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Screening of hibernation-related genes in the brain of Rhinolophus ferrumequinum during hibernation
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Comparative Biochemistry and Physiology, Part B 149 (2008) 388 – 393 www.elsevier.com/locate/cbpb
Screening of hibernation-related genes in the brain of Rhinolophus ferrumequinum during hibernation Jinping Chen a,b , Lihong Yuan d , Min Sun c , Libiao Zhang a,b , Shuyi Zhang e,⁎ a
South China Institute of Endangered Animals, Guangzhou, 510260, China b Guangdong Entomological Institute, Guangzhou, 510260, China c Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, 101300, China d Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080, China e School of Life Science, East China Normal University, Shanghai 200062, China Received 2 September 2007; received in revised form 29 October 2007; accepted 29 October 2007 Available online 4 November 2007
Abstract The greater horseshoe bat (Rhinolophus ferrumequinum) is a widely distributed small mammal that hibernates annually. A systematic study was initiated to identify differentially expressed genes in hibernating and aroused states of the greater horseshoe bat brain by using suppressed subtractive hybridization technique and dot blot. Forty-one over-expressed ESTs in the hibernating state were found and 17 were known genes reported in NCBI. Among these 17 genes, three were further checked by real time PCR. The bioinformatics analysis suggests that the major overexpressed ESTs may be responsible for the regulation of cell cycle and apoptosis, the growth of neurons, signal transduction and neuroprotection, gene expression regulation, and intracellular trafficking. © 2007 Elsevier Inc. All rights reserved. Keywords: cDNA subtraction; Hibernation; Rhinolophus ferrumequinum
1. Introduction Hibernation, with dramatic changes in physiology, such as body temperature, neural activity and metabolic activity, helps mammals to survive harsh environment (Carey et al., 2003). These change are precisely controlled by internal molecular driven mechanisms, which represent a natural model for studying human-related disease (Vikhliantsev and Podlubnaia, 2004; Depre and Vatner, 2005; Kondo et al., 2006). It has been reported that hibernation occurs in seven different orders of mammals, including primates (Srere et al., 1992; Andrews et al., 1998; Storey, 2003; Dausmann et al., 2004). Although the evolutionary origin of mammal hibernation is still unknown and hibernation is a complex physiological phenomenon, a detailed understanding of hibernation will pave the way for a variety of
⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2007.10.011
hypometabolic strategies that can help to improve human and animal health (Drew et al., 2001). To date, a few genes closely related to hibernation have been identified. PDK4 and PKL may be associated with metabolism switch to stored triacylglycerols as their primary source of fuel during mammal hibernation (Andrews et al., 1998; Buck and Barnes, 2000). Kondo et al. (Kondo et al., 2006) recently reported that HP complex in the brain circannually controls chipmunk (Tamias sibiricus) hibernation, and HP20c may modulate brain functions in order to develop a capacity for tolerating low body temperature (Tb) during hibernation. Although many works have been carried out on animal hibernation (Srere et al., 1992; O'Hara et al., 1999; Epperson and Martin, 2002), the molecular mechanism of this phenomena is still unknown. The brain, as the key component of the central nervous system, plays an important regulatory role during hibernation (Drew et al., 2001). Several brain regions, such as the hippocampus, septum, hypothalamus and suprachiasmatic nucleus,
J. Chen et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 388–393 Table 1 Primers used for real time PCR Genes
Primer sequences(5'–3')
CaMKK2
PF: TGC TGG CGT CTC ACC TC PR: CAC ACC CGC CCG TTC AGC CCND2 PF: GAGATCCCCAAGAACTCT PR: AGTCAGTCTCGTAGGGTG EE489253 PF: CCT TTG ATG TAA TGC CTT PR: TTT CTC TTC CTA CCC CCC Β-actin PF: GACCTCTATGCCAACACAG PR: CATCTGCTGGAAGGTGGAC
Tm (°C)
Products (bp)
54.3
216
51
228
53
164
55
190
may be involved in the central control of hibernation (Pakhotin et al., 1993; Weaver, 1998; O'Hara et al., 1999). The greater horseshoe bat (Rhinolophus ferrumeuimum) is a small mammal which can hibernate annually and has a prolific population in China. In this study, we used this bat species as research target and constructed a suppression subtractive hybridization (SSH) library of hibernating and aroused brain tissues. The purpose of our research is to characterize changes of gene expression in the bat brain from hibernation to aroused states and identify the key molecular regulation mechanism at low Tb, thereby bringing us closer to a full understanding of the molecular mechanisms involved in mammal hibernation. 2. Materials and methods 2.1. Animals, RNA extraction and cDNA synthesis Six hibernating great horseshoe bats (R. ferrumequinum) were collected from caves (39°48′N, 115°42.0′E) in Fangshan (Beijing, China) in November in 2003. Whole brain tissues of three bats keeping continuous torpor (meaning that rectal temperatures were about 8.7–411.8 °C measured by Testo 735 at the time of collection) were sampled and used to represent torpid state (hibernating state). Another three bats that remained aroused during collection were transported to the laboratory and
