Cloning and pattern of expression of trehalose-6-phosphate synthase cDNA from Catantops pinguis ( Orthoptera : Catantopidae )

Trehalose is not only an important disaccharide, but also a key stress resistance factor in the development of many organisms, including plants, bacteria, fungi, and insects. To study the potential function of trehalose in development and behaviour, cDNA for a trehalose-6-phosphate synthase from Catantops pinguis (CpiTPS) was cloned and sequenced. Results revealed that the CpiTPS cDNA sequence contains an open reading frame of 2430 nucleotides encoding a protein of 809 amino acids with a predicted molecular weight of 91.13 kDa and a pI value of 6.25. Northern blot and RT-PCR analyses showed that CpiTPS mRNA expression was high in the fat body and testes, ovaries, Malpighian tubules, brain, trachea, rectum, and posterior extensor of C. pinguis. Northern blotting revealed that CpiTPS mRNA was expressed in the fat body at different developmental stages and was present at a high level in first instar larvae and adults. The results demonstrate that CpiTPS is a key gene in C. pinguis development. 355 * Corresponding authors; e-mails: SGW: sgwang@mail.hz.zj.cn; ZHZ: lgbcc@263.net level evidence for this is lacking. During this process, TPS and TPP both play an important role in the regulation of trehalose production for growth and development (Elbein et al., 2003; Wang et al., 2005b). Chen et al. (2002) and Chen & Haddad (2004) report that trehalose can serve as a source of energy and accumulates dramatically in HEK-293 cells, increasing tolerance to hypoxia and anoxia and protecting cells and cellular proteins against damage. To date, although many TPS genes have been cloned or deduced from genomic sequences, there are few such studies on insects, mainly Drosophila melanogaster (Chen et al., 2002, 2003; Chen & Haddad, 2004). TPP and TPS genes have been cloned from plants, bacteria, and fungi, but none have been identified in mammals. In arthropods, TPS and TPP have been cloned or deduced from genomic sequences (according to GenBank) and are known to be very important for insect development; however, there are only a few studies on TPS genes and the details of protein characteristics in insects: D. melanogaster (Chen et al., 2002), Callinectes sapidus (Chung, 2008), Locusta migratoria (Cui et al., 2009), Helicoverpa armigera (Xu et al., 2009), and Spodoptera exigua (Tang et al., 2010). Catantops pinguis (syn. Diabolocatantops pinguis) is a worldwide pest of agriculture and is a very strong flyer which requires a lot of energy, so trehalose synthesis is important in C. pinguis. In the present study, TPS cDNA from C. pinguis was cloned (GenBank accession no. GQ389790) and the tissue distribution and pattern of expression of this gene investigated. MATERIAL AND METHODS C. pinguis were maintained in the laboratory over a period of 1 year. Larvae were reared at 26 ± 1°C, 65% humidity and an 14L : 10D photoperiod on a diet of wheatgrass. The developmental stages were synchronized at each moult by collecting new adult larvae. The brain, trachea, midgut, rectum, testes, ovaries, Malpighian tubules, posterior extensor, heart, body wall, and fat body of adults, and the fat body of different stages, from larvae to adults, were dissected in saline containing 0.75% NaCl and stored at –80°C until required (Kumar et al., 2008). RNA isolation and cDNA synthesis Total RNA was extracted from the fat body of adult C. pinguis using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski & Sacchi, 1987). Fat body (100 mg) was homogenized in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) and placed on ice for 5 min, and then sodium acetate and chloroform/isoamyl alcohol (49 : 1) were added. The mixture was centrifuged at 10,000 × g at 4°C for 20 min. The supernatant was transferred to a new tube and isopropanol added. After centrifugation, the RNA pellet was washed with 75% ethanol and then dissolved in ddH2O. A sample of 1 mg of total RNA was reverse transcribed at 42°C for 1 h in 10 μl of reaction solution containing reaction buffer, 10 mM DTT, 0.5 mM dNTPs, 0.5 μg of oligo-dT18, and 5 unit reverse transcriptase from avian myeloblastosis virus (AMV, Takara, Dalian, China). Primer design and PCR Four degenerate primers (TPS-DF1, TPS-DF2, TPS-DR1, and TPS-DR2; Table 1) were designed based on the conserved amino acid sequences of known TPS forms. The first PCR reaction was performed with primers TPS-DF1 and TPS-DR1 using the following conditions: three cycles of 40 s at 94°C, 40 s at 45°C, and 120 s at 72°C, followed by 30 cycles of 40 s at 94°C, 40 s at 48°C, and 120 s at 72°C. A second PCR was carried out using the nested primers TPS-DF2 and TPS-DR2 using the same conditions as for the first PCR (Rinehart et al., 2003; Tang et al., 2008). The PCR products were subjected to agarose electrophoresis. A strong DNA band corresponding to the expected size of approximately 1700 bp was excised from the agarose gel and purified using a DNA gel extraction kit (Takara, Dalian, China). The PCR products were cloned into the pMD18-T vector (Takara, Dalian, China) and sequenced using the dideoxynucleotide method (Takara, Dalian, China). 356 The full-length CpiTPS cDNA sequence was determined by sequencing two overlapping PCR fragments using fat body cDNA as a template. The probe primers were for Northern blot and RT-PCR experiment. a F – Forward; R – Reverse; b D – degenerate primer; G – gene specific primer; A – anchor primer. GCA AAG CCT CCG GTA CAG TG G F CpiTPS-3F3 AGC TGG ATC TGC CCA CTG TG G F CpiTPS-3F2 CTC TGA TTG AGC AGG CAG GA G F CpiTPS-3F1 3’-RACE AAG CAG TGG TAT CAA CGC AGA GT A F CTG TCT GAC CCA ATT TGC TG G R CpiTPS-5R3 TGC TGT TGA CTG CTC CTC CA G R CpiTPS-5R2 AGC AAA CAG TTC ATT CAC AC G R CpiTPS-5R1 5’-RACE CGT CAT AGT CCA GCA GCA GG G R CpiTPS-RP CTG CTG GTC GAA CAC GGT GG G F CpiTPS-FP Probe ATC YTR ATN CKT TCR CTC C D R TPS-DR2 GCC ATW CCY TTS ARV GCC D R TPS-DR1 GCT GYA ACG VNA CBT TYT GG D F TPS-DF2 TGG NNT NTG GGT NGG HTG G D F TPS-DF1 Intermediate fragments Nucleotide sequence (5’–3’) Type Direction Name Primers PCR fragment TABLE 1. The primers for CpiTPS cDNA cloned, Northern blot, and RT-PCR.


