CryoLetters 32 (3), 225-239 (2011) © CryoLetters, [email protected]

MOLECULAR CLONING AND CHARACTERIZATION OF TWO HEAT SHOCK PROTEINS IN THITARODES PUI (: )

Zhiwen Zou, Zixuan Sun, Junfeng Li and Guren Zhang*

State Key Laboratory for Biological Control, Sun Yat-sen University, Guangzhou, P. R. 510275. Corresponding author e-mail: [email protected]

Abstract

As a group of proteins present almost in all organisms, heat shock proteins (HSPs) show transient expression in response to rapid temperature increase. The larvae of Thitarodes are the host of , with high cold-tolerance. In order to study the adaptive mechanisms to temperature change, we cloned and sequenced the full-length cDNAs of two HSP genes (designated as tp-hsp90 and tp-hsp70) using the technique of rapid amplification of cDNA ends (RACE) from T. pui. The complete cDNA sequences of tp-hsp90 and tp-hsp70 are 2,842 bp and 2,169 bp long, encoding polypeptides of 712 and 651 amino acids with molecular weights of 81.57 and 71.27 kDa respectively. They show significant sequence similarity to homologous genes in insects. The inferred amino acid sequences of tp-hsp90 and tp-hsp70 are characterized by conserved features of HSP family: the proteins contain five signature sequences of HSP90 and three signatures of HSP70, respectively. Real-time quantitative reverse transcription-PCR (qRT-PCR) analyses show that tp-hsp90 expression is up-regulated in October and December, followed by a gradual rebound in January, March and May; while tp-hsp70 expression does not change significantly during the same period. These results suggest that tp-hsp90, rather than tp-hsp70, responds to temperature changes and should play a key role in cold tolerance in Thitarodes pui. Keywords: HSP90, HSP70, cold tolerance, Thitarodes pui

INTRODUCTION

Heat shock proteins (HSPs) are highly conserved and ubiquitously distributed proteins whose expression is related to the environmental changes and various kinds of stress such as: temperature, desiccation, anoxia, and exposure to a wide range of chemicals including heavy metals, ethanol and other contaminants (33). Based on molecular weight and homology, HSPs are classified into several families, including HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs (sHSPs, the molecular weight ranging from 12 to 43 kDa)(11,21,39). So far, many HSPs have been identified and cloned from insects, including HSPs from Drosophila melanogaste r(2),

225 Sarcophaga crassipalpis(19), Lymantria dispar(44), Delia antique(5), Pyrrhocoris apterus(23) and Liriomyza sativa(17). Most of these studies show that at extreme temperatures insects usually synthesize HSPs to increase cold tolerance and therefore protect organisms from thermal injury and death (8, 32)

Thitarodes pui (Zhang et al.) (Lepidoptera: Hepialidae) was first reported in China as Hepialus pui (46) and later transferred to genus Thitarodes (47, 48). Its larvae are the host for the fungus Ophiocordyceps sinensis (Berkeley) Saccardo, a kind of precious traditional Chinese medicine that is used as a special invigorant called Chinese Caterpillar Fungus (Dong Chong Xia Cao in Chinese) (6, 7). T. pui has an optimum altitude distribution of 4,100 to 5,000 m in the region of Mt. Sejila in Tibet (46). The annual average temperature in this region is below 5C and the soil is periodically frozen and thawed. In January, the frozen line of the soil varies from 0.5 to 1.5 m, and the soil remains frozen until the next April (40). Thitarodes larvae live in soil tunnels ranging from 5 to 25 cm in depth. The 1st to 3rd instar larvae favor shallower tunnels while the 4th to 6th instar larvae prefer deeper ones. Most of Thitarodes larvae can survive in the soil with a temperature of 8C to 0C, some safely overwinter at 12C(40), and a few even endure cold temperature as low as 20C (unpublished data). Like other lepidopterous species, the life cycle of T. pui contains four developmental stages: egg, larva, pupa and adult. A complete generation lasts three years, in which pupa, adult and egg stages are completed from May to August in the same year.Larvae overwinter three times in frozen status in their soil tunnels, so overwintering larvae keep frozen in frozen soil.The frozen larvae are able to move and eat immediately when the ambient temperature is higher than 0oC (47). Thus, the larvae are well adapted to the rapid change of alpine temperature and the short time for larvae to feed. Yang et al. (41, 42) found that temperature, particularly low temperature, may be one of the key factors that limit the distribution and spread of Thitarodes species. T. pui is an excellent species to study seasonal adaptation of insects inhabiting in high mountains. However, due to the difficulty in collecting the materials, no study has been conducted to explore the adaptive mechanism of Thitarodes to the temperature change. In this study, we cloned two HSP genes in T. pui and examined their expression patterns during different seasons to test the hypothesis that cold shock can induce HSP expression. Combined with the homology data from GenBank, our study provides important insights into the adaptive mechanisms to temperature change in Thitarodes.

