research article cloning and expression of a...

Research ArticleCloning and Expression of a Cytosolic HSP90 Gene inChlorella vulgaris

Zhengyi Liu1 Lei Zhang2 Yang Pu2 Zhaopu Liu1 Zhiling Li3

Yushan Zhao3 and Song Qin1

1 Key Lab of Marine Biology in Jiang Su College of Resources and Environmental ScienceNanjing Agricultural University Nanjing 210095 China

2 Yantai Institute of Coastal Zone Research Chinese Academy of Sciences Yantai 264003 China3 Shandong Oriental Ocean Sci-Tech Co Ltd Yantai 264003 China

Correspondence should be addressed to Song Qin sqinyicaccn

Received 4 December 2013 Accepted 16 January 2014 Published 13 March 2014

Academic Editor Hanzhi Lin

Copyright copy 2014 Zhengyi Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Heat shock protein 90 (HSP90) a highly conserved molecular chaperone plays essential roles in folding keeping structuralintegrity and regulating the subset of cytosolic proteins We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90)by combining homology cloning with rapid amplification of cDNA ends (RACE) Sequence analysis indicated that CvHSP90 isa cytosolic member of the HSP90 family Quantitative RT-PCR was applied to determine the expression level of messenger RNA(mRNA) in CvHSP90 under different stress conditions C vulgaris was kept in different temperatures (5ndash45∘C) for 1 h The mRNAexpression level of CvHSP90 increased with temperature from 5 to 10∘C went further from 35 to 40∘C and reached the maximumat 40∘C On the other hand for C vulgaris kept at 35∘C for different durations the mRNA expression level of CvHSP90 increasedgradually and reached the peak at 7 h and then declined progressively In addition the expression level of CvHSP90 at 40 or 45 insalinity (permil) was almost fourfold of that at 25 in salinity (permil) for 2 hTherefore CvHSP90 may be a potential biomarker to monitorenvironment changes

1 Introduction

Heat shock proteins (HSPs) that first described inDrosophilamelanogaster [1] are evolutionarily conserved protein fami-lies which are ubiquitous in all eukaryotic organisms HSPsare essential for cells under both normal and stressed con-ditions as they participate in diverse processes ranging fromcellular homeostasis and signal transduction to development[2] In addition HSPs also play key roles in defense responsesagainst various environmental stresses that could potentiallydamage the cellular and molecular structures in the cells [3]Therefore HSPs are highly expressed in all organisms andare one of the most abundant proteins contributing to 1-2 of total cellular proteins under normal conditions [4]According to molecular weight HSPs are mainly classifiedinto six families including HSP40 HSP60 HSP70 HSP90HSP100 and the small HSPs [5]

Among HSP families heat shock protein 90 (HSP90)is highly conserved member It is confirmed to locate indifferent cellular compartments including the chloroplastsmitochondria cytosol nucleoplasm and endoplasmic retic-ulum [6] Under normal conditions HSP90rsquos diverse rolesin biological processes include regulating cellular physiologysignal transduction and protein folding degradation andtransportation [2 7ndash9] And it also can be regulated by vari-ous environmental stresses such as heat salinity desiccationlight heavy metal and arsenite stresses [10ndash13]

Influenced by cyclic tide algae mainly living in intertidalzone are periodically exposed to a variety of potentiallystressful factors including heat or cold shock high light anddesiccation [14] To understand the resistant mechanism theexpression of HSPs genes have been investigated in somemacroalgae such asChondrusPorphyraUndaria LaminariaSaccharina Fucus and Ulva [15ndash22] and a few microalgae

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 487050 11 pageshttpdxdoiorg1011552014487050

2 BioMed Research International

Table 1 PCR primers used in this study

Primers Sequence (51015840-31015840) Sequence informationP1 GNGTGTTCATCATGGACAAYTGYGA Homology cloning primerP2 TTCATGATGCKYTCCATGTTNGC Homology cloning primerP3 GCCCCTCAACATCTCCCGCG 31015840-RACE primerP4 ACCTGCCCCTCAACATCTCCC 31015840-RACE primeradaptor GGCACGCGTCGACTAGTAC 31015840-RACE primerP5 TTTTGTCCTCCTCGGTCTCG 51015840-RACE primerP6 GCCCTCCTTGGTCACGCTCA 51015840-RACE primerOligo(dG)-adaptor GGCACGCGTCGACTAGTACG10 51015840-RACE primerP7 GGCCAGTTCGGCGTGGGTTT Real time gene primerP8 AAGCGACGGGTTGACGGTGT Real time gene primerP9 AACTACGAGCTGCCAGACGG Real time actin primerP10 GAAGACAAGACGATGCTGAGGG Real time actin primerM13-47 CGCCAGGGTTTTCCAGTCACGAC Sequencing primerRV-M GAGCGGATAACAATTTCACACAGG Sequencing primer

such as Chlamydomonas and Haematococcus [23ndash27] Thesestudies indicate that the algal HSPs centrally function inresisting a wide variety of environmental stressors

Chlorella vulgaris is a unicellular green alga growing infresh water [28] It can be used as live feed in fish aquaculturefor its high content of proteins and fatty acids In additionbecause of its fast growth and low cost C vulgaris becomes apromising candidate bioreactor for large-scale production ofvalue-added proteins [29]C vulgarishas important econom-ical and ecological values but often confronts environmentaladversities including high temperature and high salinityTherefore it is often used as a eukaryotic model in studieson stress responses [30] However the role of HSPs inadverse stress resistance mechanism of C vulgaris is yet tobe performed

To better understand the mechanism of response byC vulgaris to different types of environmental stimulationwe obtained a HSP90 complementary DNA (cDNA) of Cvulgaris by combining homology cloning with rapid amplifi-cation of cDNA ends (RACE) approaches analyzed in bioin-formatics the structural features homologous relationshipand phylogenetic position of CvHSP90 and investigatedthe messenger RNA (mRNA) expression levels of CvHSP90under different stress conditions using real-time quantitativeRT-PCR (qRT-PCR)

2 Materials and Methods

21 Sample Collection and Treatment Chlorella vulgaris waspresented from Institute of Oceanology Chinese Academyof Sciences and then the Chlorella was grown in Erlen-meyer flasks in F2 medium that was filter-sterilized through022120583m filters (Millipore USA) Cultures in liquid mediumor on plate were grown at 20∘C in an artificial climateincubator Cool-white fluorescent tubes were used to provideirradiance at 200120583mol photons mminus2 sminus1 in a long-daylightscheme (12 12 h light dark)

In additionC vulgariswas kept in 40∘C for 1 h for extract-ing total RNA and cloning full-length cDNA of CvHSP90gene In our heat shock temperature treatment C vulgariswas kept in different temperatures (5 10 15 20 25 30 3540 and 45∘C) for 1 h to investigate the thermal effect onthe expression level of CvHSP90 mRNA In heat shock timetreatment C vulgaris was kept at 35∘C for different times(0 h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9 h 10 h 11 h and12 h resp) to investigate the effects of heat shock times onexpression level of CvHSP90 mRNA In salt concentrationchallenge treatments C vulgariswas kept at 20∘C in differentsalt concentrations (0 5 10 15 20 25 30 35 40 and 45in permil) for 2 h to investigate the effects of salt challenges onexpression level of CvHSP90

22 RNA Extraction Total RNA extraction from C vulgariswas performed using the TRIzol reagent (Invitrogen) ThecDNA first-strand was synthesized based on M-MLV RTusage information (Promega) using RQI DNase (Promega)-treated total RNA as template cDNA mix was diluted to1 50 and stored atminus80∘C for subsequent fluorescent real-timePCR

23 CvHSP90 cDNACloning To amplify the partial fragmentof CvHSP90 gene from Chlorella vulgaris two homologouscloning primers P1 and P2 were designed based on theconserved sequence of known HSP90s (Table 1) PCR wasperformed in a 25120583L reaction volume containing 25120583L 10XPCR buffer 2120583L dNTP (25mmol Lminus1) 1 120583L of each primer(10 120583mol Lminus1) 02 120583L (1U) of Taq polymerase (Promega) 1120583Lof cDNA template and 173 120583L of PCR-grade water The PCRtemperature profile was 94∘C for 5min followed by 33 cyclesof 94∘C for 1min 56∘C for 1min 72∘C for 1min and a finalextension step at 72∘C for 10min The interesting fragment(745 bp) was excised and purified as per agarose gel DNAfragment recovery kit (TaKaRa) cloned into pMD-18T vector(TaKaRa) and sequenced (Sangon Shanghai China)

BioMed Research International 3

To clone the full-length cDNA of CvHSP90 four specificprimers sense primers P3 and P4 and reverse primers P5and P6 (Table 1) were designed based on the partial sequenceamplified Nested PCR strategy was applied to clone the 31015840end of CvHSP90 using sense primer P3 P4 and reverseprimer adapter while sense primer oligo(dG)-adaptor andreverse primer P5 P6 were used to obtain the 51015840 end ofCvHSP90 PCR amplification was performed using the samereaction system as described before The PCR productswere cloned into the pMD18-T simple vector (TaKaRa) andsequenced bidirectionally with primers M13-47 and RV-M(Table 1) The sequencing results were verified for clusteranalysis

24 Sequence Analysis of CvHSP90 CvHSP90 cDNAsequence was analyzed in the BLAST algorithm at NationalCentre for Biotechnology Information (httpwwwncbinlmnihgovblast) and the deduced amino acid sequence wasanalyzed with the Expert Protein Analysis System (httpwwwexpasyorg) Characteristic domains or motifs wereidentified using the PROSITE profile database Identitysimilarity and gap percentages were calculated usingFASTA program The Clustal W program (httpwwwebiacukclustalw) was used for multiple alignment of CvHSP90An unrooted phylogenetic tree was constructed accordingto amino acid sequences of the selected HSP90 by usingthe neighbor-joining (NJ) algorithm embedded in MEGA31 program (httpwwwmegasoftwarenet) [31] and theprograms of Clustal X 183 [32] The bootstrap trials werereplicated 1000 times to derive the confidence value for thephylogeny analysis