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fed with worms and water. These animals (mean rectal temperatures at 22–35.6 °C) were sacrificed 48 h postcollection and whole brain tissues were collected and used to represent active state (non-hibernating state). Tissues from euthermic and hibernating animals were rapidly excised, immediately frozen in liquid nitrogen, and then stored at − 80 °C until RNA extraction. Total RNA was isolated from brain tissue of bats from the hibernating and active states using the TRIzol reagent kit (GIBCO/BRL, Grand Island, NY, USA). The A260/280 ratio was found to be in the range of 1.7–1.9, and total RNA was also electrophoresed on a 1% agarose gel to assess quality visually. All total RNAs were treated with 6 U of RNase-free DNase I (Promega) for 1 h at 37 °C to avoid genomic DNA contamination. Two µg of DNaseI-treated total RNA of each sample was converted to cDNA by SuperScript III Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. The 50 µL reaction mixture contained 500 ng of random primer, 1 mM dNTP, 2 mM dithiothreitol, 80 U RNase inhibitor (Promega), 1 × firststrand buffer and 400 U SuperScript III Reverse Transcriptase. The reaction condition was to maintain the mixture at 65 °C for 5 min, incubated on ice for 5 min, then hold at 25 °C for 5 min, 50 °C for 1 h, and finally 70 °C for 15 min. 2.2. Construction of suppression subtractive hybridization library of bat brain Total RNAs of three hibernating bat brains and three aroused bat brains, each of which weighed 2 µg, were mixed separately representing hibernating state and non-hibernating state respectively. Poly(A) RNA were purified by using PolyATtract® mRNA Isolation Kit (Promega). Then the poly(A) RNA of hibernating and aroused state were used as templates, and double-strand cDNA were synthesized by Super SMART™ PCR cDNA Synthesis Kit (Clontech). After digestion by Ras I, the double-strand cDNA of the hibernating and aroused states served as tester and driver, respectively. Tester cDNA was
Fig. 1. Dot blot hybridization. A: hybridization with active probe; B: hybridization with torpor probe.
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aliquotted into two separate parts and ligated with adaptor 1 and 2, respectively, and hybridized by the excessive driver cDNA twice, and then we performed nest PCR amplification. The PCR products were ligated to PGEM-T easy vector (Promega) and constructed into the SSH library of the hibernating bat brain. The construction of SSH library was accomplished by PCRSelect™ cDNA Subtraction Kit (Clontech). One hundred recombinant clones were selected randomly and sequenced to check the quality of the library. 2.3. Screening of differentially expressed genes during hibernation Dot blot was carried out to screen highly expressed genes in the brain tissues of hibernating bats with biotin-labeled cDNA probe (SpotLight™ Random Primer Labeling Kit, Clontech) from the SSH tester and driver. After annealing, the PCR products of each active colony from SSH were dotted onto the nylon membrane. Hybridization was carried out as described by Sambrook et al. (1989) and the positive clones were sequenced. 2.4. Bioinformatic analysis of ESTs
Table 2 The unique ESTs of the brain SSH cDNA library of Rhinolophus ferrumequinum GenBank⁎ Size (bp)
Homolog
E value
EE489245 EE489248 EE489252
355 159 670
2e-48 1e-7 2e-161
EE489259 EE489260 EE489265 EE489267 EE489268 EE489270 EE489272
670 683 666 722 343 402 805
EE489273
799
EE489275 EE489276 EE489279 EE489280 EE489281 EE489285 EW968314
807 309 502 622 885 459 470
Carboxypeptidase D Ataxin-1 Calcium/calmodulin-dependent protein kinase II inhibitor alpha (CaMKIINα) Protein tyrosine phosphatase, receptor(PTPRF) Zinc finger, FYVE domain Netrin-G1 ligand (NGL-1) Cyclin D2(CCND2) Nuclear receptor coactivator 7 CaMKK beta 1 isoform Rap guanine nucleotide exchange factor (GEF) 5, transcript variant6 Similar to heterogeneous nuclear ribonucleoprotein U isoform b Receptor interacting protein kinase 5 (RIPK5) BTB(POZ) domain containing(BTBD3) Netrin-G1 ligand (NGL-1) Excitoxic amino acid transpoter 2(EAAT2) Microtubule associated protein 1B(MAP1B) Selenoprotein 1(SELI) Protein tyrosine phosphatase, receptor type, K, transcript variant 1 (PTPRK) Nuclear ubiquitous casein kinase and cyclindependent kinase substrate, transcript variant 1 (NUCKS1) Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) Eukaryotic translation initiation factor 4 gamma, 3 (EIF4G3) Quaking homolog, KH domain RNA binding isoform HQK-5 Rho-associated, coiled-coil containing protein kinase 1, transcript variant 2 (ROCK1) Methyl-CpG binding protein MBD2 Seizure related 6 homolog (mouse)-like (SEZ6L) Metastasis suppressor protein 1 (Missing in metastasis protein) (metastasis suppressor YGL-1) Plasminogen activator inhibitor 1 RNA-binding protein (PAI1 RNA-binding protein 1) (PAI-RBP1) Bone morphogenetic protein receptor type II, transcript variant 1 (BMPR2) Forkhead box N3 (FOXN3), transcript variant 1 Similar to solute carrier family 38, member 2 Phosphatase 1, regulatory (inhibitor) subunit 9A, transcript variant 2 (PPP1R9A) Similar to serine/threonine kinase Similar to Protein tyrosine phosphatase type IVA protein 2 Similar to zinc finger protein 106 homolog, transcript variant 4 Structural maintenance of chromosomes protein 3 Microtubule-associated protein 2, transcript variant 7 (MAP2) Similar to hypothetical LOC647979, transcript variant 2 Vulgare chalcone isomerase (CHI)
EW968315 1223
Sequences of 149 Expressed Sequence Tags (EST), including 100 clones which were randomly selected and 49 positive clones obtained by dot blot from the SSH, were processed successively. First, sequences were examined by cross_match software (Phil Green and Brent Ewing, http://www.phrap.org/consed/consed. html#howToGet) to screen the vector sequence, then the clean sequences were clustered by BLASTclust (http://biowulf.nih. gov/apps/blast/doc/blastclust.html) and aligned by phrap (http:// phrap.org/phredphrap/phrap.html) to reduce redundant sequences and obtain the unigenes. Next, the unigenes were submitted to NCBI NT databank (http://www.ncbi.nlm.nih.gov) to get information regarding functional annotation.