MATERIALS AND METHODS

2.1 Temperature measurement of soil with T. pui larvae Temperature of soil at 20 cm below the surface was measured with Hobo Pro temperature and RH data logger (Model H08-032-08, Eco-tech Co. LTD, USA). The data logger was set to record the temperature every 30 min and the data was downloaded every 30 days with BoxCar Pro software (version 4.3, Onset Computer Corporation, USA). The data from January 2007 to June 2009 was collected.

2.2 Collection of larvae and fat body T. pui larvae in different seasons were collected 7 times during the middle of July, August, October and December in 2008 and January, March and May in 2009,

226 respectively. Each time 15 individual 6th instar larvae were collected from the same location in Mt. Sejila (4156 m, 29°37'N, 94°37' E) of Tibet. They were rapidly anesthetized at -20°C for 30 s, and then dissected in 0.75% NaCl. Fat bodies isolated from three larvae were mixed and stored in RNA protect solution (TaKaRa, Japan) at -40°C. The same procedure was repeated five times.

2.3 RNA isolation and reverse transcription (RT) Total RNA was isolated from fat bodies and further purified with Trizol Reagent Kit (Invitrogen, USA) and RQ1 RNAse-Free DNAse (Promega, USA) according to the manufacturer’s protocols. The total concentration of RNA was estimated by measuring the absorbance at 260 nm. Total RNA (5 µg) was reversely transcribed using AMV reverse transcriptase (TaKaRa, Dalian, China). Reaction conditions were recommended by the manufacturer.

2.4 Cloning the full-length HSP cDNAs The full-length HSP cDNA of T. pui was obtained by RACE (Rapid Amplification of cDNA Ends) methods using the SMART RACE cDNA Amplification Kit (Clontech, CA, USA). The gene-specific primers hsp90GSP1 and hsp70GSP1 (Table1) were used to obtain 5’ region and hsp90GSP2 and hsp70GSP2 (Table1) for 3’ region. To determine the length difference, the PCR fragments were subjected to electrophoresis on 1.0% agarose gels. The amplified cDNA fragments were cloned into the pMD18-T vector (TaKaRa, Japan) following the manufacturer’s instructions. Recombinant bacteria were identified by blue/white screening and confirmed by PCR. Plasmids containing the inserted HSPs fragment were used as a template for DNA sequencing processed by Beijing Genomics Institute (BGI) Shenzhen.

Table 1. Primers used for cloning and expression analysis of two HSPs in T. pui Name Sequence(5’-3’) hsp70GSP1 TTGTCGAAGTCTTCACCTCCCA hsp70GSP2 CATCGTTCTCGTCGGTGGTT hsp90GSP1 ATGTCACGGTCACCTTGTCAGC hsp90GSP2 CTCACCAATGACTGGGAAGACC hsp90-QF ACAAGGTGACCGTGACATCC hsp90-QR TCGCACAGTAAACGACCCA hsp70-QF CATCGTTCTCGTCGGTGGTT hsp70-QR CGGGGTTGATGGATTTGTTC β-actin F TAACCCCAAAGCGAACAGAGA β-actin R GCCAAGTCCAGACGGAGAATG

2.5 Sequence analysis The homology searches of nucleotide and protein sequences were conducted by BLAST at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.gov/blast). The deduced amino acid sequences were analyzed with the Expert Protein Analysis System (http://www.expasy.org/). SignalP 3.0 program was used to predict the presence and locations of the signal peptides and the cleavage sites in amino acid sequences (http://www.cbs.dtu.dk/services/SignalP/).

2.6 Multiple sequence alignment and phylogenetic analysis The full-length multiple alignment of each HSP sequence was compared with other HSPs. Amino acid sequences from various species were retrieved from

227 the NCBI GenBank database and analyzed with the ClustalW Multiple Alignment program (http://www.ebi.ac.uk/clustalw/) and Multiple Alignment program (http://www.biosoft.net/sms/index.html). A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA software (version 3.1). The reliability of the branching was evaluated based on the bootstrap re-sampling analysis (with 1,000 pseudo replicates).