25 Quantitative Analysis of CvHSP90 mRNA ExpressionmRNA expression level of CvHSP90 was investigated byreal-time PCR amplification on a fast real-time PCR system(Applied Biosystem 7500) A product of 150 bp from cDNAwas amplified by two CvHSP90 gene-specific primers P7 andP8 (Table 1) and the PCRproduct was sequenced to verify thespecificity of RT-PCR An internal control a 109 bp fragmentwas amplified by two 120573-actin primers The real-time PCRamplifications were carried out in a total volume of 20 120583Lmixture containing 10 120583L SYBR Premix Ex Taq (TaKaRaTokyo Japan) 2 120583L cDNA 04 120583L each forward and reverseprimer (10 120583molL) 04120583L ROX Reference Dye (50X) and68 120583L PCR-grade water The reactions conditions were 94∘Cfor 5min followed by 40 cycles of 5 s at 94∘C and 30 s at 60∘CFive independent biological replicateswere carried out At theend of each PCR reaction dissociation curve of ampliconwasanalyzed to confirm if only one PCR product was amplifiedand detected After the PCR program SDS software V201(Applied Biosystems) was used to analyze the data duringwhich a baseline was set automatically for consistency Theexpression level of CvHSP90 was analyzed by the 2minusΔΔCtmethod [33] All data were expressed as mean plusmn SE (119899 =5) in terms of relative mRNA Furthermore the data wereanalyzed by ANOVA (one-way analysis of variance) followedby an unpaired two-tailed t-test Difference was consideredsignificant at 119875 lt 005

3 Results

31 cDNA Cloning and Sequencing of CvHSP90 Gene ACvHSP90 fragment (745 bp) was amplified by homologouscloning primers P1 and P2 and confirmed highly similarto other known HSP90s Two pairs of CvHSP90-specificprimers (P3-P4 and P5-P6) that designed in the abovesequencewere used to clone the full-length cDNA RACE andnested PCR were performed to amplify the two fragmentscorresponding to the 31015840 and 51015840 end of the CvHSP90 cDNAThe full-length cDNA sequence of CvHSP90 was determined3678 bp by cluster analysis of the above fragments

32 Characterization of CvHSP90 The cDNA sequenceof CvHSP90 was submitted in GenBank under accessionnumber JQ655149 The full-length cDNA of CvHSP90 was3678 bp with a 107 bp 51015840 untranslated region (51015840UTR) a1459-bp 31015840 untranslated region (31015840UTR) with a poly (A)tail and a 2112 bp open reading frame (ORF) encoding apolypeptide that contained 703 amino acids with estimatedmolecular mass of 8071 kDa and an estimated isoelectricpoint of 448 The five typical amino acid blocks of HSP90protein family (NKEIFLRE[LI]ISN[AS]SDALDKIRLGTIARSGT IGQFGVGFYSAYLVA[ED] IKLYVRRVFIG[VI]VDSEDLPLNISRE) and the consensus MEEVD atthe C-terminus were highly conserved as indicated in theCvHSP90 sequence (Figure 1) [34] Meanwhile SMARTprogram revealed a typical histidine kinase-like ATPasesdomain in the position 27-182 which is ubiquitous in allHSP90 family members

33 Homology Analysis of CvHSP90 Shown by Clustal Wprogram the deduced amino acid sequence of CvHSP90shared high homology with those of other known HSP90s(Figure 2) For example CvHSP90 shared 85 similar-ity to Micromonas sp RCC299 HSP90 (XP 002499727)and Micromonas sp RCC299 HSP90 (XP 002499727) 79similarity to Haematococcus pluvialis HSP90 (JN627245)Chlamydomonas reinhardtii HSP90 (XP 001695264) andVolvox carteri f nagariensis HSP90 (XP 002947115) and78 similarity to Pennisetum glaucum HSP90 (ADP89125)Hordeum vulgare HSP90 (BAJ86355) Triticum aestivumHSP90 (ADF31758) Triticum urartu HSP90 (ADF31773)Vitis viniferaHSP90 (CAN62488) andThellungiella halophilaHSP90 (BAJ33984) and so forth (Table 2) Multiple sequencealignment exhibited high conservation between CvHSP90and other known HSP90 proteins especially in the regionsof HSP90 family signatures (Figure 2)

To evaluate the molecular evolutionary relationshipsof CvHSP90 against other HSP90s a phylogenetic treebased on the protein sequences (Figure 3) was constructedby neighbor-joining method In the phylogenetic tree theHSP90s are clustered into two major groups in the Strep-tophyta and Chlorophyta origin CvHSP90 is clusteredfirst with HSP90 from Mamiellophyceae (Micromonas spRCC299 and Micromonas pusilla CCMP1545) and thenformed a sister group with those of Chlorophyta and furtherwith those of Streptophyta Relationships displayed in thephylogenic tree agree in overall with traditional taxonomy


1 GAGCTTGCGTTTCGCAGTCACTTTGCAAAAGAAGAACACGGCAACGTCAGACGTGCAGTG

1 M A E P E 61 TGGGATTAGATATTTGTGAAAACTAGAGCCTGGTGAGTAGCAAGCATGGCAGAGCCCGAG 6 T F A F Q A E I N Q L L S L I I N T F Y 121 ACCTTCGCCTTCCAGGCTGAGATCAACCAGCTGCTCAGTCTGATCATTAACACGTTCTAC

26 S N K E I F L R E L I S N A S D A I D K Signature motif-1

181 TCCAACAAGGAAATCTTCTTGAGGGAACTGATCAGCAATGCCTCGGACGCTATTGACAAA

46 V R F Q S L T D K S V L E S N P E L Y I 241 GTCCGTTTCCAGTCCCTCACTGACAAGTCGGTGCTGGAGAGCAACCCAGAGCTTTACATC

66 H I T P D K A N N T L T I T D S G V G M 301 CACATCACGCCTGACAAGGCCAACAACACGCTCACGATCACAGACTCTGGTGTCGGCATG

86 T K A D L V N N L G T I A R S G T K A F Signature motif-2

361 ACAAAGGCGGATCTGGTGAACAACTTGGGAACCATCGCACGCTCGGGGACCAAGGCGTTC

106 M E A L S A G A D I S M I G Q F G V G FSignature motif-3

421 ATGGAGGCTCTGAGTGCCGGCGCGGACATCAGCATGATTGGCCAGTTCGGCGTGGGTTTC

126 Y S A Y L V A D R V S V I T K H N D D E 481 TACTCCGCCTACCTGGTTGCAGACCGCGTGAGCGTCATCACAAAGCACAACGATGACGAG

146 Q Y I W E S Q A G G S F T I A R D T V N 541 CAGTACATATGGGAGAGCCAGGCCGGCGGCTCTTTCACCATCGCGCGTGACACCGTCAAC

166 P S L G R G T Q I T L H L K E D Q M E Y 601 CCGTCGCTTGGCCGCGGCACGCAGATCACCCTCCACCTCAAGGAGGACCAGATGGAGTAC

186 L E E R R L K D L I K K H S E F I S Y P 661 CTCGAGGAGCGCCGCCTCAAGGACTTGATCAAGAAGCACAGCGAGTTCATCTCGTACCCG

206 I S L W V E K T T E K E V D D D E E E E 721 ATCAGCCTGTGGGTGGAGAAGACCACCGAGAAGGAGGTGGATGATGACGAGGAGGAGGAG

226 P K D D D E E G K V E E I K E E E E E E 781 CCCAAGGACGACGACGAGGAGGGCAAGGTTGAGGAGATCAAGGAGGAGGAGGAGGAGGAG

246 K K E K K K K K V K E V S H E W Q L V N 841 AAGAAGGAGAAGAAGAAGAAGAAGGTGAAGGAGGTGTCGCACGAGTGGCAGCTGGTGAAC

266 K Q K P I W M R N P E E I S K E E Y E A 901 AAGCAGAAGCCCATCTGGATGCGCAACCCCGAGGAGATCTCCAAGGAGGAGTACGAGGCC

286 F Y K S L T N D W E E P L A Q K H F A V 961 TTCTACAAGTCCCTCACCAATGACTGGGAGGAGCCCCTGGCGCAGAAGCACTTTGCTGTG

306 E G Q L E F K S I L F V P K R A P F D L 1021 GAGGGCCAGCTAGAGTTCAAGTCCATCCTCTTCGTGCCCAAGCGCGCCCCCTTCGACCTC

326 F D T R K K S N N I K L Y V R R V F I M Signature motif-4

1081 TTCGACACCAGGAAGAAGTCGAACAACATCAAGCTGTATGTGAGGCGGGTGTTCATCATG

346 D N C E E L I P E W L G F M K G I V D S

1141 GACAACTGCGAGGAGCTCATCCCCGAGTGGCTCGGCTTCATGAAGGGCATCGTGGACAGC

366 E D L P L N I S R E M L Q Q N K I L K V Signature motif-5

1201 GAGGACCTGCCCCTCAACATCTCCCGCGAGATGCTCCAGCAGAACAAGATCCTCAAGGTC

386 I K K N L I K K S I E L F N E I A E N K 1261 ATCAAGAAGAACCTCATCAAGAAGAGCATTGAGCTCTTCAATGAGATTGCCGAGAACAAG

406 D D Y N K F Y E S F G K N L K L G V H E 1321 GATGACTACAACAAGTTCTACGAGTCCTTCGGCAAGAACCTGAAGCTGGGAGTGCACGAG

426 D S A N R S K L A E L L R Y H S T K S G 1381 GACAGCGCCAACCGCTCCAAGCTGGCAGAGCTGCTCCGCTACCACTCCACCAAGTCTGGT

446 E E L T S L K D Y V T R M K E S Q K D I 1441 GAGGAGCTGACGAGTCTGAAGGACTATGTGACCCGGATGAAGGAGAGCCAGAAGGACATC

466 Y Y I T G E S R K A V E N S P F I E R L 1501 TACTACATCACCGGCGAGTCGCGCAAGGCCGTCGAGAACTCCCCCTTCATCGAGCGCCTC

486 K K K N L E V L F L V D P I D E Y A V Q 1561 AAGAAGAAGAACCTCGAGGTGCTGTTCCTGGTGGACCCCATCGATGAGTATGCGGTGCAG

506 Q L K E Y D G K K L V S V T K E G L T I 1621 CAGCTGAAGGAGTACGACGGCAAGAAGCTGGTGAGCGTGACCAAGGAGGGCCTGACTATC

526 D E T E E D K K R L E E L K A S Y E P L 1681 GACGAGACCGAGGAGGACAAAAAGCGGCTGGAGGAGCTCAAGGCCTCCTACGAGCCCCTC

546 C G L I K D I L S D K V E K V V V G E R

(a)

