.23 kDa. The recombinant protein

was expressed in E. coli and induced by 1.5 mM IPTG for

5 h. Obvious band at *115 kDa appeared on the SDS-

PAGE electrophesis (7.5 %) and western blotting detection

(Fig. 5), which coincided with the theoretical molecular

mass, and demonstrated the successful heterologous

expression of SjaTPS. Further work are needed to purify

the recombinant SjaTPS protein, and Km, Vm and Kcat

parameters are warranted to test its properties.

Acknowledgments This research was supported by National Natural

Science Foundation of China (No. 40976085) and Shandong Agriculture

Breeding Engineering Biological Resources Innovation of Research

Project and National High Tech 863 Project (2012AA10A406). Sincerely

thanks are due to Lin Xiao, Jin Zhao, Ge Liu for their help with the

experiments. The authors acknowledged the anonymous reviewers for

the critical comments and suggestions for the manuscript.

References

1. Steneck RS, Graham MH, Bourque BJ, Corbett D, Erlandson

JM, Estes JA, Tegner MJ (2002) Kelp forest ecosystems:

534 Mol Biol Rep (2014) 41:529–536

123

biodiversity, stability, resilience and future. Environ Conserv

29:436–459

2. Elbein AD (1974) The metabolism of a, a-trehalose. Adv Car-

bohyd Chem Bi 30:227–256

3. Crowe JH, Crowe LM, Chapman D (1984) Preservation of

membranes in anhydrobiotic organisms: the role of trehalose.

Science 223:701–703

4. Goddijn OJM, van Dun K (1999) Trehalose metabolism in plants.

Trends Plant Sci 4:315–319

5. Thevelein JM, Hohmann S (1995) Trehalose synthase: guard to

the gate of glycolysis in yeast? Trends Biochem Sci 20:3–10

6. Bonini BM, Van Vaeck C, Larsson C, Gustafsson L, Ma P,

Winderickx J, Van Dijck P, Thevelein JM (2000) Expression of

Escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant

restores trehalose 6-phosphate levels and partly restores growth

and fermentation with glucose and control of glucose influx into

glycolysis. Biochem J 350:261–268

7. Noubhani A, Bunoust O, Rigoulet M, Thevelein JM (2000)

Reconstitution of ethanolic fermentation in permeabilized

spheroplasts of wild-type and trehalose-6-phosphate synthase

mutants of the yeast Saccharomyces cerevisiae. Eur J Biochem

267:4566–45576

8. Eastmond PJ, van Dijken AJ, Spielman M, Kerr A, Tissier AF,

Dickinson HG, Jones JD, Smeekens SC, Graham IA (2002)

Trehalose-6-phosphate synthase 1, which catalyses the first step

in trehalose synthesis, is essential for Arabidopsis embryo mat-

uration. Plant J 29:225–235

9. Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on

trehalose: a multifunctional molecule. Glycobiology 13(4):17R–27R

10. Cabib E, Leloir LF (1958) The biosynthesis of trehalose-6-

phosphate. J Biol Chem 231:259–275

11. Giaever HM, Styrvold OB, Kaasen I, Strøm AR (1988) Bio-

chemical and genetic characterization of osmoregulatory treha-

lose synthesis in Escherichia coli. J Bacteriol 170:2841–2849

12. Strøm AR, Kaasen I (1993) Trehalose metabolism in Escherichia

coli: stress protection and stress regulation of gene expression.