2.7 Quantification of HSP mRNA expression The expression of HSP mRNA was determined by a SYBR Green real-time quantitative RT-PCR analysis in an ABI StepOne Sequence Detection System (Applied Biosystems, USA). The real-time PCR amplifications were carried out following the manufacturer’s instructions of SYBR PremeScriptTM RT-PCR kit (TaKaRa, Dalian, China). The gene-specific primer pairs (hsp90-QF, hsp90-QR; hsp70-QF, hsp70-QR) (Table 1) were used to amplify the HSP90 and HSP70 transcripts, respectively. The primers, β-actin F and β-actin R were used to amplify the β-actin fragments, the positive control. The PCR temperature profile was 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. To confirm that only one PCR product was amplified and detected, dissociation curve analysis of amplification products was performed at the end of each PCR. The two ratios of HSP/β-actin levels of gene expression were calculated. The relative copy numbers of the two HSPs mRNA were calculated according to the 2−ΔΔCt method (26). The threshold cycle value difference (ΔCT) between the two HSPs mRNA and β-actin RNA of each reaction was used to normalize the level of total RNA. The assay was repeated for three times with separately extracted total RNA samples. Three replicates were performed for each reaction to account for intra-experiment variation.

2.8 Statistical analysis Statistical analysis was performed with SAS program (version 8.01, SAS Institute Inc., Cary, NC, USA). The expression levels of the two HSP genes in July were set as the control. All data were shown as mean ± S.D. of the relative mRNA expression. The results were compared with variance (ANOVA) and Tukey’s Studentized range test, and the level of significant difference was set at p < 0.05. The correlation coefficient between the habitat temperature and HSP expression was analyzed by the correlation analysis of SAS

RESULTS

3.1 Temperature dynamics of T. pui habitat Figure 1 shows annual temperature dynamics of T. pui habitat. Although the average month temperature varies across years, the trend is consistent.Every year soil temperature slowly increases from March with a thawing start and reaches the peak (about 12oC) between July and August. Then the temperature decreases to about 0oC in December when the frozen soil begins to form, and it maintains below 0oC until the next March during which the soil keeps frozen. Thus, the habitat temperature of T. pui larvae is below 12oC, and the soil keeps frozen for 4 months during a year.

3.2 Sequence characterization of HSPs Using RACE and nested PCR approaches, we cloned two heat shock genes from T. pui, namely tp-hsp90 and tp-hsp70. The full lengths of tp-hsp90 and tp-hsp70

228 cDNA are 2,842 bp and 2,169 bp, respectively. Fig. 2 shows the nucleotide sequences and the deduced amino acid sequences.The nucleotide/amino acid sequences have been deposited in NCBI GenBank with accession numbers GU205815/ADA61011 and GU205816/ ADA61012, respectively. The full-length tp-hsp90 cDNA contains 122 bp in the 5’-untranslated region (UTR), 2,139 bp in the open reading frame (ORF) and 581 bp in 3’-UTR with three A-U rich regulatory motifs (AUUUA), a canonical polyadenylation signal sequence (AATAAA) and a poly (A) tail of 25 bp. The ORF encodes a polypeptide of 712 amino acids. The inferred molecular mass of the mature protein (712 amino acids) is 81.57 kDa with an estimated pI of 5.0. The tp-hsp70 cDNA includes 90 bp in 5’-UTR, 123 bp in 3’-UTR and 1,956 bp in an ORF encoding a polypeptide of 651 amino acids with molecular weight of 71.27 kDa. There are two A-U rich regulatory motifs (AUUUA), a canonical polyadenylation signal sequence (AATAAA) and a poly (A) tail of 28 bp in 3’-UTR of tp-hsp70 cDNA.

Figure1. Seasonal changes of habitat temperature in T. pui 3.3 Homology and phylogenetic analysis of HSPs The sequence analysis shows that the tp-hsp90 protein shares 88.1%, 87.9% and 87.4% sequence similarity to HSP90 of Spodoptera frugiperda, Bombyx mori and Chilo suppressalis HSP90, respectively; and the similarity of tp-hsp70 is more than 95% in all the matches from vertebrates or invertebrates. These results confirm that the cloned genes indeed encode HSP90 and HSP70 proteins. The multiple alignment of tp-hsp90 and tp-hsp70 with HSP90 and HSP70 of different lepidopterous insects shows that they are highly conserved. For HSP90 protein family, five conserved amino acid blocks define an HSP90 protein family signature (positions Asn34 to Arg54, Gly102 to Lys110, Gly126 to Lys141, Asn336 to Ile352, and Gly369 to Gly385), three non-conserved domains (positions Met1 to Val12, Glu217 to Glu 271, and Met686 to Ala705) and one consensus sequence MEEVD at the C-terminus (Fig. 2-A). For HSP70 protein family, there are three signature sequences (positions Ile10 to Ser17, Ile198 to Leu211, and Ile335 to Gln348) (Fig. 2-B). In addition, SMART program analysis reveals that the typical histidine kinase-like ATPase domain, which is ubiquitous in all HSP70 family members, exists from amino acid positions 34 to 187. We also found other conserved motifs of the HSP70 family, such as the Bipartite Nuclear Targeting Sequence (from positions Ala132 to Thr139) and ATP/GTP-Binding Site Motif A (P-loop) (from positions Arg148 to Arg263) using the InterPro software(Fig. 2-B).