EW968317 1085 EW968349 1213 EW968325 390 EW968326 1479 EW968328 559 EW968336 1441 EW968347 364
2.5. Real-time quantitative PCR
EW968355 889
The single-strand cDNAs was used as the templates. Primers used for the PCR reaction were specific for each of the randomly selected ESTs from the SSH. The sequences and annealing temperatures of primer pairs are shown in Table 1. The housekeeping β-actin gene of a greater horseshoe bat was used as a native control and sequences of primer pair were shown in Table 1. RT-PCR was performed using PTC-200 (MJ Research) and the fluorescence threshold value was calculated using Opticon2.0 system software. PCR was performed in a two-step method under the following conditions: pre-denaturation at 94 °C for 1 min, then 45 cycles: 94 °C, 10 s, annealing for 20 s, plate read after each cycle, then melting curve. A 20 μL PCR mixture contained 10 μL SYBR Green I Premix Ex Taq (TakaRa), 2.5 μL cDNA template and 0.4 μM of each primer. The 2− ΔΔCT method was used for quantity calculations (Livak and Schmittgen, 2001). All data were expressed as means ± SD and analyzed by oneway ANOVA to determine the statistical significance (SPSS software 10.0). P b 0.05 was taken to represent a statistically significant difference between means.
EW968365 1267 EW968372 1042 EW968375 450 EW968376 1223 EW968379 1224 EW968380 885 EW968381 1059 EW968382 901 EW968384 914 EW968388 1019 EW968390 1262
⁎Clones from EE489245 to EE489285 were obtained by dot blot.
0.0 3e-18 0.0 8e-41 8e-20 1e-22 4e-05 0.0 1e-45 4e-86 0.0 7e-79 3e-49 1e-10 1e-91 2e-15
0.0 1e-158 3e-11 2e-77 4e-14 5e-19 1e-143
6e-39
5e-31 6e-21 1e-114 4e-13 1e-107 1e-121 2e-26 2e-48 3e-59 2e-23 0.0
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3. Results
4. Discussion
3.1. Screening and informatic analysis of the bat brain SSH library
Hibernation is a key survival strategy for many mammals living in climates with harsh winter conditions where limited or no food sources are available. The interspersed phylogenetic distribution of hibernating and non-hibernating species has led to the hypothesis that hibernation phenotype results from the differential expression of existing genes rather than the creation of novel genes (Srere et al., 1992; Andrews et al., 1998; Carey et al., 2003; Storey, 2003; Dausmann et al., 2004). According to this hypothesis, many differentially expressed genes of known function have been identified in different hibernating species at both the mRNA and protein levels and most of them play a role in energy metabolism (Hittel and Storey, 2001; Hittel and Storey, 2002; Carey et al., 2003; Eddy et al., 2005). Because these molecular studies on hibernation only focused on the analysis of one or a few candidate genes at a time, broader unbiased cDNA screening has recently been used in order to globally analyze the expression pattern of genes in different hibernating species (Andrews et al., 1998; Fahlman et al., 2000; Epperson and Martin, 2002; Hittel and Storey, 2002; Brauch et al., 2005; Williams et al., 2005; Yan et al., 2006). In our study, we used the whole brain tissue of greater horseshoe bats in hibernating and active states to construct a SSH library in order to obtain the differentially expressed genes during hibernation and explore the molecular mechanism of hibernation. After the SSH cDNA library of bat brain tissues was successfully constructed, 100 clones were sequenced, and then the biotin-labeled cDNA probes were used to further screened the differentially expressed genes during hibernation. In total, 49 over-expressed clones of a total of 718 positive ESTs in hibernating state were identified by hybridization. After eliminating the redundant clones, 120 unigenes were obtained and 41 unigenes of these unique unigenes were over-expressed during hibernation, including 17 characterized and 24 unknown ESTs. The rate of over-expressed genes is about 5.71% (41/ 718). Yan et al detected the differentially expressed genes in brown adipose tissue (BAT) of hibernating ground squirrels by microarrays, and the percent of over-expressed genes versus totally differential expressed genes was 9.78% (65/665) (Yan et al., 2006), and higher than that found in our study. The difference of the rate of over-expressed genes may be caused by the different tissues used in the different studies. Bio-informatic analysis showed that the known overexpressed genes are mainly involved in five processes: the regulation of cell cycle and apoptosis (Protein tyrosine phosphotase receptor, Cyclin D2, Rap guanine nucleotide exchange factor 5 and Receptor interacting protein kinase 5); the growth of neurons (Netrin-G1 ligand and Microtubule associated protein 1B); signal transduction and neuroprotection (CaMK II inhibitor alpha, CaMKK beta, excitoxic amino acid transpoter 2 and Selenoprotein I); gene expression regulation (Nuclear receptor coactivator 7 and Similar to heterogeneous nuclear ribonucleoprotein U isoform b) and intracellular traffic (carboxypeptidase D) (Diamond et al., 1994; Sicinski et al., 1996; Bito et al., 1997; Chang et al., 1998; Darimont et al., 1998; Gao et al., 2001; Kalinina and Fricker, 2003; Lin et al., 2003; Opal et al., 2003; Howell et al., 2004; Zha et al., 2004;