Figure 1 Continued


1741566180158618616061921626198146620416662101686

Cytosolic HSP90-specifc motif2161222122812341240124612521

2641 2701276128212881 2941300130613121318132413301336134213481354136013661

2581

TGCGGCCTCATCAAGGACATCCTCTCCGACAAGGTCGAGAAGGTGGTGGTGGGCGAGCGC

A V D S P C V L V T G E Y G W S A N M E GCGGTGGACTCGCCATGCGTGCTGGTGACGGGCGAGTACGGCTGGAGCGCCAACATGGAG

R I M K A Q A L R D S S M S S Y M T S K CGCATCATGAAGGCGCAGGCCCTGCGCGACTCCTCCATGTCCTCCTACATGACCTCCAAG

K T L E I N P E N A I V S E L K K R A D AAGACCCTGGAGATCAACCCCGAAAACGCCATCGTCTCGGAGCTGAAGAAGCGCGCTGAC

V D K S D K T V K D L V L L L F E T A L GTGGACAAGAGCGACAAGACGGTGAAGGACCTGGTGCTGCTGCTGTTTGAGACTGCGCTG

L T S G F S L D E P N T F G T R I H R M CTGACTAGCGGCTTCTCCCTGGACGAGCCCAACACCTTTGGCACGCGCATCCACCGCATG

I K L G L S I D E D D L P V E G D D E D ATCAAGCTCGGCCTCTCCATCGACGAGGACGACCTGCCCGTGGAGGGTGATGACGAGGAC

L P P L E E D V D E G S R M E E V D

CTGCCACCCCTTGAGGAGGATGTGGATGAGGGCTCCCGCATGGAGGAGGTTGACTAAGTG

CAGCACAGACAACAAAGACAGCAGCCGGTGGGGAGCAGTCAGTGTGGTTGTTCCTTCCCA

AGCGCATGCCTGGATGCTTTGAGCCGCAGAAGAGACGGGCAAAGTGCCAAGATTGCTGTG

CATTTGACAAGAGGCTGCAGTGTGCCCCAGAAAGGGGGAGGCTTGGCTGGCTTGGTGCCA

ATGGCACAGTGGAAGTGTAGCAATATAAGGAGTTGTCCTCTAGCAGAGGGTTTGTCCTTG

AACATCCTGCCCGTGTGAGAATGTTGATTCGTGTGTGATGGGTTTCCTCACAGAGTATTC

TTCACTTTTCCACAGAACAATTCATGCTGTCATAGGAAGGAATTTAGTACCGGAACAAGC

GATACGTGCTGTGGTTGAATCTTAAGCCTCGCCTAAGGTGCAGTGCCCTTTGGCTGTGTA

ATACAGCTTGAGCAAAGTCACAGACATGCATGAATATATTGTTTGCGAGCTGTTTTACTC

ACAGTTCCACTTGCAACTTGCCTGAGATCATTTCGCCACAATCATGCCACCCTTCAAGGC

AGTGTCCATTCTGAGCCATCATCATGATACACACCAAATCCAGACTGGAGGTCAAGAACA

TGGACATCCTCTCTTAAAAAACCCAGTACATTTGTCACAATTCTGTTCTTCCCTACCATT

GCGAGCGAAGCTTAAAGTCCAAAGCTTAGTTCTGCTTGCTGATCTGTCAATAGTGCATGC

CACAGACTCCTTGCCACTGTTGTCCATGCGAAGAACCTCACAATGGCAGCAAAAGGCTCT

TCCAGAGCATGTGCTCTTCCATCATAATAATATACAATAGCATTGAGGCACGCGTCAATG

TGTGAGCCTGGCGACATCAATTCTCCTGTCCATCCATCGTCCAGACCTTACGCCATGACA

GGGATGTTGAAGACGACAACACCGTGGCCCAGGTTGATGAGCTTGTCTGGTCTTTTGTAC

ACCACAAAATTATAAAACAATTGATGATGGAACTTGTACAACGCCCTCCAAGTCAAGCCC

TTCTTTCCAGAGAGGTCGGCGTGGCGGGACATACGGAACTTGCTGCAGCACAGCTCCTGC

CATAAAGAATCGTCACTCAGGACAGCTTCCCTTGAGAACTTGCACACTTGTTCAAAGGTG

CACAAGTCTCTCGCGTTGTCAAATTTACACGCCACCAAGTGCACAATGTGCAATGGAAGG

TTCTCTAATTTCACGGGACCCTTCAGCGGTGTCTGCTCAGGCAGAGTGTGCAGAAATTGC

GGGTGCACTATTAAAGCTTTCTTACCCATCGTCCTGAATGACCATATAAAACAGGATGCT

GCTTCTCAATGCCAAGCTAGCTCGCGAGAACCAATAGTTTTACATCCTGACCGTAAAAAAA

AAAAAAAAAAAAAAA

lowast

ACATCATTTTGTTCTAAATCCTTGCCAATGAGATATCGATATAGAGGCAGAGCCAAACGT

(b)

Figure 1The full length cDNA sequence of CvHSP90 and its deduced amino acid sequenceThenucleotide and deduced amino acid sequenceof the open reading frame and flanking region were numbered on the left The start and stop codons were bold HSP90 signature motifs andcytosolic HSP90-specific motif were underlined

34 Expression Levels of CvHSP90 under Different HeatShock Temperatures The expression level of CvHSP90 wasinvestigated by real-time quantitative PCR under differentstressful conditions As the optimal growth temperature forC vulgaris is 20ndash25∘C different heat shock temperatures (5ndash45∘C) was scheduled to study the mRNA expression levelsof CvHSP90 (Figure 4) In 20∘C and 25∘C treatment groupsthe expression levels of CvHSP90 were relatively low while in5∘C and 35∘C treatment groups were almost 25-fold of that in20∘C group (119875 lt 005)The highest expression level (3-fold ofthat in 20∘Cgroup)was determined at 40∘C for 1 h (119875 lt 001)

35 Expression Levels of CvHSP90 at Different Heat ShockTimes In this study we found that a long time (over 8 h)exposure at over 40∘Cwould be fatal toC vulgarisTherefore

the organism was kept at 35∘C for different hours to findthe impact of different heat shock times on the expressionlevel of CvHSP90 As shown in Figure 5 in 35∘C CvHSP90mRNA expression level increased gradually and reached themaximum (42-fold of that of blank group) at 7 h (119875 lt 001)and then declined progressively to the original level at 12 hUnder a heat shock the expression level of CvHSP90 wasobserved significantly different at 1 2 3 4 5 8 and 9 h fromthat of the blank group (119875 lt 005)

36 Expression Levels of CvHSP90 under Different Salt Con-centrations The expression levels of CvHSP90 at differentsalt concentrations for 2 h were detected (Figure 6) Theexpression level of CvHSP90 at 10 15 20 and 30 in salinity


T D K S R L E A Q P E L F I H I V P D K T N N T L T I I D S G I G M T K A D L V N N L G T I A R S G T K A F M E A L T A 112T D K S R L E G Q P E L F I H I I P D K T N N T L T I I D S G I G M T K A D L V N N L G T I A R S G T K S F M E A L T A 112T D K S V L E S N P E L Y I H I T P D K A N N T L T I T D S G V G M T K A D L V N N L G T I A R S G T K A F M E A L S A 111T D K S K L D A Q P E L F I H I V P D K T N N S L T I I D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D A Q P E L F I H I I P D K T N N S L T I I D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D A Q P E L F I H I I P D K T N N S L T I V D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D A Q P E L F I H I I P D K T N N T L T I I D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D G Q P E L F I H I I P D K T N N T L T I I D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D G Q P E L F I H I I P D K T N N T L T I V D S G I G M T K A D L V N N L G T I A R S G T K E F M E A L A A 111T D K S K L D A Q P E L F I H I V P D K A N N T L T I I D S G I G M T K S D L V N N L G T I A R S G T K E F M E A L A A 112T D K S K L D A Q P E L F I H I V P D K A N N T L T I I D S G I G M T K S D L V N N L G T I A R S G T K E F M E A L A A 112T D K S K L D A Q P E L F I H I V P D K A S N T L T I I D S G I G M T K S D L V N N L G T I A R S G T K E F M E A L A A 112T D K S K L D A P P E L F I H I I P D K A T N T L T L I D S G I G M T K S D L V N N L G T I A R S G T K D F M E A L A A 112T D K S K L D A Q P E L F I R I I P D K A T N T L T L I D S G I G M T K S D L V N N L G T I A R S G T K D F M E A L A A 112T D K S K L D A Q P E L F I H I I P D K A T N T L T L I D S G I G M T K S D L V N N L G T I A R S G T K D F M E A L A A 112T D K S K M D G Q P E L F I H I V P D K A N N T L S I I D S G I G M T K A D M V N N L G T I A R S G T K E F M E A L T A 115T D K S V L D N N P E L Y I H L Q P N K A D G T L A I T D S G I G M T K A D L I N N L G T I A R S G T K A F M E A L S A 114T D K S V L D S N P E L Y I H L V P N K S D G S L A I I D S G I G M T K A D L I N N L G T I A R S G T K A F M E A L S A 117T D K T Q L D S H P E L H I R L L P N K Q E G T L A I L D S G I G M T K P D L V N N L G T I A R S G T K S F M E A L S A 112