229 1 attgtcagaaaacctttgttgcgtgtgaacgtgcgaacagtgagctt 48 acttggtttgaccaagtgcggttatatttaataataattgatttaagcacattgtgaaataatacggaatttaag 123 atgccggaattaaatcaaggcgccggcgatgtggagaccttcgcgtttcaggctgaaattgctcagctcatgtcc 1 M P E L N Q G A G D V ETFA FQAEIAQL MS 198 ttgatcatcaacacattctactcgaacaaagaaattttcctccgagagctgatatcaaattcatcagatgcattg 26 L I I N T FYS NKE IFLRELISN SSDAL 273 gacaaaatacgctatgaatctctcacggatccatcgaagctcgacaatggtaaagaactttacatcaaactcata 51 DKIR YESLTD PSKLDNGKELYIKL I 348 cctaacaagagtgagggtacactgacaatcatagataccggtattggtatgactaaagcagatcttgtcaacaac 76 PNKSEGTLTIIDTGI GMTKADLVN N 423 cttggaacaattgcaaaatctggtactaaggcattcatggaggcacttaatgctggtgcagatatcagtatgatc 101 L GTIA KSGTK AFMEALNAGA DI SM I 498 ggtcaatttggtgtaggtttctattctgcatacttggtggctgacaaggtgaccgtgacatccaaacataacgat 126 GQFGVGFYSA YLVADK VTVTSKH ND 573 gatgagcagtatctgtgggaatcttcagcaggtgggtcgtttactgtgcgatctgacgatggagaaccactgggc 151 D EQYLWESSAGGSFT VRSDDGEP LG 648 cgtggtacaaaaatagttttacatatgaaggaggctttggatgaatttttggaagagcgcaaaatcaaagacatt 176 RGTKI VLHMKEALDEFLEER KIKD I 723 gttaaaaaacactctcaattcattggatatccaatcaaattgttggttgagaaagaacgtgaaaaggagttgtct 201 VKKHSQFIGY PIKLLV E K E R E K E L S 798 gatgaagaagaagaggaagccaaggaagatgataaggacactaaaccaaaaatagaagatgttggagaagatgaa 226 D E E E E E A K E D D K D T K P K I E D V G E D E 873 gaatctacaaaggagaagaagaagaagaaaactatcaaagagaagtacactgaagacgaagaattgaacaaaaca 251 E S T K E K K K K K T I K E K Y T E D E E LNK T 948 aaacctatatggacaaggaatgctgatgatattacacaggaagaatatggcgaattctataaatcactcaccaat 276 KPIWTRNADD ITQEEYGEFYKSLT N 1023 gactgggaagaccatcttgctgtaaaacatttctctgtagaaggtcagttggaattcagagcactcctgtttgtc

230 301 DWEDHLAVKHFSVEG QLEFRAL L FV 1098 ccgagaagacttccatttgatctgtttgaaaataagaaacgcaaaaacaacatcaaattgtatgtacgcagagtc 326 PRRLP FDLFE NKKRKNNIKL YVRR V 1173 tttattatggacaactgtgaagaaataattcctgaatatctcaacttcatcaagggtgtggtagacagtgaagat 351 F I MDNCEEII PEYLNFIK GVVDSE D 1248 ttacctcttaacatctcccgtgaaatgttgcaacaaaataagattgttaaagtaatccgtaagaatttagttaag 376 LPLNISREML QQNKI VKVIRKNLV K 1323 aaatgtctggagctcttcgaagaactttccgaggacaaagagggctacaagaagttttatgaattgtttagcaag 401 KCLEL FEELSEDKEGYKKFY ELFS K 1398 aatctcaagttgggcattcatgatgattcacaaaatagagctaaattggctgaattcctcagattccacacttca 426 NLKLGIHDDS Q NRAKLAEFLRFHT S 1473 gcatcgggtgatgaagcttgctctctcaaagaatatgtatctcgaatgaaagaaaaccagaaacacatctatttc 451 ASGDEACSLKEYVSR MKENQKHI YF 1548 atcactggtgaaaataaagaacaagttgcaaactcttcctttgtcgagcgggtaaagaagcgtggttttgaagtt 476 ITGEN KEQVANSSFVERVKK RGF EV 1623 atttacatgactgagcctattgatgagtatgtagtacaacagatgaaggaatacgatggtaaacaattggtatct 501 IYMTEPIDEY VVQQMKEYDGKQ L VS 1698 gttaccaaagagagcctggagttaccagaggacgaggaagagaaaaagaagatggaagaagacaaaactaaattt 526 VTKESLELPEDEEEK KKMEEDKT KF 1773 gaaggtttatgcaaagtaatgaaaaacattttggacaataaagtggaaaaagtggttgtgagtaatagattggta 551 E GLCK VMKNILDNKVEKVVV SNR LV 1848 gagtctccgtgttgtattgttacttcacaatatggctggactgccaacatggagcgcataatgaaggcacaggct 576 ESPCCIVTSQ YGWTANMERIMKA QA 1923 ctcagagacacatctaccttgggttacatggccgctaagaagcacttggaagttaatcctgatcattcaatcata 601 LRDTSTLGYMAAKKH LEVNPDHSI I 1998 gaaactttacggcagaaggctgatgtcgataaaaatgataaagctgttaaggatttggtaattcttctttttgaa

