Deep sequencing of the tilapia (Oreochromis niloticus )liver transcriptome response to dietary protein to starch ratio
Yuanyan Xiong b ,c ,1,Junfeng Huang b ,1,Xiaoxia Li a ,1,Liliu Zhou a ,Fang Dong b ,Hui Ye a ,Lian Gan a ,?
a Animal Science College,South China Agricultural University,Guangzhou,China
b School of Life Science,Sun Yat-Sen University,Guangzhou,China
c
SYSU-CMU Shunde International Joint Research Institute,Shunde,China
a b s t r a c t
a r t i c l e i n f o Article history:
Received 10March 2014
Received in revised form 9June 2014Accepted 12June 2014
Available online 28June 2014Keywords:
Dietary protein Starch
Transcriptome Tilapia
To comprehensively understand the effects of dietary protein to starch ratio on alternations in physiologic status of tilapia,?sh were fed with high protein to starch ratio (HP,33.5%protein and 16.62%starch content)and low protein to starch ratio isoenergetic diets (LP,25.2%protein and 26.82%starch content)for 8weeks.Our results indicated that the ?sh fed with LP diet had a signi ?cantly poor growth performance and feed utilization.Lower dietary protein to starch ratio also resulted in markedly higher plasma cholesterol,plasma triacylglycerol,liver lipid and muscle lipid content,which indicated more fat deposition in ?sh.RNA-seq was employed to evaluate the tilapia hepatic transcriptome response to LP diet.RNA-seq data showed that 71genes were signi ?cantly up-regulated and 26genes were signi ?cantly down-regulated by LP diet.Different expression genes were mapped to 47signaling pathways including glycolysis,gluconeogenesis,biosynthesis of amino acids,lipogenesis and lipolysis etc.The present study gains a comprehensive understanding of the molecular mechanisms under-lying the effects of dietary protein and starch ratio on alternations in physiologic of tilapia.
?2014Elsevier B.V.All rights reserved.
1.Introduction
Dietary proteins are used continuously by ?sh for maintenance,growth and reproduction functions.By feeding representation of over 50%of the operational costs of aquaculture,protein has been the most expensive nutrient source in aquaculture feeds.Reducing dietary protein content is an important way to formulate cost effective and low pollution diets.Carbohydrates are the lowest cost energy source in practical diet ingredients and ef ?ciently used by ?sh.Some re-searchers demonstrated that inexpensive carbohydrate inclusion in the diet can improve growth performance,decrease ammonia excretion and spare some proteins in many ?sh species (Azaza et al.,2013;Mohanta et al.,2007;Peres and Oliva-Teles,2002),which may be help-ful to reduce ?sh feed cost.However,excess dietary carbohydrate has been demonstrated to cause negative effect on ?sh health through met-abolic disturbances (Polakof et al.,2012),?sh physiological alterations were observed,such as prolonged hyperglycemia (Hatlen et al.,2005),triggering hepatic anti-oxidative response (Azaza et al.,2013),high fat deposition in whole body and liver (Hemre et al.,2002),low red blood cells and hemoglobin (Abdel-Tawwab et al.,2010),increased liver
histopathology (Russell et al.,2001)and impaired bone development (Tan et al.,2009).
Tilapia are the most important commercial cichlids,which have been farmed extensively in many tropical and subtropical regions of the word due to their large size,rapid growth,and palatability (EI-Sayed,2006).By 2010yearly global aquaculture production of Oreochromis niloticus had risen to nearly 2.538million tonnes (FAO,2010).Tilapia still continues to increase production.The protein requirement of tilapia is 28%–56%,which varies with ?sh size and age (Abdel-Tawwab et al.,2010).The omnivorous Nile tilapia can utilize about 40–50%carbohydrate content in the diets (Hemre et al.,2002).Some research had demonstrated that tilapia never compromise growth performance when fed with low protein and high carbohydrate diet (Azaza et al.,2013;Li et al.,2013).But a clear molecular response remains scarcely known when tilapia fed with low protein and high carbohydrate diet.Identi ?cation of genes and pathways altered by dietary protein to carbohydrate ratio may contribute towards a better understanding of nutrient metabolism pathways and assessing the effect of nutrition control on ?sh physiology.