G A D I S M I G Q F G V G F Y S A Y L V A E K V I V Y T K H N D D E Q Y R W E S Q A G G S F T V T K D N S E P - M G R G 171G A D I S M I G Q F G V G F Y S A Y L V A E K V V V Y T K H N D D E Q Y R W E S Q A G G S F T V T K D N A E A - M G R G 171G A D I S M I G Q F G V G F Y S A Y L V A D R V S V I T K H N D D E Q Y I W E S Q A G G S F T I A R D T V N P S L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A E K V I V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E S L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A E K V I V T A K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E S L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A E K V I V T S K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E V L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E N L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A D K V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E T L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A D K V I V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E S L G R G 171G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E Q L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E Q L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T T K H N D D E Q Y V W E S Q A G G S F T V T R D T S G E P L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T S K H N D D E Q Y V W E S Q A G G S F T V T R D T T G E P L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E R V I V T S K H N D D E Q Y V W E S Q A G G S F T V T R D T T G E P L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E R V V V T S K H N D D E Q Y V W E S Q A G G S F T V T R D T T G E P L G R G 172G A D V S M I G Q F G V G F Y S A Y L V A E K V V V T S K H N D D E Q Y I W E S Q A G G S F T I T R D T S G E Q L G R G 175G A D V S M I G Q F G V G F Y S A Y L V A D R V T V V T K H N D D E Q Y V W E S Q A G G S F S I R R D T E G E P L G R G 174G A D V S M I G Q F G V G F Y S A Y L V A D K V T V V T K H N D D E Q Y V W E S Q A G G S F S I R R D T D G E P L G R G 177G A D V S M I G Q F G V G F Y S A Y L V A D K V T V V T K H N D D E Q Y V W E S Q A G G S F T V T Q D T S G H S M G R V 172

T K M V L H L K D D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W T E K T T E K E V S D D E - A E E D D A A E 230T K M V L H L K D D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W T E K T T E K E V S D D E - E E E T D E K E 230T Q I T L H L K E D Q M E Y L E E R R L K D L I K K H S E F I S Y P I S L W V E K T T E K E V D D D E - E E E P K D D D 230T K I T L Y L K E D Q L E Y L E E R R L K D L I K K H S E F I S Y P I S L W I E K T T E K E I S D D E - D E E D K K D - 229T K I T L Y L K E D Q L E Y L E E R R V K D L I K K H S E F I S Y P I S L W I E K T T E K E I S D D E - D E E D K K D - 229T K I T L F L K E D Q L E Y L E E R R L K D L I K K H S E F I S Y P I S L W V E K T T E K E I S D D E - D E E E K K D - 229T K I T L F L K E D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W V E K T I E K E I S D D E - D E E E K K D - 229T K M V L Y L K E D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W I E K T I E K E I S D D E - E E E E K K D - 229T K M T L H L K E D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W V E K T I E K E I S D D E - D E E E K K D - 229T K M T L Y L K D D Q L E Y L E E R R L K D L I K K H S E F I S Y P I S L W I E K T T E K E I S D D E - D E E D K K D - 230T K M T L Y L K D D Q L E Y L E E R R L K D L I K K H S E F I S Y P I S L W I E K T T E K E I S D D E - D E E D K K D - 230T K M T L Y L K D D Q L E Y L E E R R L K D L I K K H S E F I S Y P I S L W T E K T T E K E I S D D E - D E E D K K D - 230T K I T L Y L K D D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W T E K T T E K E I S D D E - D E D E K K D T 231T K I T L Y L K D D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W T E K T T E K E I S D D E - D E D E K K D T 231T K I T L Y L K D D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W T E K T T E K E I S D D E - D E D E K K D T 231T H I K L Y L K E D Q L E Y L E E R R L K D L V K K H S E F I S Y P I S L W S E K T T E K E V S D D E - E D E E K K E E 234T K I I L H L K E D Q K E Y L E E R R I K D L V K K H S E F I S Y P I S L W T E K T V D K E V S D D E A E E E E K K E E 234T K I I L H L K E D Q K E Y L E E R R L K D L V K K H S E F I S Y P I E L W T E K T V D K E V S D D - - - E E E V K E D 234T K I I L H R K E D Q K E Y L E E R R L K D L V K K H S E L I S Y P I L W T E K S V D K E V S D E E - D E A M A D A T 231

E E G K I T E I K D E D E - - K K E K K K K T V K E V S H E W A L M N K Q K P I W M R A P E E I S K D E Y S A F Y K S L 288

E E G K I T E I K D E D E - - K K E K K T K K V K E V S H E W A I M N K Q K P I W M R N P E E I S K D E Y S A F Y K S L 288E E G K V E E I K E E E E E E K K E K K K K K V K E V S H E W Q L V N K Q K P I W M R N P E E I S K E E Y E A F Y K S L 290E E G K V E E V D E E K E K E E - K K K K K - I K E V S H E W S L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L 287E E G K V E E V D E E K E K E E - K K K K K - I K E V S H E W S L V N K Q K P I W M R K P E E I T K E E Y S A F Y K S L 287E E G K V E D V D E E K E K E E - K K K K K - I K E V S H E W S L V N K Q K P I W M R K P E E I T K E E Y S A F Y K S L 287E E G K V G E V D E E K E K E E - K K K K K - I K E V S N E W S L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L 287E E G K V E E V D E E K E K E E - K K K K K - I K E V S H E W D L V N K Q K P I W M R K P E E I N K E E Y A A F Y K S L 287E E G K V E E V D E E K E K E E - K K K K K - I K E V S N E W D L V N K Q K P I W M R K P E E I N K E E Y A A F Y K S L 287E E G K V E D V D E E K E E K E - K K K K K - I K E V S H E W Q L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L 288E E G K V E D V D E - K E E K E - K K K K K - I K E V S H E W Q L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L 287E E G K V E E I D E E K E E K E - K K K K K - I K E V S H E W Q L V N K Q K P I W M R K P E E I T K E E Y A A F Y K S L 288E E G K V E E I D E E K E E K E - K K K K K - I K E V S H E W N L I N K Q K P I W M R K P E E I T K D E Y A A F Y K S L 289E E G K V E E I D E E K E E K E - K K K K K - I K E V S H E W N L I N K Q K P I W M R K P E E I T K D E Y A A F Y K S L 289E E G K V E E I D E E K E E K E - K K K K K - I K E V S H E W N L I N K Q K P I W M R K P E E I T K D E Y A A F Y K S L 289E E G K I E E V D E E K E K E K E K K K K K - V K E V S H E W A L M N K Q K P I W M R K P E D V T K E E Y A A F Y K S L 293E E G K V E E V K E E K - - - - - E K K T K K V Q E V Q H E W D L L N K Q K P I W M R N P D E V T K E E Y A A F Y K S I 289E E G K V E E V K E E K - - - - - E K K K K K V K E V Q H E W S L L N K Q K P I W M R N P D E V T K E E Y A A F Y K S I 289E E G K V E E V K E K K G Q - - - D K K R K K V K E V Q H E W N L L N K Q K P I W M R A P E T V A K E E Y A A F Y K S L 288

T N D W E E Q L A V K H F A V E G Q L E F K S V L F V P K R A P F D M F D G K K K A N N I K L Y V R R V F I M D N C E D 348T N D W E E Q L A V K H F A V E G Q L E F K S V L F V P K R A P F D M F D G K K K S N N I K L Y V R R V F I M D N C E D 348T N D W E E P L A Q K H F A V E G Q L E F K S I L F V P K R A P F D L F D T R K K S N N I K L Y V R R V F I M D N C E E 350T N D W E E H L A V K H F S V E G Q L E F K A I L F V P K R A P F D L F D T R K K P N N I K L Y V R R V F I M D N C E E 347T N D W E E H L A V K H F S V E G Q L E F K A I L F V P K R A P F D L F D T R K K P N N I K L Y V R R V F I M D N C E E 347T N D W E E H L A V K H F S V E G Q L E F K A I L F V P K R A P F D L F D T R K K P N N I K L Y V R R V F I M D N C E E 347T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T K K K P N N I K L Y V R R V F I M D N C E E 347S N D W E E H L A V K H F S V E G Q L E F K A I L F V P K R A P F D L F D T K K K P N N I K L Y V R R V F I M D N C E D 347S N D W E E H L A V K H F S V E G Q L E F K A I L F V P K R A P F D L F D T K K K P N N I K L Y V R R V F I M D N C E D 347T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N I K L Y V R R V F I M D N C E E 348T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N I K L Y V R R V F I M D N C E E 347T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K Q N N I K L Y V R R V F I M D N C E E 348T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N I K L Y V R R V F I M D N C E E 349T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N I K L Y V R R V F I M D N C E E 349T N D W E E H L A V K H F S V E G Q L E F K A V L F V P K R A P F D L F D T R K K L N N I K L Y V R R V F I M D N C E E 349S N D W E E H L A V K H F S V E G Q L E F K S V L F V P K R A P F D L F D S R K K Q N N I K L Y V R R V F I M D N C E E 353S N D W E D H L A V K H F S V E G Q L E F K S I L Y L P K R A P F D M F D Q R K K P N N I K L Y V R R V F I M D N C E D 349S N D W E D Y L S V K H F S V E G Q L E F K C I L F L P R R A P F D M F D Q R K K P N N I K L Y V R R V F I M D N C E E 349S N D W E D H L A C K H F S V E G Q L E F K S I L F I P K R A P F D L F D Q H K K R N N I K L Y V R R V F I M D N C E E 348

lowastlowast

lowast lowast lowast lowast

lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

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lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

Micromonas sp RCC299 - - - M A E - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E G L 52Micromonas pusilla - - - M A E - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E G L 52

Chlorella vulgaris - - - - M A - - - - - E P E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A I D K V R F Q S L 51Vitis vinifera - - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E I I S N S S D A L D K I R F E S L 51

Vitis pseudoreticulata - - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 51Glycine max - - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 51

Nicotiana benthamiana - - - - M A - - - - - E A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51Arabidopsis thaliana - - - - M A - - - - - D A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51

Thellungiella halophila - - - - M A - - - - - D A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51Sorghum bicolor - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52

Zea mays - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52Pennisetum glaucum - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52

Triticum urartu - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52Triticum aestivum - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52Hordeum vulgare - - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52

Physcomitrella patens M A D G D A - - - - - Q V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 55Chlamydomonas reinhardtii - - - M A S - - - E A P V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y M S L 54

Volvox carteri - - - M A T T V D A P E V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y M S L 57Haematococcus pluvialis