231 626 ETLRQ KADVDK NDKAVKDLV ILLF E 2073 actgctttgctttcatctggtttcaccttggatgaacctggagttcatgcatcccgcatttacagaatgatcaaa 651 TALLSSGFTL DEPGVHASRIYRMI K 2148 ctcggcttgggcatcgatgaggatgagccaatggctgcggaggagacctctgctgaagttccacctctggagggt 676 LGLGIDEDEP M A A E E T S A E V P P L E G 2223 gatgctgacgatgcatcaagaatggaagaagtagattaagtcgatgtaatttagtttgaacctcgttttagcggt 701 D A D D A SR MEEVD * 2298 acatttataaatctcaaaacctgctttcatttgttgttttcaatgaaagcagcttttgggtgtctcaaaactggc 2373 tttcatttgatattccatcacacaccatctcagcctgctttggtgaatttaatctataaatttgaggcggttcaa 2448 ttaagaatgttaagaggaatgaaaatcaacaagtgatccgtcagcaacccaggcaatccacctctagttttggcc 2513 tgtatgaacgtagatggtcttagttatcgcattaaagccggaattggatataatggatgtggataactttggttt 2588 caacgagacatacttatcacactgcggctgaaacaatgcaattattgcagatgaagtttcctggtcgtgctatct 2663 ggccctggggagaggtaaatcggccatcaagatcgtgccatttgactaggcgcaatattgtgttccgcatgaaag 2738 ccggttttgagactcgcatttcttattactttatccgcaaataaagacttattactcgtatttaatatataaaaa 2813 aaaaaaaaaaaaaaaaaaaa A Family signature sequences are underlined. Three non-conserved domains are boxed. MEEVD at the C-terminal is shadowed. In nucleotide sequences, AATAAA is underlined and A-U rich regulatory motifs are boxed

1 ttttaattctcaagc 16 aagcgatcactagacacacgtttctactattactcatttttagtctgcgatttgaaacactactagaaacaaaag 91 atggcaacccctgctcccgcagttggtatcgatttgggtactacgtactcttgcgtgggcgttttccagcatgga 1 MATPAPAVG IDLGTT YS CVGVFQH G 166 aaggtcgaaattattgcaaacgaccagggcaacaggaccacaccttcctatgttgcatttactgacacagagcgt 26 K V E I I ANDQGNRTTPSYVAF TDTE R 241 ctcatcggagacgccgctaagaaccaggtggccatgaatcccaacaacaccatctttgatgccaagagacttatt 51 LIGDAAKNQV AMNPNNTIFDAKRL I 316 ggtcgtaagtttgaagatgccaccgtccaagcagacatgaaacattggccgttcgaagtggtcagcgacggtggc 76 GRKFEDATVQAD MKH WPFEVVSD GG 391 aaacctaaaattaagatcgaatataagggtgaatccagaaccttctcaccagaagaggtcagctccatggtgctc 101 KPKIK IEYKGESRTFSPEEV SSMV L 466 acaaaaatgaaggaaactgccgaggcttacctcggcaagactgtgcaaaatgctgtcataacagttcctgcatat