For this reason,a global analysis of gene expression in response to dietary protein to starch ratio is essential for understanding the bio-logical mechanisms.The objective of this study was to identify hepatic genes differentially expressed among tilapia fed with different protein to starch ratio isoenergetic diets.
Aquaculture 433(2014)299–306
?Corresponding author.Tel.:+8602085283529.E-mail address:ganlian@https://www.doczj.com/doc/595204064.html, (L.Gan).1
Co-?rst
authors.https://www.doczj.com/doc/595204064.html,/10.1016/j.aquaculture.2014.06.0090044-8486/?2014Elsevier B.V.All rights
reserved.
Contents lists available at ScienceDirect
Aquaculture
j o u rn a l h o m e p a g e :w w w.e l s e v i e r.c o m/l o c a t e /a q u a -o n l i n e
2.Material and methods
2.1.Fish and rearing experiment
The two isoenergetic practical experimental diets,a33.5%protein and16.62%starch diet(HP)and a25.2%protein and26.82%starch diet(LP),were prepared as shown in Table1.The wheat?our and cellulose content in the LP diet were increased to make it isoenergetic. All experimental diets were prepared as previously described by Gan et al.(2013).After drying,the diets were packed into plastic bags and stored at?20°C until use.Juvenile tilapia from our facilities were used and their initial wet weight was15.2±0.06g.After acclimated to the experimental conditions for2weeks with32%protein and4% lipid(wet weight)to satiation,?sh were randomly divided into2 groups in a recirculated aquaculture system,and each group had three tanks containing20tilapia each.The?sh were respectively fed with 7%of body weight thrice a day for8weeks.During the trial period, the diurnal cycle was12-h light/12-h dark.Water quality parameters monitored weekly as follows:temperature,27.1±1.1°C;pH,7.22±0.17,dissolved oxygen,6.08±0.10mg L?1;total ammonia–nitrogen, 0.05±0.01mg L?1,respectively.
2.2.Sampling and analytical methods
At the beginning of the feeding trial,18?sh were randomly sampled from the initial?sh and killed for analyses of whole body composition. After feeding experiment,three?sh from each tank were randomly collected for analysis of whole-body composition and six?sh were anesthetized with tricaine methane sulphonate(MS222)(50mg L?1) for blood collection from tail vein and to obtain weights of individual whole body,viscera,liver and intraperitoneal fat.Blood and tissue samples were obtained from fasted(12h)animals.Serum isolated was stored at?70°C until analyzed.Liver and white muscle from both sides of the?lets without skin were dissected,frozen immediately in liquid nitrogen and then stored at?70°C until used.
Diets and?sh samples(including white muscle and liver)were analyzed in triplicate for proximate composition.Crude protein,crude lipid,moisture,crude ash and gross energy(GE)were determined following standard methods(AOAC,1984).The concentrations of plasma cholesterol(CHO),triacylglycerol(TG),high density lipoprotein (HDL)and low density lipoprotein(LDL)were determined using an automatic blood analyzer(Hitachi7170A,Japan)from a clinical laboratory.
2.3.RNA extraction,cDNA library construction and RNA-seq
Total liver tissue RNA was extracted using TRIzol Reagent(Life Tech-nologies,US)according to the manufacturer's instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100(Agilent Technologies,US).Quali?ed total RNA was puri?ed by RNeasy micro kit(QIAGEN,Germany)and RNase-Free DNase Set (QIAGEN,Germany).Poly(A)mRNA was isolated from quali?ed total RNA using Oligotex mRNA mini kit(QIAGEN,Germany)and then broken into short fragments which were used as templates for synthesis of the?rst-and the second-strand cDNA.Two paired-end libraries were synthesized by using the Genomic Sample Prep kit(Illumina, US)following the manufacturer's instructions.Short fragments were puri?ed with the Qubit?dsDNA HS Assay kit(Invitrogen,US)and connected with different sequencing adapters.Each of the two libraries had an average insert size of400bp and was sequenced by Shanghai Biotechnology Corporation(Shanghai,China)using Illumina HiSeq?2000.