Micromonas sp RCC299Micromonas pusilla

Chlorella vulgarisVitis vinifera

Vitis pseudoreticulataGlycine max

Nicotiana benthamianaArabidopsis thaliana

Thellungiella halophilaSorghum bicolor

Zea maysPennisetum glaucum

Triticum urartuTriticum aestivumHordeum vulgare

Physcomitrella patensChlamydomonas reinhardtii

Volvox carteriHaematococcus pluvialis





Thellungiella halophila

Nicotiana benthamiana


Sorghum bicolorZea mays

Pennisetum glaucumTriticum urartu

Triticum aestivumHordeum vulgare






Arabidopsis thaliana
























- - - M A S - - - - - E T E T Y A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y L G L 52

- - - M A E - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E G L 52- - - M A E - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E G L 52- - - - M A - - - - - E P E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A I D K V R F Q S L 51- - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E I I S N S S D A L D K I R F E S L 51- - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 51- - - - M A - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 51- - - - M A - - - - - E A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51- - - - M A - - - - - D A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51- - - - M A - - - - - D A E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 51- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 52- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52- - - M A S - - - - - E T E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R F E S L 52M A D G D A - - - - - Q V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N S S D A L D K I R F E S L 55- - - M A S - - - E A P V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y M S L 54- - - M A T T V D A P E V E T F A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y M S L 57- - - M A S - - - - - E T E T Y A F Q A E I N Q L L S L I I N T F Y S N K E I F L R E L I S N A S D A L D K I R Y L G L 52

I I P E F L S F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I K K N I V K K C L E M F N E I A E N K D D Y T K 408I I P E Y L S F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I K K N I V K K C L E M M N E I A E N K D D Y T K 408L I P E W L G F M K G I V D S E D L P L N I S R E M L Q Q N K I L K V I K K N L I K K S I E L F N E I A E N K D D Y N K 410L I P E Y L G F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I R K N L V K K C L E L F F E I A E N K D D Y N K 407L I P E Y L G F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K D D Y N K 407L I P E Y L G F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I R K N L V K K C L E L F F E I A E N K E D Y N K 407L I P E Y L S F V K G I V D S E D L P L N I S R E M L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y D K 407I I P E Y L G F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C L E L F F E I A E N K E D Y N K 407I I P E Y L G F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C L E L F F E I A E N K E D Y N K 407L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y N K 408L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y N K 407L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K D D Y N K 408L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y N K 409L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y N K 409L I P E W L S F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I R K N L V K K C I E L F F E I A E N K E D Y T K 409L I P E Y L G F V K G V V D S E D L P L N I S R E T L Q Q S K I L K V I R K N L V K K C M E M F A E V A E N K E D Y Q K 413L I P E W L N F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I K K N I V K K C L E L F A E V A E N K D D Y N K 409L I P E W L N F V K G I V D S E D L P L N I S R E T L Q Q N K I L K V I K K N I V K K C L E L F A E V A E N K D D Y A K 409L I P E W P S F V K G V V D S E D L P L N I S R E T L Q Q N K I L K V I R K N V V K K C L E L F G E V A E N K D D Y A K 408










lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

(a)

Figure 2 Continued


F Y E A F G K N L K L G I H E D A Q N R S K L A E L L R Y H S T K S G E E M T S L K D Y V T R M K E N Q K D I Y Y I T G 468F Y E S F G K N L K L G I H E D S Q N R T K L A E L L R Y H S T K S G D E M T S L K D Y V T R M K E N Q K D I Y Y I T G 468F Y E S F G K N L K L G V H E D S A N R S K L A E L L R Y H S T K S G D E L T S L K D Y V T R M K E S Q K D I Y Y I T G 470F Y E A F S K N L K L G I H E D S Q N K G K L A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I Y Y I T G 467F Y E A F S K N L K L G I H E D S Q N K T K L A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q S D I Y Y I T G 467F Y E A F S K N L K L G I H E D S Q N K G K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q S D I Y Y I T G 467F Y E A F S K N L K L G I H E D S Q N R S K F A E L L R Y H S T K S G D E M T S L K D Y V T R M K E G Q N D I Y Y I T G 467F Y E A F S K N L K L G I H E D S Q N R T K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I F Y I T G 467F Y E A F S K N L K L G I H E D S Q N R T K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I F Y I T G 467F Y E A F S K N L K L G I H E D S T N R T K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I Y Y I T G 468F Y E A F S K N L K L G I H E D S T N R N K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I Y Y I T G 467F Y E A F C K N L K L G I H E D S Q N R N K I A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q S D I Y Y I T G 468F Y E A F S K N L K L G V H E D S T N R T K L A E L L R Y H S T K S G D E L T S L K D Y V T R M K E G Q N D I Y Y I T G 469F Y E A F S K N L K L G V H E D S T N R T K L A E L L R Y H S T K S G E E L T S L K D Y V T R M K E G Q N D I Y Y I T G 469F Y E A F S K N L K L G V H E D S T N R T K L A E L L R Y H S T K S G E E L T S L K D Y V T R M K E G Q N D I Y Y I T G 469F Y E A F A K N L K L G I H E D S Q N R S K L A D L L R Y H S T K S G E E M T S L K D Y V T R M K E G Q K D I Y Y I T G 473F Y E S F G K N L K L G V H E D S Q N R A K L A D L L R Y H S T K S G D E T T S L K D Y V T R M K E G Q K A I Y Y I T G 469F Y E A F G K N L K L G V H E D S Q N R A K L A D L L R Y H S T K S G E E L T S L K D Y V T R M K E G Q K S I Y Y I T G 469F Y E A F S K N L K L G V Y E D S Q N R A K L A D L L R F H S T K S G D E A T S L K D Y V T R M K E G Q K D V Y Y I T G 468

E S R K A V E N S P F I E K L K K R G L E V L F M V D P I D E Y A V Q Q L K E Y D G K K L V C C T K E G L Q L D E T E E 528E S R K A V E N S P F I E K L K K R G L E V L F M V D P I D E Y A V Q Q L K E Y D G K K L V C C T K E G L T L D E T D E 528E S R K A V E N S P F I E R L K K K N L E V L F L V D P I D E Y A V Q Q L K E Y D G K K L V S V T K E G L T I D E T E E 530E S K K A V E N S P F L E K L K K K G I E V L F M V D A I D E Y A V G Q L K E F E G K K L V S A T K E G L K L D E S E D 527E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y A V G Q L K E F E G K K L V S A T K E G L K L D E S E D 527E S K K A V E N S P F L E K L K K K G Y E V L F M V D A I D E Y A V G Q L K E F E G K K L V S A T K E G L K L D E S E D 527E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y S V G Q L K E F E G K K L V S A T K E G L K L D E S E D 527E S K K A V E N S P F L E K L K K K G I E V L Y M V D A I D E Y A I G Q L K E F E G K K L V S A T K E G L K L D E T E D 527E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y A I G Q L K E F E G K K L V S A T K E G L K L E E S E D 527E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y A I G Q L K E F E G K K L V S A T K E G L K L D E S E D 528E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y A I G Q L K E F E G K K L V S A T K E G L K L D E S E D 527E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y A I G Q L K E F E G K K L V S A T K E G L K L D E S E D 528E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y S I G Q L K E F E G K K L V S A T K E G L K L D D S E E 529E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y S I G Q L K E F E G K K L V S A T K E G L K L D D S E E 529E S K K A V E N S P F L E K L K K K G Y E V L Y M V D A I D E Y S I G Q L K E F E G K K L V S A T K E G L K L D D S E E 529E S K K A V E N S P F L E K L K R R G Y E V L Y M V D A I D E Y A V G Q L K E Y D G K K L V S A T K E G L V L E E T E E 533E S R K A V E N S P F L E R L K K M G Y E V L F M V D P I D E Y A V Q Q L K E Y D G K K L V C C T K E G L D L D E S E E 529E S R K A V E N S P F L E R L K K K G Y E V L F M V D P I D E Y A V Q Q L K E Y D G K K L V C C T K E G L D L D D S E E 529E S R K A V E N S P F V E K L R R K G L E V L F M V D P I D E Y V T Q Q L K E Y D G K K L V C C T K E G L K L E E S E E 528

E K A K K E E V K A Q Y E A L C R L M K D I L G D K V E K V L V S D R V V D S P C V L V T G E Y G W S A N M E R I M K A 588E K A K K E E V K S T F E A L C R L M K D I L G D K V E K V L V S D R V V D S P C V L V T G E Y G W S A N M E R I M K A 588D K K R L E E L K A S Y E P L C G L I K D I L S D K V E K V V V G E R A V D S P C V L V T G E Y G W S A N M E R I M K A 590E K K K Q E A L K E K F E G L C K V I K D V L G D R V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K Q Q E A L K E K F E G L C K V M K D V L G E R V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K K Q E E L K E K F D N L C K V I K D V L G D K V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K K H E E L K E K F E G L C K V I K D V L G D K V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K K K E E L K E K F E G L C K V I K D V L G D K V E K V I V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K K K E E L K E K F E G L C K V I K D V L G D K V E K V I V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K K K E E L K E K F E G L C K V I K E V L G D K V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 588E K K R K E E L K E K F E G L C K V I K E V L G D K V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 587E K K R K E E L K E K F E G L C K V I K E V L G D K V E K V V V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 588E K K R K E E L K E K F E G L C K V I K E V L G D R V E K V I V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 589E K K R K E E L K E K F E G L C K V I K E V L G D R V E K V I V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 589E K K R K E E L K E K F E G L C K V I K D V L G D R V E K V I V S D R V V D S P C C L V T G E Y G W T A N M E R I M K A 589E K K K K E E T K A R F E P L C K A V K D I L G D K V E K V V V S D R I V D S P C V L V T G E Y G W S A N M E R I M K A 593E K K R K E E L A S Q F E P L C R L M K D I L G D K V E K V M V S H R V V D S P C V L V T G E Y G W S A N M E R I M K A 589E K K R K E E L A S Q F E P L C R L M K D I L G D K V E K V T V S H R V V D S P C V L V T G E Y G W S A N M E R I M K A 589E K K A W E E L K A Q T E P L C K V M K D I L G D K V E K V A V S D R L V D S P C I L V T G E Y G W S A N M E R I M K A 588

