GENE-39391; No. pages: 7; 4C: Gene xxx (2014) xxx–xxx Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress Hua Wang a, Yan Fang a, Zhongzan Bao a, Xing Jin a, Wenjuan Zhu a, Lipeng Wang a, Teng Liu a, Haipeng Ji a, Haiying Wang a, Shiqing Xu a,b, Yanghu Sima a,b,⁎ b Department Applied Biology, School Biology and Basic Medical Sciences, Medical College Soochow University, China National Engineering Laboratory for Modern Silk, Soochow University, China r t i c l e i n f o Article history: Accepted 6 January 2014 Available online xxxx Keywords: Bombyx mori Small heat shock protein Prokaryotic expression Immunofluorescence b s t r c t Elucidating mechanisms underlying response and resistance to high-temperature stress in Lepidoptera is essential for understanding effect high-temperature on regulation gene expression. tag (CATGAACGTGAAGAGATTCAG) matching predicted gene BGIBMGA005823-TA in SilkDB identified most significant response to high-temperature stress in screen heat-treated digital gene expression library Bombyx mori (B. mori) (Unpublished data). BLAST and RACE showed that gene is located on chromosome 5 and has an open reading frame (ORF) 741 bp. Phylogenetic analysis found that B. mori small heat shock protein 27.4 (BmHSP27.4) is in an evolutionary branch separate from other small heat shock proteins. Expression analysis showed that BmHsp27.4 is highly expressed in brain, eyes and fat bodies in B. mori. Its mRNA level was elevated at high-temperature and this increase was greater in females. The ORF without signal peptide sequence was cloned into vector pET-28a(+), transformed and over-expressed in Escherichia coli Rosetta (DE3). Western blotting and immunofluorescence analysis with polyclonal antibody, confirmed that level protein BmHSP27.4 increased at high-temperature, in accordance with its increased mRNA level. In this study, BmHsp27.4 was identified as novel B. mori gene with an important role in response to high-temperature stress. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Heat shock proteins (Hsps) are members class proteins highly expressed in organisms and cells in response to variety external stresses, including high-temperature, viral infection and starvation. Hsps, which are highly expressed in range tissues under hightemperature stress conditions, have an important role in protecting cells from damage (Cara et al., 2005; Carranco et al., 1997; Landais et al., 2001; Tammariello et al., 1998). Bombyx mori is an economically important insect and serves as valuable model organism. Hsps are divided according to their molecular size, Abbreviations: ACD, α-Crystallin protein; B. mori, Bombyx mori; BCA, Bicinchoninic acid; BmHsp27.4, Bombyx mori small heat shock protein 27.4; cAMP, Cyclic Adenosine monophosphate; cDNA, Complementary deoxyribonucleic acid; cGMP, Guanosine 3′,5′cyclophosphate; DAPI, 4′,6-diamidino-2-phenyindole; ELISA, enzyme linked immunosorbent assay; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GRAVY, grand average hydropathicity; IPTG, isopropyl-β-d-thiogalactopyranoside; mRNA, messenger ribonucleic acid; NCBI, National Center for Biotechnology Information; ORF, open reading frame; PDB, Protein Data Bank; PVDF, polyvinylidene difluoride membrane; qRT-PCR, quantitative real-time RT-PCR; RACE, rapid amplification cDNA ends; sHsp, small heat shock protein; SilkDB, Silkworm Genome Database; WB, Western blotting. ⁎ Corresponding author at: Department Applied Biology, School Biology and Basic Medical Sciences, Medical College Soochow University, Suzhou 215123, China. Fax: +86 0512 65880255. E-mail address: simyh@suda.edu.cn (Y. Sima). structure and function into Hsp60, -70, -90 and -110 and small Hsp (sHsp), low molecular mass protein 15–30 kDa that represents large class functional proteins widespread