2.4.Mapping reads to the tilapia genome
The Nile tilapia genome was produced by Broad Institute (https://www.doczj.com/doc/595204064.html,/)and downloaded from the Ensem-ble website(version Orenil1.0.72).Clean reads were aligned to the reference genome using Tophat(Trapnell et al.,2009)with default parameters.To reduce the in?uence of low quality bases in the tail and maximize the read utilization,we set up an iteratively mapping step.After each round's mapping,the paired un-mapped reads were extracted and trimmed the?nal10bp,these processed reads were used for the next round's mapping.The process was stopped while the reads shorter than30bp,and all the BAM?les were merged as the?nal mapping output.
2.5.Detecting novel transcripts
A genome-free strategy for transcriptome reconstruction was im-plemented as described by cuf?inks suite(Roberts et al.,2011).This novel assembly was compared to the known transcripts recorded in Ensemble.Those that were not overlapped with the known transcripts were extracted as candidate novel transcripts for further analysis.Four criterions must also be met for novel transcripts:(a)length N200bp, (b)away10kb upstream or downstream from any known transcripts to reduce the possibility that these sequences derived from extended exons of the known one,(c)with two or more exons,and (d)predicted to be‘coding’both by CPC(Kong et al.,2007)and CNCI (Sun et al.,2013)programs.
2.6.Differentially expressed gene analysis
Both known and novel genes were used to detect the differential expression.Read counts for each gene were assigned by HTSeq (http://www-huber.embl.de/users/anders/HTSeq/).Fish fed with
Table1
Experimental formulation and composition(%).
Diet HP LP
Ingredient
Soybean meal a3612
Canola meal a1717
Cotton meal a1111
Rice bran meal a99
Wheat?our a19.1634.77
Mineral mix b0.50.5
Vitamin mix c0.50.5
Soy oil a33
Choline chlorine(50%)a0.30.3
Monocalcium phosphate a22
78%lysine-HCL d00.17
84%MHA-Ca e0.440.24
98%L-Threonine f00.12
Cellulose08.3
Phospholipid a11
VC ascorbic acid0.10.1
Total100100
Proximate analysis(%DM)
Moisture8.02 6.03
Crude protein33.525.2
Crude fat 5.40 5.23
Starch16.6226.82
Gross energy,kJ g?114.414.4
a Zhuhai Shihai Feed Corporation Ltd.,Zhuhai,China.
b Mineral mix(mg kg?1of diet):MgSO
4
.7H2O,4061.5;ZnSO4.7H2O,525.46;FeSO4.7-
H2O,238.83;MnSO4.H2O,49.22;CoCl2.6H2O,0.2;KI,5.23;CuSO4.5H2O,11.82;Na2SeO3,
0.66;KCl,600;NaCl,400(Guangzhou Chengyi Aquatic Technology Ltd.,Guangzhou,
China).
c Vitamin mix(mg kg?1of diet):thiamin,20;ribo?avin,20;vitmin A,2;vitamin E,50;
vitamin D3,0.05;menadione,10;pyridoxine,10;cyanocobalamin,0.02;biotin,1;calcium
pantothenate,50;folic acid,5;niacin,100;inositol,500(Guangzhou Chengyi Aquatic
Technology Ltd.,Guangzhou,China).
d
L-Lysine.HCL contained L-lysine≥78%(CJ Co.,Ltd.,Liaocheng,China).
e MHA.Ca contained DL-HMTBA(2-hydroxy-4-methylthio butanoic acid)≥84%
(Novus International Inc.,Zhibo,China).
f Supplied as L-form(Shanghai Cangda Amino acid Company Ltd.,Shanghai,China).
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HP diet was used as control sample.The genes with more than10reads in both samples were considered to active transcribed and used for following DESeq(Anders and Huber,2010)differentially expressed detection.
2.7.GO annotation and pathway analysis
The gene annotation and GO enrichment analysis of tilapia genes were achieved by Blast2GO(Conesa et al.,2005).WEGO software was used to perform GO functional classi?cation(Ye et al.,2006).