Mol Microbiol 8:205–210

13. De Virgilio C, Burckert N, Bell W, Jeno P, Boller T, Wiemken A

(1993) The role of trehalose synthesis for the acquisition of

thermotolerance in yeast. I. Genetic evidence that trehalose is a

thermo protectant. Eur J Biochem 212:315–323

14. Blazquez MA, Santos E, Flores CL, Martinez-Zapater JM, Sali-

nas J, Gancedo C (1998) Isolation and molecular characterization

of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate

synthase. Plant J 13:685–689

15. Vandesteene L, Ramon M, Roy KL, Van Dijck P, Rolland F

(2010) A single active trehalose-6-phosphate synthase (TPS) and

a family of putative regulatory TPS-like protein in Arabidopsis

thaliana. Mol Plant 3:406–419

16. Zentella R, Mascorro-Gallardo JO, Van Dijck P, Folch-Mallol J,

Bonini B, Van Vaeck C, Gaxiola R, Covarrubias AA, Nieto-

Sotelo J, Thevelein JM, Iturriaga GA (1999) Selaginella lepido-

phylla trehalose-6-phosphate synthase complements growth and

stress-tolerance defects in a yeast tps1 mutant. Plant Physiol

119:1473–1482

17. Kosmas SA, Argyrokastritis A, Loukas MG, Eliopoulos E, Tsa-

kas S, Kaltsikes PJ (2006) Isolation and characterization of

drought-related trehalose 6-phosphate-synthase gene from culti-

vated cotton (Gossypium hirsutum L.). Planta 223:329–339

18. Cai Z, Peng G, Liu Y, Cao Y, Jin K, Xia Y (2009) Trehalose-6-

phosphate synthase 1 from Metarhizium anisopliae: clone, expres-

sion and properties of recombinants. J Biosci Bioeng 107:499–505

19. Wang G, Zhao G, Feng Y, Xuan J, Sun J, Guo B, Jiang G, Weng

M, Yao J, Wang B, Duan D, Liu T (2010) Cloning and com-

parative studies of seaweed trehalose-6-phosphate synthase gene.

Mar Drugs 8:2065–2079

20. Xuan J, Feng Y, Weng M, Zhao G, Shi J, Yao J, Wang X, Guo B, Qiao

L, Duan D, Wang B (2012) Expressed sequence tag analysis and

cloning of trehalose-6-phosphate synthase gene from marine alga

Laminaria japonica (Phaeophyta). Acta Oceanol Sinica 31(6):1–10

21. Cock JM, Streck L, Rouze P, Scornet D, Allen AE, Amoutzias G,

Anthouard V, Artiguenave F, Aury JM, Badger JH, Beszteri B,

Billiau K, Bonnet E, Bothwell JH, Bowler C, Boyen C, Brownlee

C, Carrano CJ, Charrier B, Cho GY, Coelho SM, Collen J, Corre

E, Da Silva C, Delage L, Delaroque N, Dittami SM, Doulbeau S,

Elias M, Farnham G, Gachon CM, Gschloessl B, Heesch S,

Jabbari K, Jubin C, Kawai H, Kimura K, Kloareg B, Kupper FC,

Lang D, Le Bail A, Leblanc C, Lerouge P, Lohr M, Lopez PJ,

Martens C, Maumus F, Michel G, Miranda-Saavedra D, Morales

J, Moreau H, Motomura T, Nagasato C, Napoli CA, Nelson DR,

Nyvall-Collen P, Peters AF, Pommier C, Potin P, Poulain J,

Quesneville H, Read B, Rensing SA, Ritter A, Rousvoal S, Sa-

manta M, Samson G, Schroeder DC, Segurens B, Strittmatter M,

Tonon T, Tregear JW, Valentin K, von Dassow P, Yamagishi T,

Van de Peer Y, Wincker P (2010) The Ectocarpus genome and

the independent evolution of multicellularity in brown algae.

Nature 465:617–621

22. Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D,

Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M, Brzezinski

MA, Chaal BK, Chiovitti A, Davis AK, Demarest MS, Detter JC,

Glavina T, Goodstein D, Hadi MZ, Hellsten U, Hildebrand M,

Jenkins BD, Jurka J, Kapitonov VV, Kroger N, Lau WWY, Lane

TW, Larimer FW, Lippmeier JC, Lucas S, Medina M, Montsant

A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM,

Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Val-

entin K, Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome

of the diatom Thalassiosira pseudonana: ecology, evolution, and

metabolism. Science 306:79–86

23. Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A,

Maheswari U, Martens C, Maumus F, Otillar RP, Rayko E,

Salamov A, Vandepoele K, Beszteri B, Gruber A, Heijde M,

Katinka M, Mock T, Valentin K, Verret F, Berges JA, Brownlee

C, Cadoret J-P, Chiovitti A, Choi CJ, Coesel S, De Martino A,

Detter JC, Durkin C, Falciatore A, Fournet J, Haruta M, Huysman

MJJ, Jenkins BD, Jiroutova K, Jorgensen RE, Joubert Y, Kaplan

A, Kroger N, Kroth PG, La Roche J, Lindquist E, Lommer M,

Martin-Jezequel V, Lopez PJ, Lucas S, Mangogna M, Mcginnis

K, Medlin LK, Montsant A, Oudot-Le Secq M-P, Napoli C,

Obornik M, Parker MS, Petit J-L, Porcel BM, Poulsen N, Robison

M, Rychlewski L, Rynearson TA, Schmutz J, Shapiro H, Siaut M,

Stanley M, Sussman MR, Taylor AR, Vardi A, Von Dassow P,

Vyverman W, Willis A, Wyrwicz LS, Rokhsar DS, Weissenbach

J, Armbrust EV, Green BR, Van De Peer Y, Grigoriev IV (2008)

The Phaeodactylum genome reveals the evolutionary history of

diatom genomes. Nature 456:239–244

24. Yao JT, Fu WD, Wang XL, Duan DL (2009) Improved RNA

isolation for Laminaria japonica Aresch (Laminariaceae, Pha-

eophyta). J Appl Phycol 21:233–238

25. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z,

Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a

new generation of protein database search programs. Nucleic

Acids Res 25:3389–3402

26. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR,

Appel RD, Bairoch A (2005) In: Walker JM (ed) The proteomics

protocols handbook. Humana Press, New Jersey

27. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP

4.0: discriminating signal peptides from transmembrane regions.