INTRODUCTION
Abiotic stress is the most important limiting constraint on crop productivity and yield, and could compromise food supply for the ever-increasing human population in the future.Organisms have evolved two different strategies to cope with abiotic stress (Avonce et al., 2006).First, some species that live in extreme environments can modify features of their metabolic capability such as optimal enzyme activity and membrane stability (Madigan & Oren, 1999).Second, some organisms have evolved a drastically different adaptation to cope with stress when exposed to extreme conditions.They evolved biosynthetic pathways for compounds such as mannitol, sorbitol, sucrose, and trehalose, which are utilized for survival until conditions become favourable again (Yancey et al., 1982).In the case of insects, they accumulate large amounts of trehalose, a blood sugar, in extreme environments.
There are three main ways for organisms to synthesize trehalose in vivo (Kong et al., 2001).The first is via catalysis by trehalose synthase, as in some bacteria (Thermus aquaticus), which can convert maltose to trehalose by transglycosidation (Tsusaki et al., 1997;Kong et al., 2001).The second is via the reaction of ß-glucose 1-phosphate and glucose catalyzed by trehalose-6phosphate phosphatase, as in Micrococcus varians (Kizawa et al., 1995).Many organisms have adapted the latter mechanism to synthesize trehalose via a twoenzyme system of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which requires a high-energy intermediate of glucose metabolism as a substrate (Kong et al., 2001).
In insects, trehalose is a major blood sugar.It is synthesized mainly by the fat body (an organ analogous in function to a combination of the liver and adipose tissue in vertebrates) and is released rapidly into the hemolymph and other tissues in larvae, pupae, and adults (Candy & Kilby, 1959, 1961;Elbein, 1974;Becker et al., 1996;Tang et al., 2008).It is reported that trehalose is synthesized from glucose phosphate.Two enzymes are directly involved in this: TPS forms trehalose 6-phosphate from glucose 6-phosphate and UDP-glucose, after which TPP removes the phosphate to release trehalose into the hemolymph (Candy & Kilby, 1959Kilby, , 1961)).However, molecular level evidence for this is lacking.During this process, TPS and TPP both play an important role in the regulation of trehalose production for growth and development (Elbein et al., 2003;Wang et al., 2005b).Chen et al. (2002) and Chen & Haddad (2004) report that trehalose can serve as a source of energy and accumulates dramatically in HEK-293 cells, increasing tolerance to hypoxia and anoxia and protecting cells and cellular proteins against damage.
To date, although many TPS genes have been cloned or deduced from genomic sequences, there are few such studies on insects, mainly Drosophila melanogaster (Chen et al., 2002(Chen et al., , 2003;;Chen & Haddad, 2004).TPP and TPS genes have been cloned from plants, bacteria, and fungi, but none have been identified in mammals.In arthropods, TPS and TPP have been cloned or deduced from genomic sequences (according to GenBank) and are known to be very important for insect development; however, there are only a few studies on TPS genes and the details of protein characteristics in insects: D. melanogaster (Chen et al., 2002), Callinectes sapidus (Chung, 2008), Locusta migratoria (Cui et al., 2009), Helicoverpa armigera (Xu et al., 2009), and Spodoptera exigua (Tang et al., 2010).Catantops pinguis (syn.Diabolocatantops pinguis) is a worldwide pest of agriculture and is a very strong flyer which requires a lot of energy, so trehalose synthesis is important in C. pinguis.In the present study, TPS cDNA from C. pinguis was cloned (GenBank accession no.GQ389790) and the tissue distribution and pattern of expression of this gene investigated.