232 126 T K M K E T A E A Y L G K T VQNAVITVPA Y 541 ttcaatgactctcaaagacaagccaccaaagatgccggcacaatctccggactgaacgtgcttcgaattatcaat 151 FNDSQRQATKDAGTI SGLNVLRI I N 616 gagcccactgccgccgctattgcctatggtctcgacaagaagggacacggcgagcgcaacgtactaatctttgac 176 EPTAA AIAYGLDKKGHGERN VL IF D 691 ttgggtggcggtacctttgatgtgtccatcttgaccattgaggatggtatctttgaggtcaaatcaacagctgga 201 LG GGTFDVSI L TIEDGIFEVKSTA G 766 gacacccacttgggaggtgaagacttcgacaaccgcatggtgaatcatttcgtccaagagttcagaagaaagtac 226 DTHLGGEDFDNRMVN HFVQEF R R K Y 841 aagaaggacctcacaaccaacaagagggcccttcgacgtctgagaacatcctgtgagagggcgaagaggactctg 251 K K D L T T N K R A L R R LRTSCER AKRT L 916 tcttcttccactcaggccagtattgaaattgattccctctttgaaggtattgatttctacacctccataactcgt 276 SSSTQASIEI DSL FEGIDFYTSITR 991 gcgagattcgaggaactgaacgctgatctgttcagatcaaccatggaacccgtagagaagtcgctgcgtgacgct 301 ARFEELNADLFRSTM EPVEKSLRD A 1066 aagatggataagtctcaggttcatgacatcgttctcgtcggtggttcaacacgcattcccaaggtgcagaagctc 326 KMDKS QVHD IVLVGGSTRIP KVQ K L 1141 ctccaggacttcttcaacggtaaggagttgaacaaatccatcaaccccgacgaagcagtcgcctacggcgccgcc 351 LQDFFNGKEL NKSINPDEAVAYGA A 1216 gtccaggccgccatcctgcacggagacaagtccgaggaggtccaggacttgcttctgcttgatgtcacacccctg 376 VQAAILHGDKSEEVQ DLLLLDVTP L 1291 tctctcggtatcgagaccgctggcggcgtgatgactacgctcatcaagaggaacaccaccattcccaccaagcag 401 SLG IE TAGGVMTTLIKRNTT IPTK Q 1366 acgcagaccttcactacctactctgataaccaacctggtgtactcattcaggtattcgagggtgagcgtgccatg 426 TQTFTTYSDN QPGVLIQVFEGERA M 1441 accaaggacaacaacattcttggaaaattcgagctcactggcattccccccgcgcctcgcggtgtccctcaaata

233 451 TKDNNILGKFELTGI PPAPRGVPQ I 1516 gaggtcacttttgacattgatgctaacggcattctgaatgtgtcggccatcgagaagtccaccaacaaggaaaac 471 EVTFD IDANGILN VSAIEKS TNKE N 1591 aagataaccatcaccaatgataagggccgcctgtcgaaggaggagatcgagcgcatggtcaacgaagccgagaag 501 KITITNDKGR LSKEEIERMVNEAE K 1666 taccgcagcgaggatgagaaacagaaggagaccatctctgccaagaacggcctcgagtcttactgcttcaacatc 526 YRSEDEKQKETISAK NGLESYCFN I 1741 aagtccaccatcgaggacgagaagctcaaggacaaaatttcggacactgacaaacaaacaattgccgacaagtgc 551 KSTIE DEKLKDKISDTDKQTb IADK C 1816 aacgacacaatcaaatggttggactctaaccagttggctgacaaggaggagtatgagcacaagcagaaagagctg 576 NDTIKWLDSN QLADKEEYEHKQK EL 1891 gagtcggtgtgcaaccccatcatcacgaagttgtaccagagcgcgggcggcgctcccggcggtatgcctggcttc 601 ESV CNPIITKLYQSA GGAPGGMPG F 1966 cccggcggtccacccggagctggtggggctgcgcccggcgccggaggcggtgctggacccaccattgaggaagtt 626 PGGPP GAGGAAPGAGGGAGP TI E EV 2041 gattaagcattccaaaaatactttattttatttaatttatacttaaagcttaaatgaccgtctggaatctgcaac 651 D * 2116 attcaaaacaataaacgttaaccaacaaaaaaaaaaaaaaaaaaaaaaaaaaa B Family signature sequences are underlined. The bipartite nuclear targeting sequence and ATP/GTP- binding site motif are boxed. EEVD at the C-terminal is shadowed. In nucleotide sequences, AATAAA is underlined and A-U rich regulatory motifs are boxed

Figure 2. Nucleotide and deduced amino acid sequences of HSP90 (A) and HSP70 (B) cDNA of H. pui