2.8.Quantitative real-time PCR
Quantitative real-time PCR with SYBR Green dye(TaKaRa Biotech-nology,Dalian,China)was performed on an ABI7500Real-Time PCR System according to the manufacturer's instructions.First-strand cDNA was synthesized from1μg of total hepatic RNA of low protein group and high protein group,respectively,as described above and then used as template for real-time PCR with speci?c primers
(Table2).Real-time PCR was performed in a total volume of20μL and the thermal cycler program was95°C for10min,followed by40cycles of95°C for15s,60°C for60s.All reactions were performed in tripli-cate.Each gene expression level was described by relative change fold. Statistical analysis was performed by one-way ANOVA of SPSS13.0 software.
3.Results
3.1.Phenotype performance
Fish readily accepted and totally eat the experimental diets.Fish survival rate was very high during the8weeks feeding trial(Table3). Fish fed with HP diet had signi?cantly better growth performance and feed conversion ratio(P b0.05).Nitrogen retention(NR)of?sh were signi?cantly increased with reducing dietary protein content (P b0.05).Lipid content of liver and muscle,viscerasomatic index (VSI),hepatopancreasomatic index(HSI),intraperitoneal fat ratio (IPF)and condition factor(CF)were signi?cantly increased with reducing of dietary protein to starch ratio(P b0.05,Table4).Plasma cholesterol and triglyceride content were signi?cantly increased with reducing of dietary protein to starch ratio(P b0.05,Table5).
3.2.De novo assembly and gene annotation
3.2.1.Mapping results
The sequencing quality for the two samples were checked using FastQC(https://www.doczj.com/doc/595204064.html,/projects/fastqc/), the average quality is higher than30(Supplementary material1). As an iterative trimming was adopted in out mapping protocol,eight BAM outputs with gradually shortened reads were generated and then merged.So the non-iterative mapping result was equivalent to the?rst round mapping.Summary of mapping was done using SAMtools(Li et al.,2009)for both non-iterative and iterative mapping, which was presented in Table6.
The high quality sequencing data achieved85.49%and85.63% mapping rate for low and high protein samples,respectively.With iterative mapping,the mapping rates were up to94.1%and95.61%for those two samples.The major increases were from singletons mapped reads,the growth was more than1.6times.While for paired mapped reads,the increase was only about1.05times.
3.2.2.Novel transcripts and functional annotation
After standard cuf?inks work?ow,7247transcripts included in6426 genes were assembled as unknown,intergenic transcripts.Several strict criterions,including transcript length,location relative to known genes, exon number and coding potential,were employed for further?ltration. Finally,1484transcripts included in1094genes were left(Supplement material2).63.7%of these novel transcripts was distributed between 200and2999bp(Fig.1A).Moreover,793(53.4%)of the1484novel transcripts have intact3′ends,as determined by the presence of one of the eleven polyadenylation signal motifs(AAUAAA,AUUAAA,UAUA
Table2
Primers used for validation.
Gene Sequences(5′–3′)
FASN-F TGAAACTGAAGCCTTGTGTGCC FASN-R TCCCTGTGAGCGGAGGTGATTA GCK-F CCTCTCGGTTTCACCTTCTCCTT GCK-R AGTCCCCTCGTCTCTTGATAGC HSL-F GCCCTATCTAAAGAGTTGGTCC HSL-R CGATGTCGTCACACATAAGTTG IGF2-F CCCCTGATCAGCCTTCCTA
IGF2-R GACAAAGTTGTCCGTGGTGA β-Actin-F CCACACAGTGCCCATCTACGA β-Actin-R CCACGCTCTGTCAGGATCTTCA Table3
Effect of different dietary protein level on growth performance of tilapia.
Diet HP LP
IBW15.2±0.0615.0±0.11 FBW105±0.78b92.3±2.90a WG596±3.72b517±17.1a SGR 3.46±0.01b 3.25±0.05a Survival100±0.0098.3±1.67 FCR 1.01±0.01a 1.19±0.02b NR35.9±0.21a40.7±0.57b LR128±3.53a139±4.64b
Means±S.E.M.of three replicates,Means followed by different letters differ signi?cantly (P b0.05).IBW(g?sh?1),initial body weight.FBW(g?sh?1),?nal body weight.