The SSH cDNA library of bat brain tissues were constructed successfully. If the signal produced was more than three times the background, then a hybridized spot was considered to have a positive cross-reaction. Genes were considered over expressed if there was a two-fold or more difference when comparing the hibernating signal with that of the active one (Fig. 1). In total, 718 positive ESTs were obtained from SSH. One hundred and forty nine of the positive ESTs, including 100 ESTs from SSH, were selected randomly and were sequenced along with 49 highly expressed clones identified by dot blot hybridization. After processed by cross_match, BLASTclust and phrap, 120 consensus sequences were obtained. These unigenes consisted of 38 known unigenes; 17 of these known unigenes were obtained by dot blot (Table 2), Of the 82 uncharacterized unigenes, 24 unknown unigenes were identified by dot blot, by a search of the NCBI NT databank. The GenBank accession numbers of the 120 unigenes were EW968312 to EW968363, EE489241 to EE489257, EE489259 to EE489278 and EE489280 to EE489286. Many of these unigenes had high redundance, with nine clones of cyclin D2 (CCND2) and seven clones of nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate, transcript variant 1 (NUCKS1) being observed. The 17 known over-expressed unigenes identified by dot blot were mainly responsible for regulation of the growth of neurons, signal transduction and neuroprotection, gene expression regulation, and intracellular trafficking. 3.2. Real-time quantitative PCR Three of the 41 over-expressed ESTs (CCND2, CaMKK2 and one unknown EST EE489253) were selected randomly for an analysis of the expression level of these ESTs in hibernation with real-time PCR. The results showed that CCND2 overexpressed 14%, CaMKK2 over-expressed 200% and EE489253 over-expressed 50% respectively during the hibernating state compared with the aroused state, consistent with the result of hybridization, but the differences between the two states are not significant (P = 0.082, 0.483 and 0.185 respectively).(Fig. 2).
Fig. 2. Real time PCR results of CCND2, CaMKK2 and unknown EST (EE489253).
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Sitcheran et al., 2005; Venardos and Kaye, 2007). This result indicates that brain function related to metabolic suppression, the differentiation of neurons, cell signaling and adaptive neuroprotection may be active during hibernation. Three of the 41 over-expressed ESTs selected randomly included CCND2 with high redundance, CaMKK2 and one unknown EST (EE489253). Real-time PCR was performed to quantify the levels of differential expression of these ESTs in hibernating bat brains. The results showed that all the three ESTs were expressed at a higher level, from 14% to 200%, in the hibernating state (Fig. 2). This observation was consistent with the result of the dot blot. However, statistical analysis indicated that the differences between the two states were not significant. This result may be due to a lack of statistical power stemming from the small sample size. In addition, Yuan et al. (2007) found that the four isoforms of CaMKK2 showed differential expression patterns in a hibernating state compared with an active state. Only the isoform 3 of CaMKK2, a non-kinase activity isoform, increased in the hibernating state and was statistically significant, and another three isoforms all decreased in hibernation. In our study, the real time PCR product of CaMKK2 is the common component of the four isoforms of CaMKK2. Thus, the differential expression level of CaMKK2 shown here represents the integration of the four CaMKK2 isoforms; the lack of observed statistical significance may be the result of the down-regulation of other three isoforms. In summary, in the present study, we successfully constructed the SSH cDNA library of bat brain tissues. We obtained forty-one over-expressed ESTs from the hibernation state which may have important functions in the differentiation of neurons, cell signaling and adaptive protection. However, the differentially expressed levels of these ESTs in proteins and their regulatory functions are still unclear and require further investigation. Acknowledgements The work was supported by the Special Foundation for Young Scientists of Guangdong Province Academy of Science (grant: 200605), and The Scientific Research Starting Foundation from Guangdong Entomological Institute. We thank Liang B, Zhang JP, Zhang JS for helping to collect samples. We are very grateful to Jessica Tuchmann for her helpful comments and English editing. References Andrews, M.T., Squire, T.L., Bowen, C.M., Rollins, M.B., 1998. Lowtemperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. Proc. Natl. Acad. Sci. U. S. A. 95, 8392–8397. Bito, H., Deisseroth, K., Tsien, R.W., 1997. Ca2+-dependent regulation in neuronal gene expression. Curr. Opin. Neurobiol. 7, 419–429. Brauch, K.M., Dhruv, N.D., Hanse, E.A., Andrews, M.T., 2005. Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal. Physiol. Genomics 23, 227–234. Buck, C.L., Barnes, B.M., 2000. Effects of ambient temperature on metabolic rate, respiratory quotient, and torpor in an arctic hibernator. Am. J. Physiol., Regul. Integr. Comp. Physiol. 279, R255–R262.