in prokaryotes and eukaryotes (Kim et al., 1998; Norimine et al., 2004; Sugiyama et al., 2000). Sciandra and Subjeck (1983) suggested that sHsp serves as chaperone in processes such as protein folding, aggregation, membrane transport and decomposition (Boston et al., 1996; Xu et al., 2011). BmHsp19.9, -21.4, -23.7, -25.4, -27.4 and Hsp1 genes have been cloned and confirmed to have important functions in vivo. Their expression can be induced to various extents by exposure to high-temperature (Li et al., 2009; Sakano et al., 2006; Sheng et al., 2010). B. mori was treated with ecdysone and rutin and transcriptional level BmHsp19.9 was changed, indicating sHsp acted as molecular chaperone and underwent an increase in expression levels in response to heat stimulation (Xia et al., 2007). BmHsp23.7 was highly expressed in midgut animals infected by cytoplasmic polyhedrosis viruses. Moreover, different types virus, time infection and varieties B. mori tested led to differences in expression responsive genes (Wu et al., 2011). The BmHsp genes encode B. mori sHsps and have important roles in thermal reactions (Howrelia et al., 2011; Li et al., 2012). In this study, we isolated new gene that encodes 27.4 kDa B. mori sHsp by screening differential gene expression library B. mori at high-temperature and at common temperature. We then cloned gene and performed bioinformatics and expression analysis. 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.021 Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 2 H. Wang et al. / Gene xxx (2014) xxx–xxx 2. Materials and methods 2.1. Bioinformatics web sites and software SilkDB (silkworm.genomics.org.cn/) was used for chromosomal localization gene. EST and gene sequence alignment were done with BLAST on National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov/), and open reading frame (ORF) was searched using ORF Finder at NCBI (www. ncbi.nlm.nih.gov/gorf/gorf.html). The amino acid composition, molecular mass and isoelectric point were predicted using Protparam tool (web.expasy.org/protparam/). The transmembrane domain protein was predicted using DAS (www.sbc.su.se/~miklos/ DAS/); signal peptide was predicted using SignalP 3.0 Server (www.cbs.dtu.dk/services/SignalP/); and protein secondary structure was predicted using an online tool SAS (www.ebi.ac.uk/ thornton-srv/databases/sas/). Post-translational modification sites protein were predicted using Motif-scan (hits.isb-sib.ch/cgi-bin/ motif_scan). Protein modeling and prediction its tertiary structure were done with Swiss-model (swissmodel.expasy.org// SWISS-MODEL.html) and its subcellular distribution was predicted using PSORT Prediction (psort.hgc.jp/form2.html) and TargetP (www. cbs.dtu.dk/services/TargetP/). The amino acid accumulation map was made with Weblogo (weblogo.berkeley.edu/). The EST-based gene expression profile was obtained from EST Profile (www.ncbi. nlm.nih.gov/unigene/). 2.2. Phylogenetic analysis Phylogenetic analysis was performed on multiple alignments heat shock protein amino acid sequences. Sixteen Hsps from other species and nine BmHsps from B. mori were included. The analysis was performed using ClustalX and manual refinement. Phylogenetic trees were constructed using Neighbor-Joining method, provided by MEGA5.1 software, under Poisson correction amino acid substitution model. Boot-strapping was performed 1000 times to obtain support values for each branch. The Pfam program was used to identify motifs found in Hsp proteins (Lu et al., 2012). The sequence logos for conservative analysis α-Crystallin protein (ACD) were obtained by submitting alignment sequences to website http:// weblogo.berkeley.edu/logo.cgi (Crooks et al., 2004). 