Q A L R D N S M S G Y M A S K K T L E I N P D N A I M Q E L R K R A D A D K S D K T V K D L V L L L F E T A L L T S G F 648Q A L R D N S M S G Y M A S K K T M E I N P D N A I M Q E L R K R A D A D K S D K T V K D L V L L L F E T S M L C S G F 648Q A L R D S S M S S Y M T S K K T L E I N P E N A I V S E L K K R A D V D K S D K T V K D L V L L L F E T A L L T S G F 650Q A L R D S S M A G Y M S S K K T M E I N P E N P I M E E L R K R A D A D K N D K S V K D L V L L L F E T A L L T S G F 647Q A L R D S S M A G Y M S S K K T M E I N P E N P I M E E L R K R T E V D K N D K S V K D L V L L L F E T S L L T S G F 647Q A L R D N S M A G Y M S S K K T M E I N P E N P I M E E L R K R A D A D K N D K S V K D L V L L L F E T A L L T S G F 647Q A L R D T S M A G Y M S S K K T M E I N P E N A I M E E L R K R A D A D K N D K S V K D L V L L L F E T A L L T S G F 647Q A L R D S S M A G Y M S S K K T M E I N P E N S I M D E L R K R A D A D K N D K S V K D L V L L L F E T A L L T S G F 647Q A L R D S S M G G Y M S S K K T M E I N P E N S I M D E L R K R A E A D K N D K S V K D L V L L L F E T A L L T S G F 647Q A L R D S S M S G Y M S S K K T M E I N P E N A I M E E L R K R A E A D K N D K S V K D L V M L L F E T A L L T S G F 648Q A L R D S S M S G Y M S S K K T M E I N P D N A I M E E L R K R A E A D K N D K S V K D L V M L L F E T A L L T S G F 647Q A L R D S S M S G Y M S S K K T M E I N P E N A I M E E L R K R A E A D K N D K S V K D L V M L L F E T A L L T S G F 648Q A L R D T S M G G Y M S S K K T M E I N P E N A I M E E L R K R A D A D K N D K S V K D L V M L L F E T S L L T S G F 649Q A L R D T S M G G Y M S S K K T M E I N P E N A I M E E L R K R A D A D K N D K S V K D L V M L L F E T S L L T S G F 649Q A L R D T S M G G Y M S S K K T M E I N P E N A I M E E L R K R A D A D K N D K S V K D L V M L L F E T S L L T S G F 649Q A L R D S S M S S Y M S S K K T M E I N P D N Q I M E E L R K R A E V D K N D K S V K D L V L L L F E T A M L T S G F 653Q A L R D N S M A A Y M T S K K T L E I N P E N A I M N E L K K R S D A D K S D K T V K D L V L L L F E T A L L S S G F 649Q A L R D N S M A A Y M T S K K T L E I N P E N P I M S E L K K R S D A D K S D K T V K D L V L L L F E T A L L S S G F 649Q A L R D T S M S A Y M T S R K T L E V N P S N A I I Q E L R K R T E A D K S D K T V R D L T L L L F D T A L L T S G F 648

S L E E P N T F G G R I H R M I K L G L S I D - D D I G L D - D D D H D L P P L - - - E E D V D E G S R M E E V D 700S L D E P N T F G G R I H R M I K L G L S I D - E D L G L D - D D E A D L P P L - - - E E D V D E G S R M E E V D 700S L D E P N T F G T R I H R M I K L G L S I D E D D L P V E - G D D E D L P P L - - - E E D V D E G S R M E E V D 703S L D E P N T F G N R I H R M M K L G L S I D - E D G - - - - - P E A D M P P L - E E A D A D A E G S K M E E V D 697S L D E P N T F G N R I H R M L K L G L N I D - E E A G D - - - V D V D M P P L - E E A D A E A E G S K M E E V D 699S L D E P N T F G N R I H R M L K L G L S I D - E D A A E A - D A D A D M P P L E E E A E A D A E G S K M E E V D 702S L D E P N T F G N R I H R M L K L G L S I D - E D C G D - - - A E A D M P P L - E D P E A D A E G S K M E E V D 699S L D E P N T F G N R I H R M L K L G L S I D - D D D A V - - E A D A E M P P L - - E D D A D A E G S K M E E V D 699S L D E P N T F G S R I H R M L K L G L S I D - E D D T V - - E A D A E M P P L - - E D D A D A E G S K M E E V D 699S L D D P N T F G S R I H R M L K L G L S I D - E D - E A P - E A D T D M P P L - - - - E D D A G E S K M E E V D 698S L D D P N T F G G R I H R M L K L G L S I D - E D - E A P - E A D T D M P P L - - - - E D D A G E S K M E E V D 697S L D D P N T F G S R I H R M L K L G L S I D - E D - E T A - E A E T D M P P L - - - - E E D A G E S K M E E V D 698S L D D P N T F G T R I H R M L K L G L S I D - E D E E A A - E A D T D M P P L - - - - E E D A G E S K M E E V D 700S L D D P N T F G T R I H R M L K L G L S I D - E D E E A A - E A D T D M P P L - - - - E E D A G E S K M E E V D 700S L D D P N T F G T R I H R M L K L G L S I D - E D D E P A - E A D T D M P P L - - - - E E D A G E S K M E E V D 700S L E E P N T F G N R I H R M L K L G L S I D - D D V T - - - E A D A D M P P L - - E A D A E E E G S K M E E V D 704S L D E P N T F A S R I H R M I K L G L S I D E E V E E G L G A A D D D L P P L - E E D A A A G E G S R M E E V D 705S L D E P N T F A S R I H R M I K L G L S I D E D V E E V L - - Q D D D L P P L - E E D A G A G E G S R M E E V D 703S L D E P N T F A G R I H R M I K L G L S L D E V E G E E E - L A D K D M P P L - E E A E G A - V G S S M E E V D 702









Chlamydomonas reinhardtii









Physcomitrella patens





























lowast lowast lowast lowast lowast

(b)

Figure 2 Multiple sequence alignment of the CvHSP90 with other registered counterparts from Haematococcus pluvialis (JN627245)Chlamydomonas reinhardtii (XP 001695264) Volvox carteri f nagariensis (XP 002947115) Micromonas sp RCC299 (XP 002499727)Pennisetum glaucum (ADP89125) Hordeum vulgare (BAJ86355) Triticum aestivum (ADF31758) Triticum urartu (ADF31773) Vitis vinifera(CAN62488) Thellungiella halophila (BAJ33984) Micromonas pusilla CCMP1545 (XP 003058104) Physcomitrella patens (XP 001777414)Sorghum bicolor (XP 002444804) Arabidopsis thaliana (AAN31859) Vitis pseudoreticulata (ABW96308) Nicotiana benthamiana(AAR12194) Glycine max (ADC45395) and Zea mays (NP 001170475) The black shaded regions represent identical amino acids amongthe different species while the gray shaded regions represent conservative replacements The HSP90 signature motifs and cytosolic HSP90-specific motif were indicated with asterisks

(permil) for 2 h showed no significant difference from that at 25in salinity However the expression level at salinity over 25increased significantly (119875 lt 005) The expression levels at40 or 45 salinity were almost 4-fold of that at 25 salinity for2 h (119875 lt 001) The results show that C vulgaris respondedstrongly to high salinity stress

4 Discussion

HSP90 as an important member of HSPs centrally functionsin various biological processes in the presence and absenceof stresses including biogenesis folding transport degrada-tion and prevention ofmisfolding and aggregation of cellular


Table 2 Sequences used for multiple alignment and phylogenetic analysis

Species Taxonomy Accession SimilarityArabidopsis thaliana Streptophyta Magnoliophyta Eudicotyledons AAN31859 77Chlamydomonas reinhardtii Chlorophyta Chlorophyceae XP 001695264 79Chlorella vulgaris Chlorophyta Trebouxiophyceae JQ655149 100Glycine max Streptophyta Magnoliophyta Eudicotyledons ADC45395 77Haematococcus pluvialis Chlorophyta Chlorophyceae JN627245 79Hordeum vulgare subsp vulgare Streptophyta Magnoliophyta Liliopsida BAJ86355 78Micromonas pusilla CCMP1545 Chlorophyta Mamiellophyceae XP 003058104 85Micromonas sp RCC299 Chlorophyta Mamiellophyceae XP 002499727 85Nicotiana benthamiana Streptophyta Magnoliophyta Eudicotyledons AAR12194 77Pennisetum glaucum Streptophyta Magnoliophyta Liliopsida ADP89125 78Physcomitrella patens subsp patens Streptophyta Bryophyta XP 001777414 77Sorghum bicolor Streptophyta Magnoliophyta Liliopsida XP 002444804 77Thellungiella halophila Streptophyta Magnoliophyta Eudicotyledons BAJ33984 78Triticum aestivum Streptophyta Magnoliophyta Liliopsida ADF31758 78Triticum urartu Streptophyta Magnoliophyta Liliopsida ADF31773 78Vitis pseudoreticulata Streptophyta Magnoliophyta Eudicotyledons ABW96308 77Vitis vinifera Streptophyta Magnoliophyta Eudicotyledons CAN62488 78Volvox carteri f nagariensis Chlorophyta Chlorophyceae XP 002947115 79Zea mays Streptophyta Magnoliophyta Liliopsida NP 001170475 76

Nicotiana benthamianaGlycine maxVitis pseudoreticulataVitis viniferaThellungiella halophilaArabidopsis thalianaPennisetum glaucumHordeum vulgareTriticum aestivumTriticum urartuZea maysSorghum bicolorPhyscomitrella patensChlamydomonas reinhardtiiVolvox carteriHaematococcus pluvialisChlorella vulgarisMicromonas pusilla CCMP1545Micromonas sp RCC299

Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100

Figure 3 A phylogenetic tree constructed with the neighbor-joining method The common names and the GenBank accession numberswere the same as those in Figure 2 Numbers at each branch indicate the percentage of times a node was supported in 1000 bootstrappseudoreplication by neighbor joining

proteins and signal transduction [2 9 35] In macroalgae theexpression of the HSP genes has been investigated in severalgenus as they usually live in intertidal zone which character-ized by regular and extreme changes in abiotic conditionsbased on tidal influence [14] However the studies of HSP90in algae are relatively limited [21 27] Chlorella vulgarisas an important economical species is also an importantmodel species in studies on stress responses [30] Therefore

the role of Chlorella vulgaris HSP90 (designated CvHSP90)in response to adverse environment was investigated in thiswork