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Comparative Biochemistry and Physiology, Part B 149 (2008) 388 – 393 www.elsevier.com/locate/cbpb
Screening of hibernation-related genes in the brain of Rhinolophus ferrumequinum during hibernation Jinping Chen a,b , Lihong Yuan d , Min Sun c , Libiao Zhang a,b , Shuyi Zhang e,⁎ a
South China Institute of Endangered Animals, Guangzhou, 510260, China b Guangdong Entomological Institute, Guangzhou, 510260, China c Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, 101300, China d Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080, China e School of Life Science, East China Normal University, Shanghai 200062, China Received 2 September 2007; received in revised form 29 October 2007; accepted 29 October 2007 Available online 4 November 2007
Abstract The greater horseshoe bat (Rhinolophus ferrumequinum) is a widely distributed small mammal that hibernates annually. A systematic study was initiated to identify differentially expressed genes in hibernating and aroused states of the greater horseshoe bat brain by using suppressed subtractive hybridization technique and dot blot. Forty-one over-expressed ESTs in the hibernating state were found and 17 were known genes reported in NCBI. Among these 17 genes, three were further checked by real time PCR. The bioinformatics analysis suggests that the major overexpressed ESTs may be responsible for the regulation of cell cycle and apoptosis, the growth of neurons, signal transduction and neuroprotection, gene expression regulation, and intracellular trafficking. © 2007 Elsevier Inc. All rights reserved. Keywords: cDNA subtraction; Hibernation; Rhinolophus ferrumequinum
1. Introduction Hibernation, with dramatic changes in physiology, such as body temperature, neural activity and metabolic activity, helps mammals to survive harsh environment (Carey et al., 2003). These change are precisely controlled by internal molecular driven mechanisms, which represent a natural model for studying human-related disease (Vikhliantsev and Podlubnaia, 2004; Depre and Vatner, 2005; Kondo et al., 2006). It has been reported that hibernation occurs in seven different orders of mammals, including primates (Srere et al., 1992; Andrews et al., 1998; Storey, 2003; Dausmann et al., 2004). Although the evolutionary origin of mammal hibernation is still unknown and hibernation is a complex physiological phenomenon, a detailed understanding of hibernation will pave the way for a variety of
⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2007.10.011
hypometabolic strategies that can help to improve human and animal health (Drew et al., 2001). To date, a few genes closely related to hibernation have been identified. PDK4 and PKL may be associated with metabolism switch to stored triacylglycerols as their primary source of fuel during mammal hibernation (Andrews et al., 1998; Buck and Barnes, 2000). Kondo et al. (Kondo et al., 2006) recently reported that HP complex in the brain circannually controls chipmunk (Tamias sibiricus) hibernation, and HP20c may modulate brain functions in order to develop a capacity for tolerating low body temperature (Tb) during hibernation. Although many works have been carried out on animal hibernation (Srere et al., 1992; O'Hara et al., 1999; Epperson and Martin, 2002), the molecular mechanism of this phenomena is still unknown. The brain, as the key component of the central nervous system, plays an important regulatory role during hibernation (Drew et al., 2001). Several brain regions, such as the hippocampus, septum, hypothalamus and suprachiasmatic nucleus,
J. Chen et al. / Comparative Biochemistry and Physiology, Part B 149 (2008) 388–393 Table 1 Primers used for real time PCR Genes
Primer sequences(5'–3')
CaMKK2
PF: TGC TGG CGT CTC ACC TC PR: CAC ACC CGC CCG TTC AGC CCND2 PF: GAGATCCCCAAGAACTCT PR: AGTCAGTCTCGTAGGGTG EE489253 PF: CCT TTG ATG TAA TGC CTT PR: TTT CTC TTC CTA CCC CCC Β-actin PF: GACCTCTATGCCAACACAG PR: CATCTGCTGGAAGGTGGAC
Tm (°C)
Products (bp)
54.3
216
51
228
53
164
55
190
may be involved in the central control of hibernation (Pakhotin et al., 1993; Weaver, 1998; O'Hara et al., 1999). The greater horseshoe bat (Rhinolophus ferrumeuimum) is a small mammal which can hibernate annually and has a prolific population in China. In this study, we used this bat species as research target and constructed a suppression subtractive hybridization (SSH) library of hibernating and aroused brain tissues. The purpose of our research is to characterize changes of gene expression in the bat brain from hibernation to aroused states and identify the key molecular regulation mechanism at low Tb, thereby bringing us closer to a full understanding of the molecular mechanisms involved in mammal hibernation. 2. Materials and methods 2.1. Animals, RNA extraction and cDNA synthesis Six hibernating great horseshoe bats (R. ferrumequinum) were collected from caves (39°48′N, 115°42.0′E) in Fangshan (Beijing, China) in November in 2003. Whole brain tissues of three bats keeping continuous torpor (meaning that rectal temperatures were about 8.7–411.8 °C measured by Testo 735 at the time of collection) were sampled and used to represent torpid state (hibernating state). Another three bats that remained aroused during collection were transported to the laboratory and