2.3. Insects and culture conditions B. mori variety 7532 with resistance to high-temperature was used in this study. Larvae were reared on fresh mulberry leaves (Morus sp.) at 26–27 ºC with photoperiod 12 h light/12 h dark. The 5th instar larvae were selected and males and females were reared separately. The experimental larvae were exposed to high-temperature (34 ºC) for 24 h and control group was maintained at 26 ºC. The fat body was dissected and then ground and stored in liquid nitrogen. Table 1 Primers for preparation cloned and prokaryotic expression. Primer name Sequence (5′ → 3′) EST-S EST-A 3′ RACE outer 3′ RACE GSP1 3′ RACE inner 3′ RACE GSP2 5′ RACE outer 5′ RACE GSP1 5′ RACE inner 5′ RACE GSP2 qPCR-S qPCR-A Prokaryotic expression-S Prokaryotic expression-A Actin 3 (A3)-S Actin 3 (A3)-A GTCTTACTAGCCGTTGCG ACAAGGGCGTAGTCCAAT TACCGTCGTTCCACTAGTGATTT GAAGTGCGAGCGATTTACAA CGCGGATCCTCCACTAGTGATTTCACTATAGG AAAGCCAGAAGCCACAACAG CATGGCTACATGCTGACAGCCTA TCATTTGCGATTATTTGCGG CGCGGATCCACAGCCTACTGATGATCAGTCGATG CGACCTTGTGTCTTCTGCTCT ACCGCCACCACCAGAAAGA GTCGCCTCAGCCAAATCCA CG GAATTC CAGAGCAGAAGACACAAGGT EcoRI CG AAGCTT TCACTCGGAATCTGGTT HindIII CTGCGTCTGGACTTGGC CGAGGGAGCTGCTGGAT Quantitative real-time RT-PCR (qRT-PCR) was used to analyze levels mRNA from BmHsp27.4 gene after heat treatment. SYBR Premix Ex Taq (Perfect Real Time; TaKaRa) kit was used, in accordance with manufacturer's instructions. The reaction volume was 20 μL, and cycling conditions were as follows: denaturation for 1 min at 95 ºC and 45 cycles 95 ºC for 5 s, 55 ºC for 10 s and 72 ºC for 10 s. Three replicates were tested for each sample and data were corrected using Sequence Detection program (v.1.3.1). 2.5. Construction and expression prokaryotic expression plasmid pET28a-BmHsp27.4 The prokaryotic expression primers excluding signal peptide sequence (amino acids 1–19) were designed (Table 1) according to complete ORF sequence BmHsp27.4 gene. The upstream and downstream primers contained EcoRI and HindIII restriction sites, respectively. Two endonucleases were used to cleave BmHsp27.4-T-vector and pET28a (+). The target bands were cloned into pET28a (+) via T4 DNA ligase (Fermentas, Canada) and transformed to competent Rosetta (DE3). Verified clones were selected and transferred to 500 mL Fresh LB medium and incubated for another 3 h, then isopropyl-β-Dthiogalactopyranoside (IPTG; Sango, China) was added to final concentration 1 mM and incubated for 8 h at 25 °C. The fusion proteins were recovered and purified by an affinity Ni column specific to His-tailed proteins. The polyclonal antibody was recovered, purified and prepared as previously described (Yang et al., 2012). 2.6. Western blotting (WB) 2.4. Cloning complete cDNA sequence and production gene expression profiles EST sequence amplification primers were used to amplify EST sequences (Table 1). The 5′ and 3′ rapid amplification cDNA ends was done using 5′ and 3′ Full RACE kits (TaKaRa, Japan) with 5′ and 3′ RACE outer and inner primers. Total RNA was isolated from fat body using Trizol® (Life Technologies, Carlsbad, CA) reagent and RNA concentrations were determined by spectrophotometry (Beckman, USA). The PCR products were examined by electrophoresis in 1% (w/v) agarose gel and stained with ethidium bromide. The selected PCR products were purified with Agarose Gel DNA Purification Kit (TaKaRa, Japan), cloned into vector pMD19-T (TaKaRa, Japan) and sequenced by Invitrogen (Carlsbad, CA). Proteins were extracted from fat body at different time points. Protein concentrations were measured using BCA Protein Assay Kit (Beyotime, China) and microplate reader. The extracts were subjected to SDS-PAGE (12% (w/v) polyacrylamide gel) and transferred electrophoretically to polyvinylidene difluoride membrane (PVDF). The membrane was blocked with blocking solution (Beyotime, China), followed by incubation with purified anti-BmHsp27.4 antibody or anti-β-tubulin antibody, washed and then incubated with horseradish peroxidase (HRP)-labeled anti-mouse IgG (Bioworld Technology, USA). Proteins were visualized using EZ-ECL Chemiluminescence Detection Kit for HRP (Biological Industries, Israel). 