In photosynthetic eukaryotes Hsp90 family proteinsare divided into five types with localization in differentcellular compartments including nucleoplasm chloroplastsmitochondria ER and cytoplasm [6] Sequence analysis ofthe deduced AA sequence showed that CvHSP90 contains


0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)

Figure 4 CvHSP90 mRNA expression levels under different heatshock temperatures (5∘C 10∘C 15∘C 20∘C 25∘C 30∘C 35∘C 40∘Cand 45∘C) for 1 h were analyzed by real-time quantitative reversetranscriptase-polymerase chain reactionThe 120573-actin gene was usedas an internal control to calibrate the cDNA template for all thesamples Vertical bars represented the mean plusmn SE (119873 = 5)

005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

Figure 5 CvHSP90mRNA expression levels in different heat shocktimes (0 h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9 h 10 h 11 h and 12 h)at 35∘C were analyzed by real-time quantitative RT-PCR CvHSP90mRNA expression was normalized to the control group and 120573-actingene was used as internal control to calibrate the cDNA templatefor all the samples Each bar represents the mean value from fivedeterminations with standard error Significant differences acrosscontrol were indicated with an asterisk at 119875 lt 005 and two asterisksat 119875 lt 001

the five typical signature motifs of the HSPs [10 11 36]Moreover the MEEVD motif which is cytoplasmic Hsp90-specific is identified at the C-terminus The MEEVD motifparticipates in the formation of a Hsp90HopHsp70 proteincomplex concerning the assembly of steroid hormone recep-tors by associating with cytoplasmic Hsp70 through a Hsp-organizing protein (Hop) In addition multiple alignmentand phylogenetic analysis showed high similarities amongthe deduced amino acid sequence of CvHSP90 and otherknownHSP90s (more than 75 similarity in all thematches)especially with those fromMamiellophyceaeMicromonas spRCC299 and Micromonas pusilla CCMP1545 (85 similarityin each match) In terms of sequence alignment structurecomparison and phylogenetic analysis CvHSP90 was con-firmed to be a cytoplasmic Hsp90

0

1

2

3

4

5

6

7

0 5 10 15 20 25 30 35 40 45Salt concentration ()

lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

Figure 6 CvHSP90 mRNA expression levels relative to 120573-actinmRNA levels under stress of different salt concentrations analyzedby real-time quantitative RT-PCR The 120573-actin gene was used as aninternal control to calibrate the cDNA template for all the samplesVertical bars represented the mean plusmn SE (119873 = 5) Significantdifferences across control were indicated with an asterisk at119875 lt 005and two asterisks at 119875 lt 001

It is a main character for almost all organisms studiedthat the maximum expression of HSPs is 10ndash15∘C aboveoptimum growth temperature [18] Moreover it has beenproved that the temperature treatment in the range 10ndash15∘Cbelow optimum growth temperature may also lead to themaximum expression of HSP90 [27] Given that the normalgrowing temperature range of C vulgaris is 20ndash25∘C inwhich the expression level of HSP90s is always low it can beexpected that the maximum CvHSP90 expression would bein the ranges of 35ndash40∘C and 5ndash10∘C which was consistentwith our results In macroalgae short time treatment intemperature higher than normal growth temperature caninduce the expression of HSPs which is upregulated byincreased thermal stresses and then decreased progressivelyafter the expression profile reached themaximum [37] In thiswork we observed that 45∘C treatment which is 15∘C overthe optimum temperature led to the decrease of CvHSP90expression which may be caused by immediate deactivationor strong inhibition of enzyme-related mRNA synthesisand expression in C vulgaris [19] Furthermore the time-dependent pattern of CvHSP90 expression in a heat shockwas observed In heat shock at 35∘C the expression increasedprogressively in 1 h and reached the maximum in 7 h andthen declined gradually Variance analysis indicated that theCvHSP90 gene expression in 6 or 7 hours after challengewas significantly higher (119875 lt 001) than those in other timepoints These results suggested that the CvHSP90 expressionis related to the response to thermal stress andheat shock timeand may play an important role in the mechanism againstthe adverse environmental stresses In natural environmentC vulgaris usually grows in salinity 20ndash30permil Its utilizationin wastewater treatment suggests that it is highly resistantto a range of salinity In this work the result showed thatthe mRNA expression levels of CvHSP90 varied with salinityand suggested its function in resistance to salinity stress Inaddition the CvHSP90 expression at different times under afixed level of salinity stress needs to be further studied


5 Conclusions

TheCvHSP90 gene can be expressed in response to challengesin thermal stress heat shock time and salinity In a stressfulenvironment changes in HSP90s expression level are moresensitive than those of growth rate death rate and reproduc-tive rate for monitoring environmental stresses ThereforeCvHSP90 can be used as a potential biomarker in practice tomonitor environment changes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the National Key TechnologyRampD Program of China (Grant no 2013BAB01B01)

References

[1] F Ritossa ldquoA new puffing pattern induced by temperature shockand DNP in drosophilardquo Experientia vol 18 no 12 pp 571ndash5731962

[2] C Prassinos K Haralampidis D Milioni D SamakovliK Krambis and P Hatzopoulos ldquoComplexity of Hsp90 inorganelle targetingrdquo Plant Molecular Biology vol 67 no 4 pp323ndash334 2008

[3] J G Soslashrensen T N Kristensen and V Loeschcke ldquoTheevolutionary and ecological role of heat shock proteinsrdquo EcologyLetters vol 6 no 11 pp 1025ndash1037 2003

[4] P Csermely T Schnaider C Soti Z Prohaszka and GNardai ldquoThe 90-kDa molecular chaperone family structurefunction and clinical applications A comprehensive reviewrdquoPharmacology andTherapeutics vol 79 no 2 pp 129ndash168 1998

[5] F U Hartl A Bracher andM Hayer ldquoMolecular chaperones inprotein folding and proteostasisrdquo Nature vol 475 no 7356 pp324ndash332 2011

[6] H Hao Y Naomoto X Bao et al ldquoHSP90 and its inhibitorsrdquoOncology Reports vol 23 no 6 pp 1483ndash1492 2010

[7] G Schatz and B Dobberstein ldquoCommon principles of proteintranslocation across membranesrdquo Science vol 271 no 5255 pp1519ndash1526 1996

[8] S L Rutherford and S Lindquist ldquoHsp90 as a capacitor formorphological evolutionrdquo Nature vol 396 no 6709 pp 336ndash342 1998

[9] J C Young IMoarefi and FUlrichHartl ldquoHsp90 a specializedbut essential protein-folding toolrdquo Journal of Cell Biology vol154 no 2 pp 267ndash273 2001

[10] DMilioni and PHatzopoulos ldquoGenomic organization of hsp90gene family in Arabidopsisrdquo PlantMolecular Biology vol 35 no6 pp 955ndash961 1997

[11] D Cao J E Froehlich H Zhang and C L Cheng ldquoThechlorate-resistant and photomorphogenesis-defective mutantcr88 encodes a chloroplast-targeted HSP90rdquo Plant Journal vol33 no 1 pp 107ndash118 2003

[12] D Liu X Zhang Y Cheng T Takano and S Liu ldquorHsp90 geneis in response to several environmental stresses in rice (Oryzasativa L)rdquo Plant Physiology and Biochemistry vol 44 no 5-6pp 380ndash386 2006

[13] X Xu H Song Z Zhou N Shi Q Ying and H WangldquoFunctional characterization of AtHsp903 in Saccharomycescerevisiae and Arabidopsis thaliana under heat stressrdquo Biotech-nology Letters vol 32 no 7 pp 979ndash987 2010

[14] I R Davison and G A Pearson ldquoStress tolerance in intertidalseaweedsrdquo Journal of Phycology vol 32 no 2 pp 197ndash211 1996

[15] J Collen I G Marsollier J J Leger and C Boyen ldquoResponseof the transcriptome of the intertidal red seaweed Chondruscrispus to controlled and natural stressesrdquo New Phytologist vol176 no 1 pp 45ndash55 2007

[16] G A Pearson G Hoarau A Lago-Leston et al ldquoAn expressedsequence tag analysis of the intertidal brown seaweeds Fucusserratus (L) and F vesiculosus (L) (Heterokontophyta Phaeo-phyceae) in response to abiotic stressorsrdquoMarine Biotechnologyvol 12 no 2 pp 195ndash213 2010

[17] W Fu J Yao X Wang F Liu G Fu and D Duan ldquoMolec-ular cloning and expression analysis of a cytosolic Hsp70gene from Laminaria japonica (Laminariaceae Phaeophyta)rdquoMarine Biotechnology vol 11 no 6 pp 738ndash747 2009

[18] W D Fu Molecular cloning and expression analysis of cytosolicHSP70 genes from four kinds of seaweeds [PhD thesis] 2009

[19] W D Fu L Shuai J Yao B Zheng M Zhong and D DuanldquoMolecular cloning and expression analysis of a cytosolic Hsp70gene fromUlva pertusa (Ulvophyceae Chlorophyta)rdquo Journal ofApplied Phycology vol 23 no 4 pp 681ndash690 2011

[20] H S Park W J Jeong E Kim et al ldquoHeat shock proteingene family of the Porphyra seriata and enhancement ofheat stress tolerance by PsHSP70 in Chlamydomonasrdquo MarineBiotechnology vol 14 no 3 pp 332ndash342 2012

[21] H Tominaga D A Coury H Amano W Miki and MKakinuma ldquoCDNA cloning and expression analysis of two heatshock protein genes Hsp90 and Hsp60 from a sterile Ulvapertusa (Ulvales Chlorophyta)rdquo Fisheries Science vol 78 no 2pp 415ndash429 2012

[22] S Heinrich K Valentin S Frickenhaus et al ldquoTranscriptomicanalysis of acclimation to temperature and light stress inSaccharina latissima (Phaeophyceae)rdquo PLOS ONE vol 7 no 8pp 1ndash13 2012