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fed with worms and water. These animals (mean rectal temperatures at 22–35.6 °C) were sacrificed 48 h postcollection and whole brain tissues were collected and used to represent active state (non-hibernating state). Tissues from euthermic and hibernating animals were rapidly excised, immediately frozen in liquid nitrogen, and then stored at − 80 °C until RNA extraction. Total RNA was isolated from brain tissue of bats from the hibernating and active states using the TRIzol reagent kit (GIBCO/BRL, Grand Island, NY, USA). The A260/280 ratio was found to be in the range of 1.7–1.9, and total RNA was also electrophoresed on a 1% agarose gel to assess quality visually. All total RNAs were treated with 6 U of RNase-free DNase I (Promega) for 1 h at 37 °C to avoid genomic DNA contamination. Two µg of DNaseI-treated total RNA of each sample was converted to cDNA by SuperScript III Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. The 50 µL reaction mixture contained 500 ng of random primer, 1 mM dNTP, 2 mM dithiothreitol, 80 U RNase inhibitor (Promega), 1 × firststrand buffer and 400 U SuperScript III Reverse Transcriptase. The reaction condition was to maintain the mixture at 65 °C for 5 min, incubated on ice for 5 min, then hold at 25 °C for 5 min, 50 °C for 1 h, and finally 70 °C for 15 min. 2.2. Construction of suppression subtractive hybridization library of bat brain Total RNAs of three hibernating bat brains and three aroused bat brains, each of which weighed 2 µg, were mixed separately representing hibernating state and non-hibernating state respectively. Poly(A) RNA were purified by using PolyATtract® mRNA Isolation Kit (Promega). Then the poly(A) RNA of hibernating and aroused state were used as templates, and double-strand cDNA were synthesized by Super SMART™ PCR cDNA Synthesis Kit (Clontech). After digestion by Ras I, the double-strand cDNA of the hibernating and aroused states served as tester and driver, respectively. Tester cDNA was
Fig. 1. Dot blot hybridization. A: hybridization with active probe; B: hybridization with torpor probe.
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aliquotted into two separate parts and ligated with adaptor 1 and 2, respectively, and hybridized by the excessive driver cDNA twice, and then we performed nest PCR amplification. The PCR products were ligated to PGEM-T easy vector (Promega) and constructed into the SSH library of the hibernating bat brain. The construction of SSH library was accomplished by PCRSelect™ cDNA Subtraction Kit (Clontech). One hundred recombinant clones were selected randomly and sequenced to check the quality of the library. 2.3. Screening of differentially expressed genes during hibernation Dot blot was carried out to screen highly expressed genes in the brain tissues of hibernating bats with biotin-labeled cDNA probe (SpotLight™ Random Primer Labeling Kit, Clontech) from the SSH tester and driver. After annealing, the PCR products of each active colony from SSH were dotted onto the nylon membrane. Hybridization was carried out as described by Sambrook et al. (1989) and the positive clones were sequenced. 2.4. Bioinformatic analysis of ESTs
Table 2 The unique ESTs of the brain SSH cDNA library of Rhinolophus ferrumequinum GenBank⁎ Size (bp)
Homolog
E value
EE489245 EE489248 EE489252
355 159 670
2e-48 1e-7 2e-161
EE489259 EE489260 EE489265 EE489267 EE489268 EE489270 EE489272
670 683 666 722 343 402 805
EE489273
799
EE489275 EE489276 EE489279 EE489280 EE489281 EE489285 EW968314
807 309 502 622 885 459 470
Carboxypeptidase D Ataxin-1 Calcium/calmodulin-dependent protein kinase II inhibitor alpha (CaMKIINα) Protein tyrosine phosphatase, receptor(PTPRF) Zinc finger, FYVE domain Netrin-G1 ligand (NGL-1) Cyclin D2(CCND2) Nuclear receptor coactivator 7 CaMKK beta 1 isoform Rap guanine nucleotide exchange factor (GEF) 5, transcript variant6 Similar to heterogeneous nuclear ribonucleoprotein U isoform b Receptor interacting protein kinase 5 (RIPK5) BTB(POZ) domain containing(BTBD3) Netrin-G1 ligand (NGL-1) Excitoxic amino acid transpoter 2(EAAT2) Microtubule associated protein 1B(MAP1B) Selenoprotein 1(SELI) Protein tyrosine phosphatase, receptor type, K, transcript variant 1 (PTPRK) Nuclear ubiquitous casein kinase and cyclindependent kinase substrate, transcript variant 1 (NUCKS1) Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) Eukaryotic translation initiation factor 4 gamma, 3 (EIF4G3) Quaking homolog, KH domain RNA binding isoform HQK-5 Rho-associated, coiled-coil containing protein kinase 1, transcript variant 2 (ROCK1) Methyl-CpG binding protein MBD2 Seizure related 6 homolog (mouse)-like (SEZ6L) Metastasis suppressor protein 1 (Missing in metastasis protein) (metastasis suppressor YGL-1) Plasminogen activator inhibitor 1 RNA-binding protein (PAI1 RNA-binding protein 1) (PAI-RBP1) Bone morphogenetic protein receptor type II, transcript variant 1 (BMPR2) Forkhead box N3 (FOXN3), transcript variant 1 Similar to solute carrier family 38, member 2 Phosphatase 1, regulatory (inhibitor) subunit 9A, transcript variant 2 (PPP1R9A) Similar to serine/threonine kinase Similar to Protein tyrosine phosphatase type IVA protein 2 Similar to zinc finger protein 106 homolog, transcript variant 4 Structural maintenance of chromosomes protein 3 Microtubule-associated protein 2, transcript variant 7 (MAP2) Similar to hypothetical LOC647979, transcript variant 2 Vulgare chalcone isomerase (CHI)
EW968315 1223
Sequences of 149 Expressed Sequence Tags (EST), including 100 clones which were randomly selected and 49 positive clones obtained by dot blot from the SSH, were processed successively. First, sequences were examined by cross_match software (Phil Green and Brent Ewing, http://www.phrap.org/consed/consed. html#howToGet) to screen the vector sequence, then the clean sequences were clustered by BLASTclust (http://biowulf.nih. gov/apps/blast/doc/blastclust.html) and aligned by phrap (http:// phrap.org/phredphrap/phrap.html) to reduce redundant sequences and obtain the unigenes. Next, the unigenes were submitted to NCBI NT databank (http://www.ncbi.nlm.nih.gov) to get information regarding functional annotation.