MATERIAL AND METHODS
C. pinguis were maintained in the laboratory over a period of 1 year.Larvae were reared at 26 ± 1°C, 65% humidity and an 14L : 10D photoperiod on a diet of wheatgrass.The developmental stages were synchronized at each moult by collecting new adult larvae.The brain, trachea, midgut, rectum, testes, ovaries, Malpighian tubules, posterior extensor, heart, body wall, and fat body of adults, and the fat body of different stages, from larvae to adults, were dissected in saline containing 0.75% NaCl and stored at -80°C until required (Kumar et al., 2008).

RNA isolation and cDNA synthesis
Total RNA was extracted from the fat body of adult C. pinguis using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski & Sacchi, 1987).Fat body (100 mg) was homogenized in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) and placed on ice for 5 min, and then sodium acetate and chloroform/isoamyl alcohol (49 : 1) were added.The mixture was centrifuged at 10,000 × g at 4°C for 20 min.The supernatant was transferred to a new tube and isopropanol added.After centrifugation, the RNA pellet was washed with 75% ethanol and then dissolved in ddH2O.A sample of 1 mg of total RNA was reverse transcribed at 42°C for 1 h in 10 µl of reaction solution containing reaction buffer, 10 mM DTT, 0.5 mM dNTPs, 0.5 µg of oligo-dT18, and 5 unit reverse transcriptase from avian myeloblastosis virus (AMV, Takara, Dalian, China).

Primer design and PCR
Four degenerate primers (TPS-DF1, TPS-DF2, TPS-DR1, and TPS-DR2; Table 1) were designed based on the conserved amino acid sequences of known TPS forms.The first PCR reaction was performed with primers TPS-DF1 and TPS-DR1 using the following conditions: three cycles of 40 s at 94°C, 40 s at 45°C, and 120 s at 72°C, followed by 30 cycles of 40 s at 94°C, 40 s at 48°C, and 120 s at 72°C.A second PCR was carried out using the nested primers TPS-DF2 and TPS-DR2 using the same conditions as for the first PCR (Rinehart et al., 2003;Tang et al., 2008).The PCR products were subjected to agarose electrophoresis.A strong DNA band corresponding to the expected size of approximately 1700 bp was excised from the agarose gel and purified using a DNA gel extraction kit (Takara, Dalian, China).The PCR products were cloned into the pMD18-T vector (Takara, Dalian, China) and sequenced using the dideoxynucleotide method (Takara, Dalian, China).