[23] M Schroda ldquoThe Chlamydomonas genome reveals its secretschaperone genes and the potential roles of their gene productsin the chloroplastrdquo Photosynthesis Research vol 82 no 3 pp221ndash240 2004

[24] F Willmund K V Dorn M Schulz-Raffelt and M SchrodaldquoThe chloroplast DnaJ homolog CDJ1 of Chlamydomonas rein-hardtii is part of amultichaperone complex containingHSP70BCGE1 and HSP90Crdquo Plant Physiology vol 148 no 4 pp 2070ndash2082 2008

[25] E Spijkerman D Barua A Gerloff-Elias J Kern U Gaedkeand S A Heckathorn ldquoStress responses and metal tolerance ofChlamydomonas acidophila in metal-enriched lake water andartificial mediumrdquo Extremophiles vol 11 no 4 pp 551ndash5622007

[26] F Willmund and M Schroda ldquoHeat shock protein 90C isa bona fide Hsp90 that interacts with plastidic HSP70B inChlamydomonas reinhardtiirdquo Plant Physiology vol 138 no 4pp 2310ndash2322 2005

[27] L Zhang Y Fan F Shi S Qin and B Liu ldquoMolecularcloning characterization and expression analysis of a cytosolicHSP90 gene from Haematococcus pluvialisrdquo Journal of AppliedPhycology pp 1ndash12 2012

[28] V Blas-Valdivia R Ortiz-Butron M Pineda-Reynoso AHernandez-Garcia and E Cano-Europa ldquoChlorella vulgaris


administration prevents HgCl2

-caused oxidative stress andcellular damage in the kidneyrdquo Journal of Applied Phycology vol23 no 1 pp 53ndash58 2011

[29] Y F NiuMH ZhangWH Xie et al ldquoA new inducible expres-sion system in a transformed green alga Chlorella vulgarisrdquoGenetics and Molecular Research vol 10 no 4 pp 3427ndash34342011

[30] Y L Wang and X D Xu ldquoConstruction of cosmid libraries ofthe Antarctic and temperate strains of Chlorella vulgarisrdquo ActaHydrobiologica Sinica vol 35 no 6 pp 1063ndash1066 2011

[31] S Kumar K Tamura andMNei ldquoMEGA3 integrated softwarefor molecular evolutionary genetics analysis and sequencealignmentrdquo Briefings in Bioinformatics vol 5 no 2 pp 150ndash1632004

[32] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[33] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-DeltaDelta C(T))MethodrdquoMethods vol 25 no 4 pp 402ndash4082001

[34] Q Gao J Zhao L Song et al ldquoMolecular cloning charac-terization and expression of heat shock protein 90 gene inthe haemocytes of bay scallop Argopecten irradiansrdquo Fish andShellfish Immunology vol 24 no 4 pp 379ndash385 2008

[35] K Richter and J Buchner ldquoHsp90 chaperoning signal trans-ductionrdquo Journal of Cellular Physiology vol 188 no 3 pp 281ndash290 2001

[36] P Krishna and G Gloor ldquoThe Hsp90 family of proteins inArabidopsis thalianardquo Cell Stress and Chaperones vol 6 no 3pp 238ndash246 2001

[37] H E Ireland S J Harding G A BonwickM Jones C J Smithand J H H Williams ldquoEvaluation of heat shock protein 70 as abiomarker of environmental stress in Fucus serratus and Lemnaminorrdquo Biomarkers vol 9 no 2 pp 139ndash155 2004

Submit your manuscripts athttpwwwhindawicom

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

Marine BiologyJournal of



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Biochemistry Research International

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Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 PCR primers used in this study

Primers Sequence (51015840-31015840) Sequence informationP1 GNGTGTTCATCATGGACAAYTGYGA Homology cloning primerP2 TTCATGATGCKYTCCATGTTNGC Homology cloning primerP3 GCCCCTCAACATCTCCCGCG 31015840-RACE primerP4 ACCTGCCCCTCAACATCTCCC 31015840-RACE primeradaptor GGCACGCGTCGACTAGTAC 31015840-RACE primerP5 TTTTGTCCTCCTCGGTCTCG 51015840-RACE primerP6 GCCCTCCTTGGTCACGCTCA 51015840-RACE primerOligo(dG)-adaptor GGCACGCGTCGACTAGTACG10 51015840-RACE primerP7 GGCCAGTTCGGCGTGGGTTT Real time gene primerP8 AAGCGACGGGTTGACGGTGT Real time gene primerP9 AACTACGAGCTGCCAGACGG Real time actin primerP10 GAAGACAAGACGATGCTGAGGG Real time actin primerM13-47 CGCCAGGGTTTTCCAGTCACGAC Sequencing primerRV-M GAGCGGATAACAATTTCACACAGG Sequencing primer

such as Chlamydomonas and Haematococcus [23ndash27] Thesestudies indicate that the algal HSPs centrally function inresisting a wide variety of environmental stressors

Chlorella vulgaris is a unicellular green alga growing infresh water [28] It can be used as live feed in fish aquaculturefor its high content of proteins and fatty acids In additionbecause of its fast growth and low cost C vulgaris becomes apromising candidate bioreactor for large-scale production ofvalue-added proteins [29]C vulgarishas important econom-ical and ecological values but often confronts environmentaladversities including high temperature and high salinityTherefore it is often used as a eukaryotic model in studieson stress responses [30] However the role of HSPs inadverse stress resistance mechanism of C vulgaris is yet tobe performed

To better understand the mechanism of response byC vulgaris to different types of environmental stimulationwe obtained a HSP90 complementary DNA (cDNA) of Cvulgaris by combining homology cloning with rapid amplifi-cation of cDNA ends (RACE) approaches analyzed in bioin-formatics the structural features homologous relationshipand phylogenetic position of CvHSP90 and investigatedthe messenger RNA (mRNA) expression levels of CvHSP90under different stress conditions using real-time quantitativeRT-PCR (qRT-PCR)

2 Materials and Methods

21 Sample Collection and Treatment Chlorella vulgaris waspresented from Institute of Oceanology Chinese Academyof Sciences and then the Chlorella was grown in Erlen-meyer flasks in F2 medium that was filter-sterilized through022120583m filters (Millipore USA) Cultures in liquid mediumor on plate were grown at 20∘C in an artificial climateincubator Cool-white fluorescent tubes were used to provideirradiance at 200120583mol photons mminus2 sminus1 in a long-daylightscheme (12 12 h light dark)

In additionC vulgariswas kept in 40∘C for 1 h for extract-ing total RNA and cloning full-length cDNA of CvHSP90gene In our heat shock temperature treatment C vulgariswas kept in different temperatures (5 10 15 20 25 30 3540 and 45∘C) for 1 h to investigate the thermal effect onthe expression level of CvHSP90 mRNA In heat shock timetreatment C vulgaris was kept at 35∘C for different times(0 h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9 h 10 h 11 h and12 h resp) to investigate the effects of heat shock times onexpression level of CvHSP90 mRNA In salt concentrationchallenge treatments C vulgariswas kept at 20∘C in differentsalt concentrations (0 5 10 15 20 25 30 35 40 and 45in permil) for 2 h to investigate the effects of salt challenges onexpression level of CvHSP90

22 RNA Extraction Total RNA extraction from C vulgariswas performed using the TRIzol reagent (Invitrogen) ThecDNA first-strand was synthesized based on M-MLV RTusage information (Promega) using RQI DNase (Promega)-treated total RNA as template cDNA mix was diluted to1 50 and stored atminus80∘C for subsequent fluorescent real-timePCR

23 CvHSP90 cDNACloning To amplify the partial fragmentof CvHSP90 gene from Chlorella vulgaris two homologouscloning primers P1 and P2 were designed based on theconserved sequence of known HSP90s (Table 1) PCR wasperformed in a 25120583L reaction volume containing 25120583L 10XPCR buffer 2120583L dNTP (25mmol Lminus1) 1 120583L of each primer(10 120583mol Lminus1) 02 120583L (1U) of Taq polymerase (Promega) 1120583Lof cDNA template and 173 120583L of PCR-grade water The PCRtemperature profile was 94∘C for 5min followed by 33 cyclesof 94∘C for 1min 56∘C for 1min 72∘C for 1min and a finalextension step at 72∘C for 10min The interesting fragment(745 bp) was excised and purified as per agarose gel DNAfragment recovery kit (TaKaRa) cloned into pMD-18T vector(TaKaRa) and sequenced (Sangon Shanghai China)





3 Results









































(a)

Figure 1 Continued


1741566180158618616061921626198146620416662101686


2641 2701276128212881 2941300130613121318132413301336134213481354136013661

2581

































AAAAAAAAAAAAAAA

lowast


(b)













lowastlowast












































































(a)

Figure 2 Continued






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results









































(a)

Figure 1 Continued


1741566180158618616061921626198146620416662101686


2641 2701276128212881 2941300130613121318132413301336134213481354136013661

2581

































AAAAAAAAAAAAAAA

lowast


(b)













lowastlowast












































































(a)

Figure 2 Continued






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































(a)

Figure 1 Continued


1741566180158618616061921626198146620416662101686


2641 2701276128212881 2941300130613121318132413301336134213481354136013661

2581

































AAAAAAAAAAAAAAA

lowast


(b)













lowastlowast












































































(a)

Figure 2 Continued






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1741566180158618616061921626198146620416662101686


2641 2701276128212881 2941300130613121318132413301336134213481354136013661

2581

































AAAAAAAAAAAAAAA

lowast


(b)













lowastlowast












































































(a)

Figure 2 Continued






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








lowastlowast












































































(a)

Figure 2 Continued






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































(b)



4 Discussion






Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Streptophyta

Chlorophyta

99

99

91

99

99

86

86

99

100

92

99

100






0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

3

35

5 10 15 20 25 30 35 40 45Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowastlowast

lowastlowast

Temp (∘C)


005

115

225

335

445

5

0 1 2 3 4 5 6 7 8 9 10 11 12Time (h)

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast



0

1

2

3

4

5

6

7


lowastlowast lowast

lowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n le

vel o

f CvH

SP90




5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 Conclusions




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article cloning and expression of a...

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