WG(weight gain,%)=100×(?nal body weight?initial body weight)/initial body weight.
SGR(speci?c growth rate,SGR,%day?1)=100×(ln?nal wt.?ln initial wt.)/56days. Survival(%)=100×(?nal?sh number)/(initial?sh number).
FCR,feed conversion ratio=Feed consumed/(FBW?IBW).
NR,nitrogen retention=100×retained nitrogen(g)/nitrogen fed(g).
LR,lipid retention=100×retained lipid(g)/lipid fed(g).
Table4
Body composition and morphometry index of?sh fed with experimental diets for56days. Diet HP LP
Whole body Composition(g kg?1)a
Moisture742±0.91b731±0.35a Protein135±0.22133±0.74 Lipid75.0±1.9586.5±0.54
Muscle
Moisture749±31.6702±37.9 Protein180±0.37180±0.59 Lipid8.73±0.32a10.3±0.32b
Liver
Moisture706±14.2b663±13.1a Protein113±0.31114±6.50 Lipid42.2±0.75a51.1±0.64b
Morphometry index b
VSI9.76±0.79a11.8±1.34b HSI 2.05±0.33a 3.69±0.61b IPF0.92±0.36a 1.43±0.50b CF 4.19±0.38b 3.89±0.29a
VSI,viscerasomatic index=100×viscerasomatic weight(g)/body weight(g).
HSI,hepatopancreasomatic index=100×liver weight(g)/body weight(g).
IPF,intraperitoneal fat ratio=100×intraperitoneal fat weight(g)/body weight(g). SSI,spleensomatic index=100×spleen weight(g)/body weight(g).
CF,condition factor=100×body weight(g)/body length(cm)3.
a Means±S.E.M.of three replicates.Means followed by different letters differ signi?cantly(P b0.05).
b Means±S.E.M.of3replicates.6?sh from each replicate.
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AA,AGUAAA,UUUAAA,CAUAAA,AAUACA,AAUGAA,AAUAUA,GAUA AA,UGUAAA)(Ulitsky et al.,2012)at 100bp before the 3′ends,which compared to 10,614(38.4%)of the 27,609RefSeq transcripts.
These novel genes were then fed to Blast2GO to assign the most possible gene ontology terms,936(85.6%)of which contained at least one GO terms.The GO terms that occupied N 5%of the associated genes were plotted (Fig.1B).
3.3.Differentially expressed genes of tilapia fed with different dietary pro-tein to starch ratio
Known and novel genes with high cumulative read mapping counts (≥10)in all samples were considered active transcribed.The differen-tial expression was performed by DESeq using the negative binomial distribution and a shrinkage estimator for the distribution's variance.In total,97genes with FDR b 0.1were detected as signi ?cant differen-tially expressed.There are 71up and 26down regulated genes when tilapia were fed with low protein diet (Supplement material 3).MA-plot (A)and Clustering of Gene Expression were shown in Supplement material 4.
3.4.Functional analysis of differentially expressed genes
To gain insights in to the biological processes regulated during the different diets and to see processes enriched in differentially expressed genes,we subjected genes with signi ?cant differential expression to gene ontology (GO)term enrichment analysis.Genes with differential expression were tested against the background set using Fisher's Exact Test with Multiple Testing Correction of FDR.We found several GO terms signi ?cantly enriched (P -value =0.01)for up regulated genes,which included several processes related to metabolic process.No GO enrichment was found for down regulated genes.Table 7represents GO terms enriched in differentially expression gene.