356
The full-length CpiTPS cDNA sequence was determined by sequencing two overlapping PCR fragments using fat body cDNA as a template.The probe primers were for Northern blot and RT-PCR experiment.a F -Forward; R -Reverse; b D -degenerate primer; G -gene specific primer; A -anchor primer.

Analysis of the CpiTPS cDNA sequence
The CpiTPS cDNA sequence was compared with other TPS sequences deposited in the GenBank using the BLAST-N and BLAST-X tools on the National Center for Biotechnology Information (NCBI) website.The amino acid sequence of CpiTPS was deduced from the corresponding cDNA sequence using the translation tool on the ExPASy Proteomics website (http://expasy.org/tools/dna.html).A phylogenetic tree was constructed using MEGA 3.1 software based on the known TPS amino acid sequences.A bootstrap analysis was carried out and the robustness of each cluster was verified using 1000 replicates.Other protein sequence analysis tools used in this study, including molecular weight, pI and N-glycosylation sites, were obtained from the ExPASy Proteomics website (http://expasy.org/).Multiple alignment of insect TPS sequences was performed using the tool on the multiple sequence alignment website http://bioinfo.genotoul.fr/multalin/multalin.html.

Northern blot and RT-PCR analysis
CpiTPS mRNA expression at different development stages and different seasons was determined by Northern blot and RT-PCR analysis.Total RNA was extracted from brain, trachea, midgut, rectum, testes, ovaries, Malpighian tubules, posterior extensor, heart, body wall, and fat body of young adults.Samples of 1 mg of total RNA were reverse transcribed at 42°C for 1 h in a 10-µl reaction solution containing reaction buffer, 10 mM DTT, 0.5 mM dNTPs, 0.5 µg of oligo-dT18, and 5 unit reverse transcriptase from avian myeloblastosis virus (AMV, Takara, Dalian, China).The product was used as the RT-PCR template.Another 25 mg of total RNA was subsequently blotted onto a Hybond-N + membrane (Amersham of GE Healthcare, Little Chalfont Buckinghamshire, UK).Membranes were prehybridized at 42°C for 4 h and then incubated with the -32 Plabelled CpiTPS probe (same probe as described for Southern blotting) at 42°C for 18 h in 5× SSPE containing 50% formamide, 5× Denhardt's solution, 0.1% SDS, and 100 mg/ml 357 salmon sperm DNA.After hybridization, the membrane was washed with 0.2× SSPE at 45°C, and exposed to X-ray film at -70°C for 24 h (Choo et al., 2007;Tang et al., 2008).
RT-PCR was performed with the CpiTPS-FP/CpiTPS-RP primers.cDNA from brain, trachea, midgut, rectum, testes, ovaries, Malpighian tubules, posterior extensor, heart, body wall, and fat body was used as templates under the following conditions: 30 cycles of 40 s at 94°C, 40 s at 55°C, and 60 s at 72°C.Each PCR product (5 µl) was subjected to electrophoresis and detected by ethidium bromide staining; the amount of S. exigua ß-actin per lane was used as a loading control (Tang et al., 2008).

Conserved sequence analysis of TPS
Multiple sequence alignment of TPS proteins showed a high degree of conservation, particularly in the middle of the putative catalytic domain (Fig. 3).CpiTPS has two motifs (HDYHL and DGMNLV) that are conserved in TPS proteins, as well as many more conserved sequences, such as VVSNRLPF, ASAGGLVTA, FWPLFHSMP, FFLHIPFPPW, LGVDRLDYTKGLVHRL, LVTPLRDG MNLVAKEFVACQIN, KLALLLDYDGTLAPIA, TYA GNHGLEILHPD, and ENKGALLTFH (Fig. 3).