Nat Methods 8:785–786

28. Krogh A, Larsson B, von Heijne B, Sonnhammer ELL (2001)

Predicting transmembrane protein topology with a hidden Mar-

kov model: application to complete genomes. J Mol Biol 305(3):

567–580

Mol Biol Rep (2014) 41:529–536 535

123

29. Geourjon C, Deleage G (1995) SOPMA: significant improve-

ments in protein secondary structure prediction by consensus

prediction from multiple alignments. Comput Appl Biosci 11:

681–684

30. Deng Y, Yao J, Wang X, Guo H, Duan D (2012) Transcriptome

sequencing and comparative analysis of Saccharina japonica

(Laminariales, Phaeophyceae) under blue light induction. PLoS

ONE 7(6):e39704

31. Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ,

Reed MW (2000) Quantitative reverse transcription-polymerase

chain reaction to study mRNA decay: comparison of endpoint

and real-time methods. Anal biochem 285:194–204

32. Lunn JE (2007) Gene families and evolution of trehalose

metabolism in plants. Funct Plant Biol 34:550–563

33. Leyman B, Dijck P, Thevelein JM (2001) An unexpected plethora

of trehalose biosynthesis genes in Arabidopsis thaliana. Trends

Plant Sci 6:510–513

34. Iordachescu M, Imai R (2008) Trehalose biosynthesis in response

to abiotic stresses. J Integr Plant Biol 50(10):1223–1229

35. Penninga D, van der Veen BA, Knegtel RMA, van Hijum SAFT,

Rozeboom HJ, Kalk KH, Dijkstra BW, Dijkhuizen L (1996) The

raw starch binding domain of cyclodextrin glycosyltransferase from

Bacillus circulans strain 251. J Biol Chem 271:32777–32784

36. Sorimachi K, Le Gal-Coeffet MF, Williamson G, Archer DB,

Williamson MP (1997) Solution structure of the granular starch

binding domain of Aspergillus niger glucoamylase bound to b-

cyclodextrin. Structure 5:647–661

37. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbo-

hydrate-binding modules: fine tuning polysaccharide recognition.

Biochem J 382:769–781

38. Eastmond PJ, Li Y, Graham IA (2003) Is trehalose-6-phosphate a

regulator of sugar metabolism in plants? J Exp Bot 54:533–537

39. Thaller MC, Schippa S, Rossolini GM (1988) Conserved

sequence motifs among bacterial, eukaryotic, and archaeal

phosphatases that define a new phosphohydrolase superfamily.

Protein Sci 7:1647–1652

40. Collet JF, Stroobant V, Pirard M, Delpierre G, Van Schaftingen E

(1998) A new class of phosphotransferases phosphorylated on an

aspartate residue in an amino-terminal DXDX (T/V) motif. J Biol

Chem 273:14107–14112

41. Fieulaine S, Lunn JE, Ferrer JL (2005) The structure of a

cyanobacterial sucrose-phosphatase reveals the sugar tongs that

release free sucrose in the cell. Plant Cell 17:2049–2058

42. Burroughs AM, Allen KN, Dunaway-Mariano D, Aravind L

(2006) Evolutionary genomics of the HAD superfamily: under-

standing the structural adaptations and catalytic diversity in a

superfamily of phosphoesterases and allied enzymes. J Mol Biol

361:1003–1034

43. Collen J, Guisle-Marsollier I, Leger JJ, Boyen C (2007) Response

of the transcriptome of the intertidal red seaweed Chondrus

crispus to controlled and natural stresses. New Phytol 176:45–55

44. Dittami SM, Scornet D, Petit J, Segurens B, Silva CD, Corre E,

Dondrup M, Glatting K, Konig R, Sterck L, Rouze P, Van de Peer

Y, Cock JM, Boyen C, Tonon T (2009) Global expression ana-

lysis of the brown alga Ectocarpus siliculosus (Phaeophyceae)

reveals large-scale reprogramming of the transcriptome in

response to abiotic stress. Genome Biol 10:R66

45. Adams RP, Kendall E, Kartha KK (1990) Comparison of free

sugars in growing and desiccated plants of Selaginella lepido-

phylla. Biochem Syst Ecol 18:107–110

536 Mol Biol Rep (2014) 41:529–536

123