2.7. Immunofluorescence After treatment for 72 h, fat body was placed onto ice and fixed in 4% (v/v) paraformaldehyde. The samples were dehydrated in an ethanol series, then embedded in paraffin and sections (8–10 μm thick) were cut with microtome. Paraffin sections silk glands were Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 H. Wang et al. / Gene xxx (2014) xxx–xxx 3 dewaxed with xylene, rehydrated in an ethanol series then subjected to an antigen unmasking procedure by heating in sodium citrate buffer (Beyotime, China) for 15 min and cooled. Sections were incubated for 1 h with blocking solution and then incubated overnight at 4 ºC with anti-BmHsp27.4 antibody or anti-β-tubulin antibody or normal rabbit serum as negative control. Sections were washed three times (5 min each time) with PBST (Beyotime, China) then incubated with an appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody (CWBIO, China) for 30 min at 37 ºC. Sections were washed three times (5 min each time) with PBST then stained with 4′,6diamidino-2-phenyindole (DAPI; Beyotime, China). Reference sections were stained with rabbit negative serum as primary antibody. All sections were examined under an OlympusBX51 fluorescence microscope (Olympus, Japan). it being located in secretory pathway was 0.845 and it was located in cytoplasm. Together, these results suggest that it is secretory protein. Analysis secondary structure BmHSP27.4 protein, by SAS, indicated that α helix, β fold and random coil contents were 32.52%, 27.64% and 39.84%, respectively (Fig. 2B). The 3D structure protein was predicted by SWISS-MODEL (Fig. 2C). We found protein homologous with alignment length 175 and corresponding PDB ID: 2ygd, which encodes 24 meric Eye Lens Chaperone α-Crystallin protein (ACD) as an important human molecular chaperone (Braun et al., 2011). The BmHSP27.4 protein and this homolog showed 32.4% identity, with 57.7% coverage. 3. Results Analysis homology sHSP27 with other species showed that BmHsp27.4 belongs to single branch with large genetic distance. Furthermore, its homology with invertebrate sHSP27 differed significantly compared to vertebrate sHSP27 (Fig. 4A). Phylogenetic analysis cloned members B. mori sHsp family showed that B. mori sHsp exhibited significant gradient branch (Fig. 4B), which might be because B. mori sHsp family evolved from highly homologous proteins. sHsp contains conserved ACD at C terminus, which can produce large number oligomers, preventing damage caused by protein aggregation when serving as chaperone (Crack et al., 2002). Our results also indicate that HSP27 different species and B. mori sHsp family both contain ACD composed most conserved amino acids (Figs. 4C and D). However, base stacking diagram (amino acids 73–149) showed ACD HSP27 in different species (Fig. 4C) was more conservative compared to B. mori sHsp family (Fig. 4D), which might be an important reason for functional differences between different sHsp species. 3.1. Gene information 709 bp target EST sequence was obtained by qRT-PCR (Fig. 1A) and cloning strategy, which was based on sequence BGIBMGA005823-TA, is shown in Fig. 2A. 410 bp 3′ RACE product and 106 bp 5′ RACE product were obtained. The gene sequence BmHsp27.4 is accession number KF547930 in GenBank. The full length gene sequence is 940 bp with 741 bp ORF (from base pair 34774) encoding protein containing 246 amino acid residues (Fig. 2B). The BLASTN analysis detected no intron in cloned gene. The gene was located at nscaf2838:1796002-1796742(+ strand) on chromosome 5. 