Tissue distribution of CpiTPS
Tissue-specific expression of CpiTPS was determined by Northern blotting and RT-PCR.CpiTPS transcripts were detected in fat body, testes, ovaries, Malpighian tubules, brain, trachea, rectum, and posterior extensor, but not in the mid-gut, heart or body wall of C. pinguis adults, which indicates that CpiTPS mRNA is expressed in many tissues of C. pinguis (Fig. 4).RT-PCR was performed to verify expression of CpiTPS mRNA in all of these tissues.PCR products of CpiTPS were consistent with the Northern blot results, confirming that CpiTPS is specifically expressed in fat body, testes, ovaries, and Malpighian tubules.

Expression of CpiTPS during development
Northern blot experiments were carried out to analyze the expression patterns of CpiTPS in fat body during the development of C. pinguis from first instar larva to adult.The results showed that CpiTPS mRNA is expressed in the fat body at different levels at different developmental stages.CpiTPS transcripts were highly expressed in fat body in early first instar larvae, as well as in early and middle second instar larvae, late fifth instar larvae, and early sixth instar larvae and adults.Transcripts were present at a lower level in fat body of late first and second instar larvae, early, middle, and late third and fourth instar larvae, early and middle fifth instar larvae, and middle sixth instar larvae (Fig. 5).These results suggest that CpiTPS mRNA was expressed in the fat body according to the need of the different developmental stages, but most markedly in the egg to first instar larva and last instar larva to adult stages.

Relative expression of CpiTPS in different seasons
RT-PCR experiments to measure CpiTPS expression in adult fat body in different seasons of the year (from 17 May to 29 October) revealed that CpiTPS mRNA levels varied.CpiTPS transcripts were highly expressed in fat body in early July and mid-October.Transcripts were present at a lower level in fat body in early and mid-June, mid-August, and early and late October.Transcripts were hardly ever expressed in mid-and late May, and late June and September (Fig. 6).The results suggest that CpiTPS mRNA is expressed at varying levels in the fat body depending on developmental and behavioural requirements.