EW968317 1085 EW968349 1213 EW968325 390 EW968326 1479 EW968328 559 EW968336 1441 EW968347 364
2.5. Real-time quantitative PCR
EW968355 889
The single-strand cDNAs was used as the templates. Primers used for the PCR reaction were specific for each of the randomly selected ESTs from the SSH. The sequences and annealing temperatures of primer pairs are shown in Table 1. The housekeeping β-actin gene of a greater horseshoe bat was used as a native control and sequences of primer pair were shown in Table 1. RT-PCR was performed using PTC-200 (MJ Research) and the fluorescence threshold value was calculated using Opticon2.0 system software. PCR was performed in a two-step method under the following conditions: pre-denaturation at 94 °C for 1 min, then 45 cycles: 94 °C, 10 s, annealing for 20 s, plate read after each cycle, then melting curve. A 20 μL PCR mixture contained 10 μL SYBR Green I Premix Ex Taq (TakaRa), 2.5 μL cDNA template and 0.4 μM of each primer. The 2− ΔΔCT method was used for quantity calculations (Livak and Schmittgen, 2001). All data were expressed as means ± SD and analyzed by oneway ANOVA to determine the statistical significance (SPSS software 10.0). P b 0.05 was taken to represent a statistically significant difference between means.
EW968365 1267 EW968372 1042 EW968375 450 EW968376 1223 EW968379 1224 EW968380 885 EW968381 1059 EW968382 901 EW968384 914 EW968388 1019 EW968390 1262
⁎Clones from EE489245 to EE489285 were obtained by dot blot.
0.0 3e-18 0.0 8e-41 8e-20 1e-22 4e-05 0.0 1e-45 4e-86 0.0 7e-79 3e-49 1e-10 1e-91 2e-15
0.0 1e-158 3e-11 2e-77 4e-14 5e-19 1e-143
6e-39
5e-31 6e-21 1e-114 4e-13 1e-107 1e-121 2e-26 2e-48 3e-59 2e-23 0.0
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3. Results
4. Discussion
3.1. Screening and informatic analysis of the bat brain SSH library
Hibernation is a key survival strategy for many mammals living in climates with harsh winter conditions where limited or no food sources are available. The interspersed phylogenetic distribution of hibernating and non-hibernating species has led to the hypothesis that hibernation phenotype results from the differential expression of existing genes rather than the creation of novel genes (Srere et al., 1992; Andrews et al., 1998; Carey et al., 2003; Storey, 2003; Dausmann et al., 2004). According to this hypothesis, many differentially expressed genes of known function have been identified in different hibernating species at both the mRNA and protein levels and most of them play a role in energy metabolism (Hittel and Storey, 2001; Hittel and Storey, 2002; Carey et al., 2003; Eddy et al., 2005). Because these molecular studies on hibernation only focused on the analysis of one or a few candidate genes at a time, broader unbiased cDNA screening has recently been used in order to globally analyze the expression pattern of genes in different hibernating species (Andrews et al., 1998; Fahlman et al., 2000; Epperson and Martin, 2002; Hittel and Storey, 2002; Brauch et al., 2005; Williams et al., 2005; Yan et al., 2006). In our study, we used the whole brain tissue of greater horseshoe bats in hibernating and active states to construct a SSH library in order to obtain the differentially expressed genes during hibernation and explore the molecular mechanism of hibernation. After the SSH cDNA library of bat brain tissues was successfully constructed, 100 clones were sequenced, and then the biotin-labeled cDNA probes were used to further screened the differentially expressed genes during hibernation. In total, 49 over-expressed clones of a total of 718 positive ESTs in hibernating state were identified by hybridization. After eliminating the redundant clones, 120 unigenes were obtained and 41 unigenes of these unique unigenes were over-expressed during hibernation, including 17 characterized and 24 unknown ESTs. The rate of over-expressed genes is about 5.71% (41/ 718). Yan et al detected the differentially expressed genes in brown adipose tissue (BAT) of hibernating ground squirrels by microarrays, and the percent of over-expressed genes versus totally differential expressed genes was 9.78% (65/665) (Yan et al., 2006), and higher than that found in our study. The difference of the rate of over-expressed genes may be caused by the different tissues used in the different studies. Bio-informatic analysis showed that the known overexpressed genes are mainly involved in five processes: the regulation of cell cycle and apoptosis (Protein tyrosine phosphotase receptor, Cyclin D2, Rap guanine nucleotide exchange factor 5 and Receptor interacting protein kinase 5); the growth of neurons (Netrin-G1 ligand and Microtubule associated protein 1B); signal transduction and neuroprotection (CaMK II inhibitor alpha, CaMKK beta, excitoxic amino acid transpoter 2 and Selenoprotein I); gene expression regulation (Nuclear receptor coactivator 7 and Similar to heterogeneous nuclear ribonucleoprotein U isoform b) and intracellular traffic (carboxypeptidase D) (Diamond et al., 1994; Sicinski et al., 1996; Bito et al., 1997; Chang et al., 1998; Darimont et al., 1998; Gao et al., 2001; Kalinina and Fricker, 2003; Lin et al., 2003; Opal et al., 2003; Howell et al., 2004; Zha et al., 2004;