3.2. Bioinformatics analysis novel gene BmHsp27.4 The BmHSP27.4 protein (molecular mass 27.4173 kDa theoretical pI 5.86) contains 246 amino acids. The instability index was 45.07, which is classified as unstable. Motif-scan analysis was used to obtain 13 hits: two N-myristoylation sites; six protein kinase C phosphorylation sites; four casein kinase II phosphorylation sites; and one cAMP and cGMP-dependent protein kinase phosphorylation site (Fig. 3). The grand average hydropathicity (GRAVY) value, as predicted by ProtScale, was −0.409, which indicated that it is hydrophilic protein. Analysis by Dense Alignment Surface showed only one transmembrane structure (6–15 amino acids) BmHSP27.4 protein. Using SignalP and TargetP, it had predicted signal peptide with 20 amino acid residues in N terminus causing them to be secreted from cell. The score 3.3. Homologous alignment and phylogenetic tree 3.4. Gene expression profile BmHsp27.4 and response to high-temperature stress The UniGene ID BmHsp27.4 is Bmo.4295 (www.ncbi.nlm.nih.gov/ UniGene/ESTProfileViewer.cgi?uglist=Bmo.4295), so we obtained corresponding EST profile gene by EST Database. Under normal circumstances, BmHsp27.4 is expressed mainly before silk spinning stage and is highly expressed in pupal and embryo stages, but expression level is low in larval stage (Fig. 1C). Expression levels gene in different tissues in decreasing order are brain, maxilla, testis, eye and fat body (Fig. 1B). Considering most Hsps can be induced by high-temperature and fat body is an important site Fig. 1. Cloning method production and EST expression profiles. (A) Results PCR amplification. (B) Digital expression profiles BmHsp27.4 in different tissues. (C) Expression levels different developmental stages. The corresponding UniGene number for BmHsp27.4 gene was Bmo.4295. Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 4 H. Wang et al. / Gene xxx (2014) xxx–xxx Fig. 2. Cloning strategy and protein structure analysis. (A) Cloning strategy. First, spliced EST sequences were amplified using PCR, and 5′ and 3′ RACE primers on both sides EST sequences were designed. The 5′ and 3′ RACE products were 106 bp and 410 bp in length, respectively, and were spliced with EST sequence to obtain gene sequence with full length 940 bp. The black frame is ORF. (B) Prediction protein secondary structure. (C) Prediction protein 3D structure based on 2ygd:T. energy metabolism in B. mori, we investigated gene expression in fat body at high-temperature. The results showed that, similar to other sHsp genes, BmHsp27.4 could be induced by high-temperature, and expression levels were increased with treatment time at high- temperature. However, expression level showed downward trend after day 6. The results also showed that, during various periods time, gene expression levels in B. mori males were higher compared to females (Fig. 6A). Fig. 3. Amino acid sequence analysis, Motif-scan was used to analyze motifs and 13 hits were found. Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 H. Wang et al. / Gene xxx (2014) xxx–xxx 5 Fig. 4. Comparison homology comparison and phylogenetic tree. (A) Phylogenetic trees sHSP27 different species. The phylogenetic tree was created by neighbor joining (NJ) method, using Mega4.0. The protein sequences BmHSP27.4 homologs were retrieved from following species (GenBank ID), Canis lupus (AAA87172.1), Mus musculus (AAA18335.1), Homo sapiens (AAB51056.1), Bos taurus (NP 001020740.1), Capra hircus (AFK93550.1), Alligator mississippiensis (BAF94137.1), Xenopus laevis (ABF17872.1), Platichthys flesus (CCO03033. 