DISCUSSION
It is reported that trehalose is exclusively synthesized by the fat body (Candy & Kilby, 1959, 1961) from glucose phosphates and UTP.Many TPS genes have been cloned and reported from plants, yeast, and bacteria.An insect TPS gene was first cloned from D. melanogaster, encoding a protein of 809 amino acids and containing conserved domains similar to both yeast Tps1 (OtsA) and Tps2 (OtsB) (Chen et al., 2002).These authors, however, did not analyze the condition of the signature motifs or some protein characteristics.But there are two signature motifs (HDYHL and DGMNLV) according to the alignment of TPS protein sequences for insects, plants, bacteria, fungi, and nematodes.In C. pinguis, CpiTPS not only contains these signature motifs (Fig. 1), but also has other conserved motifs, such as VVSNRLPF, ASAGGLVTA, FWPLFHSMP, FFLHIPFPPW, LGVDRLDYTKGLV HRL, LVTPLRDGMNLVAKEFVACQIN, KLALLLDY DGTLAPIA, TYAGNHGLEILHPD, and ENKGALL TFH (Fig. 3).
TPS and TPP genes have been cloned from bacteria, fungi, and plants.In the genome of the plant Arabidopsis thaliana, there are 21 genes homologous to TPS that may have various functions (Leyman et al., 2001).In insects, the TPS gene was first cloned from D. melanogaster (Chen et al., 2002).Soon after, many TPS and TPP genes were cloned or deduced from genomic sequences, with two functional conserved domains similar to yeast genes, namely OtsA (TPS) and OtsB (TPP) (Chen et al., 2002;Chung 2008).According to the GenBank database, TPP genes have been identified in several insects, including D. melanogaster (BT100029), Maconellicoccus hirsutus (EF092085), Polypedilum vanderplanki (AB490334), C. quinquefasciatus (XM_001850944), A. aegypti (XM_001660971), and A. gambiae (XM_317247).Although TPS is a remarkably conserved gene family, Fig. 2 shows that TPS forms from Crustacea and Insecta can be clearly differentiated.Moreover, TPS forms from different insect species can also be differentiated.
It is reported that SexTPS is specifically expressed in the fat body and ovaries in larval stages, which is consistent with the results of studies on H. armigera (Xu et al., 2009, Tang et al., 2010).In Locusta migratoria, TPS is expressed not only in the fat body, but also in gut, hemolymph, and leg muscle (Cui & Xia, 2009).However, in adult C. sapidus, CsaTPS is expressed in all tissues (Chung, 2008).In adult C. pinguis, TPS was not only expressed in fat body and ovaries, but also in the testes, Malpighian tubules, brain, trachea, rectum, and posterior extensor (Fig. 4).The distribution of CpiTPS in tissues is very similar to that of CsaTPS.These results suggest that the distribution of TPS in tissues may differ not only between larval and adult stages, but also among different arthropod species.
In non-diapausing and diapausing H. armigera, HarTPS mRNA expression was low and high, respectively (Xu et al., 2009).In addition, in S. exigua, semiquantitative RT-PCR results reveal that SexTPS mRNA is expressed in fat body at different levels from fifth instar larvae to pupae (Tang et al., 2010).Howerver, different patterns of expression of CpiTPS were found and studied (Fig. 5).The seasonal expression of CpiTPS from 17 May to 29 October was also investigated (Fig. 6).There are possibly two reasons for the variations in expression and lack of a regular pattern in different seasons.First, TPS expression is associated with development and behaviour.Second, TPS expression can be induced at particular times, just as TPS mRNA expression varies over the course of a day in nature.
In yeast, trehalose synthesis is mediated by a complex of TPS1 and TPS2.Trehalose has a dual function as a storage carbohydrate and a stress protection metabolite (Kwon et al., 2003;Thevelein, 1996).Transcriptional levels of TPS1 and TPS2 are increased by heat shock, cold or ethanol stress, salt stress, nutrient starvation, and other osmotic stresses (Winderickx et al., 1996;Wolschek & Kubicek, 1997;Soto et al., 2002;Pereira et al., 2001).Improving stress tolerance is a major goal in agriculture.Over expression of TPS or TPP genes from either E. coil or yeast in tobacco, potato, and tomato improved drought tolerance, but only a limited accumulation of trehalose is recorded (Holmström et al., 1996;Romero et al., 1997;Yeo et al., 2000;Cortina Culiáñez-Macià, 2005;Grennan, 2007).Miranda et al. (2007) report that transgenic Arabidopsis thaliana containing yeast TPS and TPP genes exhibited a significant increase in tolerance of drought, freezing, salt, and heat.Thus, a transgenic approach could be a potential tool for improving the stress tolerance of crops.Interestingly, the absence of TPS in Arabidopsis thaliana can result in the death of embryos (Eastmond et al., 2002;Gómez et al., 2006), demonstrating that AtTPS1 is essential for embryo development (Eastmond et al., 2002).In insects, particularly pests, suppressing the expression of the TPS gene inhibits development and may result in death.In D. melanogaster, a mutation in the TPS gene results in young larvae dying, which indicates that the TPS gene plays a critical role in insect development (Chen et al., 2002).SexTPS RNAi results also suggest that interference with TPS expression results in the death of larvae (Tang et al., 2010).Thus, TPS is a potential key gene that might also be used to control insect pests.
The primers for CpiTPS cDNA cloned, Northern blot, and RT-PCR.

Fig. 1 .
Fig. 1.Nucleotide and deduced amino acid sequences of CpiTPS.Underlined and bold nucleotides indicate the start and stop codons, respectively.Motifs (or signature motifs) unique to trehalose-6-phosphate synthase (TPS) (residues 163-167 and 393-398) are shaded in black.Potential N-glycosylation sites (residues 103-105 and 107-109) are boxed.The nucleotide sequence reported in this paper has been submitted to the GenBank database (accession no.GQ389790).The asterisk indicated the terminal protein.

Fig. 6 .
Fig. 6.Relative expression of CpiTPS mRNA in different seasons.RNA was extracted from fat body in different seasons from 17 May to 29 October.[ -32 P] dCTP-labelled CpiTPS cDNA, amplified using specific primers CpiTPS-FP and CpiTPS-RP, was used as a probe.-actin was labelled with [ -32 P] dCTP as a control.