234 The amino acid-based NJ phylogeny shows the evolutionary relationships among HSP90 and HSP70 of different lepidopterous species, respectively (Fig. 3, 4). The relationships in the phylogenic trees are in good agreement with the traditional , indicating that Thitarodes is a primitive group in Lepedoptera (28). Mamestra brassicae BAF03556.1 65 Helicoverpa armigera ACL31668.1 Spodoptera frugiperda AAG44630.1 27 54 46 Helicoverpa zea ACV32639.1 Helicoverpa zea ACV32641.1 49 74 Spodoptera exigua ACL77779.1 Mythimna separata ABY55233.1 69 Mamestra brassicae BAF03554.1 Sesamia nonagrioides AAY26452.2 27 98 Sesamia nonagrioides ABA54273.2 31 Trichoplusia ni ABH09732.1 Omphisa fuscidentalis ABP93404.1 Lonomia obliqua AAV91465.1 39 60 Chilo suppressalis BAE44307.1 30 33 Bombyx mori NP_001036892.1 54 Loxostege sticticalis ABW87791.1 42 Manduca sexta Q9U639.1 Bombyx mori NP_001036876.1 68 Dendrolimus punctatus ABM90803.1 Antheraea yamamai BAD15163.1 99 54 99 Dendrolimus superans ABM90551.1 61 Dendrolimus punctatus ABM90804.1 89 Dendrolimus tabulaeformis ABM90802.1 100 Dendrolimus superans ABM89112.1 73 Dendrolimus tabulaeformis ABM89111.1 Plutella xylostella BAE48743.1

Plutella xylostella BAE48742.1 56 Chilo suppressalis BAE44308.1 Tphsp90 GU205815.1 40 Omphisa fuscidentalis ABP93403.1 Locusta migratoria AAS45246.2 Tphsp70 GU205816.1 Locusta migratoria AAO21473.1 0.01

0.01 Figure 3. Phylogenetic tree for known Figure 4. Phylogenetic tree for known lepidopterous HSP90 with Locusta migratoria as lepidopterous HSP70 with Locusta migratoria as the outgroup constructed by mega 3.1 (1,000 the outgroup by mega 3.1 (1,000 bootstrap) bootstrap)

3.4 Quantitative analysis of HSP expression To investigate the function of HSPs in temperature change of Thitarodes, we measured the mRNA expression of HSPs in fat bodies responding to cold hardening by qRT-PCR with β-actin as an internal control. For both HSP and β-actin genes, there was only one peak at the corresponding melting temperature in the dissociation curve, indicating that the PCR was specifically amplified. The temporal expression of tp-hsp90 gene demonstrated a clear season-dependent expression pattern while the relative expression level of tp-hsp70 did not significantly change during the investigated duration (Fig. 5). The expression of tp-hsp90 was up-regulated in October and December, and it reached the highest level in October. Then, the expression level dropped from January to March and May. The tp-hsp90 gene expression level in October and December was significantly higher than that in other months (F6,14 = 19.37, p < 0.001). However, there was no significantly changein the expression levels of tp-hsp70 in different seasons (F6,14=1.25, p> 0.3). The correlation between the expression levels of tphsp90 and tphsp70 and habitat temperatures was not significant, and the correlation coefficient values (R) were -0.73 (p = 0.25) and -0.25 (p = 0.6), respectively.

235 Figure 5. Seasonal changes of mRNA expression of two HSP genes in T. pui “” and “”on the top of bars mean significant difference. The two hsps expression of T. pui collected in July, 2008 are set as the control group. DISCUSSION

It is well known that HSPs are capable of responding to environmental stresses in a wide range of organisms, including cold tolerance of insects (8, 32). T. pui is distributed in the regions of high altitude, and cold-tolerant mechanisms should have evolved to adapt to the highly seasonal alpine environment (40, 41).However, the impressive strategies for survival of this species at the extreme conditions are still unknown. In order to understand the adaptive mechanism to temperature changes, we cloned HSP90 and HSP70 from T. pui and examined their temporal expression levels in different seasons. The complete cDNA sequences of two HSP genes from T. pui were obtaind by RACE-PCR, while BLAST analysis revealed that the deduced amino acid sequences of the two HSPs show high similarity to other known HSP90s and HSP70s of Lepidoptera, which are 86%~88% and 92%~96%, respectively. Sequence alignment, structure comparison, and phylogenetic analysis suggested that the sequences of HSPs we obtained from T. pui belong to the HSP90 and HSP70 families, respectively. In addition, the relationships of the two HSPs in the phylogenic tree with other known lepidopterous HSPs are in good agreement with the traditional taxonomy, indicating that Thitarodes is a primitive group (28). Motif scan shows that the sequence MEEVD at the C-terminus which is a character shared by all the cytosolic HSP90 proteins and the sequence EEVD at the C-terminus of the cytosolic HSP70-specific motif are also present in tp-hsp90 and tp-hsp70 respectively, suggesting that the two HSPs we obtained are cytosolic members of HSP90 and HSP70 families, respectively(24). Furthermore, the deduced tp-hsp90 and tp-hsp70 amino acid sequences contain conserved sequences and characteristic motifs, such as HSP90 and HSP70 family signature motifs, ATP and geldanamycin binding domain, the major structural and functional domains typically in HSP90 (2, 3) as well as ATP-GTP binding site motif and a bipartite nuclear localization signal sequence in HSP70. The EEVD sequence at the N-terminus of tp-hsp90 is strictly conserved and resembles other members of HSP90 family which are recognized by TPR domains of HOP (HSP70 and HSP90 organizing protein), an adapter protein mediating the association of HSP70 and HSP90 into a multichaperone complex (24, 35). The sequences of HSP70 family show higher conservation at the N-terminus but a greater divergence at the C-terminus. This rather variable C-terminus may be related to the functional specificity of