1), Gymnocephalus cernuus (CCO03019.1), Poeciliopsis lucida (AAB46593.1), Larimichthys crocea (ADX98507.1), Thunnus orientalis (BAH59273.1), Ceratitis capitata (ACD76913.1), Drosophila melanogaster (AAA28638.1), Culex quinquefasciatus (XP 001847191.1), Brachionus ibericus (ADR79277.1), (B) Homology analysis cloned sHsp in B. mori. The other B. mori heat shock protein sequences used were: Hsp20.8 (ACM24338.1), Hsp20.4 (AAG30945.2), Hsp19.9 (BAD74195.1), Hsp20.1 (BAD74196.1), Hsp21.4 (BAD74197.1), Hsp23.7 (BAD74198.1), Heat shock protein 1 (ABF51459.1), Hsp25.4 (ACA25336.1), Hsp22.6 (ACM24354.1). (C) Conservative analysis ACD sHSP27 in different species (D) Conservative analysis ACD and sHsp cloned from B. mori. 3.5. Western blot and immunofluorescence analysis protein expression The target gene with length 687 bp after removal signal peptide was cloned and connected to high efficiency expression vector pET28a (+). SDS-PAGE and western blot detections showed that fusion target protein was detected only in supernatants after centrifugation, indicating that fusion protein was soluble (Fig. 5A). ELISA assays showed polyclonal antibodies had titer 1:51200. The western blot assays showed that polyclonal antibodies had high specificity (Figs. 5B and C). The results western blots showed that transcriptional levels were consistent with translational levels, reaching peak at day 5 and then decreasing (Fig. 6B). Similarly, protein levels in male silkworms were higher compared to females. Subsequently, localization BmHSP27.4 proteins in fat body under hightemperature was done on day 5, which showed that proteins were localized outside nuclei (Fig. 6C) and normal is only low lever (figure not shown). 4. Discussion Fig. 5. Construction and expression prokaryotic expression plasmid, pET28aBmHSP27.4. (A) The prokaryotic expression vector with target gene. Lane 1, empty vector; lane 2, vector with target gene. (B) SDS-PAGE proteins after induction. Lane 1, empty vector; lane 2, BmHSP27.4-pET28a (+) whole bacterial liquid after induction; lane 3, supernatant after sonication; and lane 4, precipitate after sonication. (C) Western blot assays proteins after induction using His-tag antibody as secondary antibody for validation. Lanes 1–4 have same material as in (B). Lanes M are molecular mass markers. The synthesis Hsps in insects can be induced by low or hightemperature. The speed acquiring heat resistance is positively correlated with rate Hsp accumulation, and decrease heat resistance is synchronized with Hsp degradation (Boston et al., 1996; Sakano et al., 2006). BmHsp27.4 has some characteristics in common with B. mori sHsps investigated earlier. First, gene sequence contains only one exon and has no intron, which is suitable for rapid expression large quantities sHsps and prevention impact severe thermal shock on pre-mRNA splicing (Sheng et al., 2010). Second, protein sequence contains conserved 77 amino acid α-Crystallin domain (MacRae, 2000), which can prevent undesired interactions between proteins and help refolding denatured protein (Haslbeck, 2006). In addition, Hsps have thermal reactivity; some proteins also have Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 6 H. Wang et al. / Gene xxx (2014) xxx–xxx Fig. 6. Expression gene and protein in response to high-temperature stress. (A) Expression levels BmHsp27.4 gene in fat body in high-temperature environment. After feeding for 24 h, B. mori 5th instar larvae in experimental group were transferred to 34 ºC (control group 26 ºC) and sampled once every 24 h. B. mori actin A3 was used as internal reference gene. The BmHsp27.4/A3 ratio represented relative gene expression level BmHsp27.4. (B) BmHSP27.4 protein expression in high-temperature environment. (C) Localization BmHSP27.4 protein in fat body under high-temperature stress. Column 1, genomic DNA labeled with DAPI; column 2, BmHSP27.4 (green fluorescence) stained by anti-BmHSP27.4 antibody and FITC; column; 3, merged images for FITC and DAPI. Diagrams 1′, 