The SSH cDNA library of bat brain tissues were constructed successfully. If the signal produced was more than three times the background, then a hybridized spot was considered to have a positive cross-reaction. Genes were considered over expressed if there was a two-fold or more difference when comparing the hibernating signal with that of the active one (Fig. 1). In total, 718 positive ESTs were obtained from SSH. One hundred and forty nine of the positive ESTs, including 100 ESTs from SSH, were selected randomly and were sequenced along with 49 highly expressed clones identified by dot blot hybridization. After processed by cross_match, BLASTclust and phrap, 120 consensus sequences were obtained. These unigenes consisted of 38 known unigenes; 17 of these known unigenes were obtained by dot blot (Table 2), Of the 82 uncharacterized unigenes, 24 unknown unigenes were identified by dot blot, by a search of the NCBI NT databank. The GenBank accession numbers of the 120 unigenes were EW968312 to EW968363, EE489241 to EE489257, EE489259 to EE489278 and EE489280 to EE489286. Many of these unigenes had high redundance, with nine clones of cyclin D2 (CCND2) and seven clones of nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate, transcript variant 1 (NUCKS1) being observed. The 17 known over-expressed unigenes identified by dot blot were mainly responsible for regulation of the growth of neurons, signal transduction and neuroprotection, gene expression regulation, and intracellular trafficking. 3.2. Real-time quantitative PCR Three of the 41 over-expressed ESTs (CCND2, CaMKK2 and one unknown EST EE489253) were selected randomly for an analysis of the expression level of these ESTs in hibernation with real-time PCR. The results showed that CCND2 overexpressed 14%, CaMKK2 over-expressed 200% and EE489253 over-expressed 50% respectively during the hibernating state compared with the aroused state, consistent with the result of hybridization, but the differences between the two states are not significant (P = 0.082, 0.483 and 0.185 respectively).(Fig. 2).
Fig. 2. Real time PCR results of CCND2, CaMKK2 and unknown EST (EE489253).
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Sitcheran et al., 2005; Venardos and Kaye, 2007). This result indicates that brain function related to metabolic suppression, the differentiation of neurons, cell signaling and adaptive neuroprotection may be active during hibernation. Three of the 41 over-expressed ESTs selected randomly included CCND2 with high redundance, CaMKK2 and one unknown EST (EE489253). Real-time PCR was performed to quantify the levels of differential expression of these ESTs in hibernating bat brains. The results showed that all the three ESTs were expressed at a higher level, from 14% to 200%, in the hibernating state (Fig. 2). This observation was consistent with the result of the dot blot. However, statistical analysis indicated that the differences between the two states were not significant. This result may be due to a lack of statistical power stemming from the small sample size. In addition, Yuan et al. (2007) found that the four isoforms of CaMKK2 showed differential expression patterns in a hibernating state compared with an active state. Only the isoform 3 of CaMKK2, a non-kinase activity isoform, increased in the hibernating state and was statistically significant, and another three isoforms all decreased in hibernation. In our study, the real time PCR product of CaMKK2 is the common component of the four isoforms of CaMKK2. Thus, the differential expression level of CaMKK2 shown here represents the integration of the four CaMKK2 isoforms; the lack of observed statistical significance may be the result of the down-regulation of other three isoforms. In summary, in the present study, we successfully constructed the SSH cDNA library of bat brain tissues. We obtained forty-one over-expressed ESTs from the hibernation state which may have important functions in the differentiation of neurons, cell signaling and adaptive protection. However, the differentially expressed levels of these ESTs in proteins and their regulatory functions are still unclear and require further investigation. Acknowledgements The work was supported by the Special Foundation for Young Scientists of Guangdong Province Academy of Science (grant: 200605), and The Scientific Research Starting Foundation from Guangdong Entomological Institute. We thank Liang B, Zhang JP, Zhang JS for helping to collect samples. We are very grateful to Jessica Tuchmann for her helpful comments and English editing. References Andrews, M.T., Squire, T.L., Bowen, C.M., Rollins, M.B., 1998. Lowtemperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. Proc. Natl. Acad. Sci. U. S. A. 95, 8392–8397. Bito, H., Deisseroth, K., Tsien, R.W., 1997. Ca2+-dependent regulation in neuronal gene expression. Curr. Opin. Neurobiol. 7, 419–429. Brauch, K.M., Dhruv, N.D., Hanse, E.A., Andrews, M.T., 2005. Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal. Physiol. Genomics 23, 227–234. Buck, C.L., Barnes, B.M., 2000. Effects of ambient temperature on metabolic rate, respiratory quotient, and torpor in an arctic hibernator. Am. J. Physiol., Regul. Integr. Comp. Physiol. 279, R255–R262.
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