236 individual HSP70s (18). Moreover, the C-terminal portion in tp-hsp70 contains a GGMP motif that may play an important role in the structure and function of the HSP family (10). HSP90 and HSP70, two dominant HSPs, are key players in cold-exposure response of many insects (9, 37). Previous studies have showed up-regulation of hsp90 and hsp70 mRNA levels in response to low temperature in several insect species, such as Hsp70 in various fruit flies, Drosophila (12, 13, 36), adult potato beetles (43), pupae of the onion fly, D. antique a(5), adult fly L. huidobrensi s(15) and S. crassipalpis(19), as well as HSP90 in Delia antique (4), C. suppressalis (37), and D. melanogaster(30). In this study, we found a negative correlation between tp-hsp90 expression and habitat temperature (r = -0.73, p = 0.25), suggesting that the tp-hsp90 expression is also up-regulated in cold environment. Some studies have detected relatively high levels of HSP mRNA during overwintering in insects(8, 14, 33, 34, 44) and in the larva of an Antarctic midge, Belgica Antarctica (32). We observed the same pattern in T. pui, that the tp-hsp90 expression was significantly higher in winter than in summer. Therefore, it is likely that up-regulation of tp-hsp90 in winter might be a major factor contributing to cold-hardiness. In contrast, the expression of hsp70 in T. pui was not affected with changes of habitat temperature and not up-regulated in cold temperature (r = -0.25, p = 0.6). Such observations suggest that tp-hsp90, rather than tp-hsp70, should play a key role in cold tolerance in T. pui. We also found the tp-hsp90 mRNA expression was the lowest in July and reached the peak in October. In addition, the tp-hsp90 expression was not at the highest level in the coldest January, which may be attributed to the transient-response characteristic of hsp90 (22, 39). Rinehart and Denlinger (31) found that hsp90 response in S. crassipalpis is strongest at 2 h post-cold shock, and it becomes substantially reduced by 4 h post-cold shock. The same changes were observed on D. antique: the hsp90 is up-regulated before the onset of winter diapause, and the expression level is decreased during the treatment duration after the first five days of stresses for cold-stressed winter diapause pupae (4). So we speculate that the long-term molecular regulation of hsp90 should be affected by both habitat temperature and the transient-response characteristic of this gene (4). Different HSPs respond to different stresses, and HSP members may deal with cold stresses of various intensities (17). In contrast to HSP90, the expression of HSP70 in T. pui was not regularly affected by the changes of habitat temperature. A similar phenomenon is revealed in C. suppressalis: hsc70 (heat shock cognate protein, hsc) expression is only slightly decreased during cold acclimation in non-diapausing individuals while the hsp90 expression is up-regulated (37). Interestingly, the opposite pattern occurred in L. huidobrensi s(15) which the hsp90 expression don’t change under cold treated.These results show that different HSPs may respond to different stress, and HSPs members may deal with cold stresses of various intensities. This phenomenon is also found in L. sativae which hsp20 and three additional HSPs (hsp19.5, hsp20.8 and hsp21.7) show different sensitivities in response to cold, but the mitochondrial hsp60 is not induced by cold (16, 17). In this study, the HSP70 of T. pui is not upgraded in cold season. It may not be related with cold stresses. The same conclusion occurred in some Drosophila, which HSP70 is not upregulated species in response to low temperatures (20, 29). HSPs can enhance cold tolerance in the organism by functioning as molecular chaperones (8, 17, 37). HSP up-regulation is closely correlated with synthesis of cryoprotectant glycerol and adaptive regulation of membrane fluidity (25, 27). According to the expression patterns of tp-hsp90 and tp-hsp7, we speculate that

237 tp-hsp90, not tp-hsp7, may be related to the enhanced cold tolerance in T. pui. However, the mechanism by which HSPs regulate cold tolerance in T. pui larvae need further investigation. In summary, T. pui up-regulated tp-hsp90 and didn’t change tp-hsp70 expression in response to cold stress. It could be therefore conjectured that tp-hsp90, rather than tp-hsp70, responded to temperature changes and should play an important role in cold tolerance in T. pui. Acknowledgements The study was financially supported by the National Key Technology Research and Development Program of China (2007BAI32B05, 2007BAI32B06, 2011BAI13B06). REFERENCES

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Accepted for publication 07/02/2011

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