2′ and 3′ show amplified sections (yellow frames) 1, 2 and 3. Yellow arrowheads indicate genomic DNA. The scale bars represent 50 μm. The expression data were downloaded from EST database and calculation was: TPM (transcript per million) expression values (pool) = Gene EST/ Total EST in pool. (For interpretation references to color in this figure legend, reader is referred to web version this article.) important roles in other adverse environments (Sakano et al., 2006). BmHSP27.4 constitutes an independent and relatively primitive branch in phylogenetic tree sHsp B. mori or other species. The prokaryotic expression BmHSP27.4 protein was found to be highly expressed only in E. coli Rosetta (DE3) strain, but not in normal BL21 strain (DE3). Rare codon analysis found that translation initiation site contained leucine residue (CTA) and could not be expressed in BL21 (DE3). However, it had good expression in Rosetta (DE3) and could be expressed only in viral supernatant, indicating good solubility. sHsps with Cys are prone to form interchain disulfide bonds that affect correct folding, leading to aggregation (Fu et al., 2003). Therefore, these proteins often exist in form inclusion bodies. The BmHSP27.4 protein sequence has only one Cys and thus cannot form disulfide bond. Both PCR and western blot assays showed that BmHsp27.4 gene transcription and expression levels at high-temperature were higher in males compared to females. Sorensen et al. (2007) found significant difference between male and female fruitflies in adaptability Hsps to high-temperature in both initial and at late stages. It is known that high-temperature resistance is significantly higher in male compared to female silkworms (Traut et al., 2007). The subcellular localization BmHsp25.4 under normal circumstances is mainly in cytoplasm and starts to appear in membrane after heat shock (39 ºC) for 3 h, suggesting that transfer BmHsp25.4 to cell membrane could maintain its mobility and integrity (Sheng et al., 2010). The subcellular localization BmHSP27.4 protein indicates that it is distributed mainly in cytoplasm at high-temperature. However, question whether BmHSP27.4 has same function as BmHsp25.4 remains to be answered. Conflict interest There is no conflict interest. Acknowledgments The authors gratefully acknowledge financial support from earmarked fund for China Agriculture Research System (CARS; grant no. CARS-22-ZJ0104), Priority Academic Program Development Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021 H. Wang et al. / Gene xxx (2014) xxx–xxx Jiangsu Higher Education Institutions, and Research Innovative Projects for average college graduate students 2011 in Jiangsu Province, China. Database: PDB ID:2ygd (http://www.rcsb.org/pdb/explore.do?structureId= 2ygd) References Boston, R.S., Viitanen, P.V., Vierling, E., 1996. Molecular chaperones and protein folding in plants. Plant Mol. Biol. 32, 191–222. Braun, N., et al., 2011. Multiple molecular architectures eye lens chaperone alphaBcrystallin elucidated by triple hybrid approach. Proc. Natl. Acad. Sci. U. S. A. 108, 20491–20496. Cara, J.B., Aluru, N., Moyano, F.J., Vijayan, M.M., 2005. Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 142, 426–431. Carranco, R., Almoguera, C., Jordano, J., 1997. plant small heat shock protein gene expressed during zygotic embryogenesis but noninducible by heat stress. J. Biol. Chem. 272, 27470–27475. Crack, J.A., Mansour, M., Sun, Y., MacRae, T.H., 2002. Functional analysis small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance. Eur. J. Biochem. 269, 933–942. Crooks, G.E., Hon, G., Chandonia, J.M., Brenner, S.E., 2004. WebLogo: sequence logo generator. Genome Res. 14, 1188–1190. Fu, X., Li, W., Mao, Q., Chang, Z., 2003. Disulfide bonds convert small heat shock protein Hsp16.3 from chaperone to non-chaperone: implications for evolution cysteine in molecular chaperones. Biochem. Biophys. Res. Commun. 308, 627–635. Haslbeck, M., 2006. Recombinant expression and in vitro refolding yeast small heat shock protein Hsp42. Int. J. Biol. Macromol. 38, 107–114. Howrelia, J.H., Patnaik, B.B., Selvanayagam, M., Rajakumar, S., 2011. Impact temperature on heat shock protein expression Bombyx mori cross-breed and effect on commercial traits. J. Environ. Biol. 32, 99–103. Kim, K.K., Kim, R., Kim, S.H., 1998. Crystal structure small heat-shock protein. Nature 394, 595–599. Landais, I., et al., 2001. Characterization cDNA encoding 90 kDa heat-shock protein in Lepidoptera Bombyx mori and Spodoptera frugiperda. Gene 271, 223–231. 7 Li, Z.W., Li, X., Yu, Q.Y., Xiang, Z.H., Kishino, H., Zhang, Z., 2009. The small heat shock protein (sHSP) genes in silkworm, Bombyx mori, and comparative analysis with other insect sHSP genes. BMC Evol. Biol. 9, 215. Li, J., Moghaddam, S.H., Du, X., Zhong, B.X., Chen, Y.Y., 2012. Comparative analysis on expression inducible HSPs in silkworm, Bombyx mori. Mol. Biol. Rep. 39, 3915–3923. Lu, W., et al., 2012. Identification and characterization fructose 1,6-bisphosphate aldolase genes in Arabidopsis reveal gene family with diverse responses to abiotic stresses. Gene 503, 65–74. MacRae, T.H., 2000. Structure and function small heat shock/alpha-crystallin proteins: established concepts and emerging ideas. Cell. Mol. Life Sci. 57, 899–913. Norimine, J., Mosqueda, J., Palmer, G.H., Lewin, H.A., Brown, W.C., 2004. Conservation Babesia bovis small heat shock protein (Hsp20) among strains and definition T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes. Infect. Immun. 72, 1096–1106. Sakano, D., et al., 2006. Genes encoding small heat shock proteins silkworm, Bombyx mori. Biosci. Biotechnol. Biochem. 70, 2443–2450. Sciandra, J.J., Subjeck, J.R., 1983. The effects glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells. J. Biol. Chem. 258, 12091–12093. Sheng, Q., Xia, J., Nie, Z., Zhang, Y., 2010. Cloning, expression, and cell localization novel small heat shock protein gene: BmHSP25.4. Appl. Biochem. Biotechnol. 162, 1297–1305. Sorensen, J.G., Kristensen, T.N., Kristensen, K.V., Loeschcke, V., 2007. Sex specific effects heat induced hormesis in Hsf-deficient Drosophila melanogaster. Exp. Gerontol. 42, 1123–1129. Sugiyama, Y., et al., 2000. Muscle develops specific form small heat shock protein complex composed MKBP/HSPB2 and HSPB3 during myogenic differentiation. J. Biol. Chem. 275, 1095–1104. Tammariello, S.P., Denlinger, D.L., 1998. Cloning and sequencing proliferating cell nuclear antigen (PCNA) from flesh fly, Sarcophaga crassipalpis, and its expression in response to cold shock and heat shock. Gene 215, 425–429. Traut, W., Sahara, K., Marec, F., 2007. Sex chromosomes and sex determination in Lepidoptera. Sex.Dev. 1, 332–346. Wu, P., et al., 2011. Microarray analysis gene expression profile in midgut silkworm infected with cytoplasmic polyhedrosis virus. Mol. Biol. Rep. 38, 333–341. Xia, Q., et al., 2007. Microarray-based gene expression profiles in multiple tissues domesticated silkworm, Bombyx mori. Genome Biol. 8, R162. Xu, Q., Zou, Q., Zheng, H., Zhang, F., Tang, B., Wang, S., 2011. Three heat shock proteins from Spodoptera exigua: gene cloning, characterization and comparative stress response during heat and cold shocks. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 159, 92–102. Yang, Y., Wang, B., Yang, D., Lu, M., Xu, Y., 2012. Prokaryotic expression woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation polyclonal antibody to wCTLA-4. Protein Expr. Purif. 81, 181–185. Please cite this article as: Wang, H., et al., Identification Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.021