The pathway analysis helped to further understand genes'biological functions.In our study,97differentially-expressed genes were annotat-ed to 47signaling pathways in KEGG.The pathways with more than three differentially-expressed genes were shown in Table 8.Differentially-expressed genes were involved in metabolic pathways,carbon metabolism,biosynthesis of secondary metabolites,fatty acid metabolism,PPAR signaling pathway,insulin signaling pathway,bio-synthesis of amino acids,citrate cycle,pyruvate metabolism,glycolysis and gluconeogenesis etc.3.5.Validation of RNA-seq data
To validate our RNA-seq results,insulin-like growth factor 2(IGF2),fatty acid synthase (FASN),hormone sensitive lipase (HSL)and glucokinase (GCK)were randomly selected for quantitative real time-PCR (qRT-PCR)analysis.The qRT-PCR results were indicated in Fig.2,which con ?rmed the data from RNA-sequencing analysis showing similar trends in down-or up-regulation of tilapia genes (Supplement material 3).4.Discussion
Carbohydrate utilization have been studied in most cultured ?sh (Hemre et al.,2002).It has been generally accepted that ?sh do not have a speci ?c requirement for dietary glucose (NRC,2011),many ?sh species showed a long postprandial hyperglycemia after feeding high digestible carbohydrates,which is associated with a retarding growth performance and sometimes a “fatty liver ”(Enes et al.,2009;Hemre et al.,2002).Our results also indicated that tilapia fed with LP diet had poor growth performance and high lipid accumulation in body.Dietary protein and starch content affect ?sh growth and reproduction.A
Table 5
Biochemical compositions of plasma from tilapia fed with experimental diets.Diet
HP LP CHO mmol L ?1 2.44±0.34a 3.90±0.55b TG mmol L ?1 2.00±0.15a 4.12±1.07b HDL mmol L ?10.56±0.070.43±0.14LDL mmol L ?1
0.13
±0.03
0.28
±0.15
Means ±S.E.M.of three replicates.Means followed by different letters differ signi ?cantly (P b 0.05).
Table 6
RNA-seq reads and their physical mapping result in tilapia.
Non-iterative mapping Iterative mapping LP
HP LP HP all_reads
32,634,11640,450,05632,634,11640,450,056mapped_reads (all)27,897,52534,639,78330,709,67138,673,560unique_mapped
23,446,92031,470,71125,476,98431,470,711mapped_reads (paired)24,884,43630,705,34625,857,46232,314,464mapped_reads (singletons)3,013,0893,934,4374,852,2096,359,096mapping_rate
85.49%
85.63%
94.1%
95.61%
Fig.1.Novel transcript length (A)and the gene functional distribution (B).
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number of dietary protein responsive physical signs have previously been identi?ed by conventional approaches(Abdel-Tawwab et al., 2010),but majority of the responsive genes to dietary protein and starch nutritional state have not previously been identi?ed.Regulation of gene expression is one of the key controlling mechanisms used by living cells to sustain and execute their functions.Analyses of gene expression at mRNA levels have provided many useful insights to gene function.
RNA-seq is a recently developed approach to transcriptome pro?ling that uses deep-sequencing technologies,which have been applied in many organisms ranging from plants to animals under different exper-imental conditions(Wang et al.,2009).RNA-seq is a very useful tool for nutrigenomics research.RNA-seq supply the promise of comprehensive discovery of novel transcripts in less time and at considerably lower cost than ESTs from conventional Sanger sequencing.An important applica-tion of RNA-seq was to improve the existing genome annotations (Denoeud et al.,2008).The genome of many model organisms had been published for years,but novel transcripts still could be detected (Jakhesara et al.,2013).1484novel transcripts of tilapia were detected in the present study,which were contributed to complete annotations of tilapia genomes based on RNA-seq(Roberts et al.,2011).
In the present study,the effects of dietary protein to starch ratio on the hepatic transcriptome pro?le of tilapia was compared.Our results revealed that97genes were up or down regulated more than1.5fold when tilapia were fed with LP diet.The altered genes were involved in a wide range of physiologic functions,such as cell structure,metabolism and signal transduction,although the direct relationship to protein nutrition is unknown for many of them(Table7).
Low protein isoenergetic diet had high carbohydrate content in our study.Glucose is completely catabolized by glycolysis,Citrate cycle and the respiratory chain for ATP production or through the pentose phosphate pathway leading to the production of nicotinamide adenine dinucleotide phosphate for biosynthesis under aerobic conditions. Excess glucose may be stored as glycogen or converted into lipids (Enes et al.,2009).Our results indicated glycolysis,Citrate cycle and the pentose phosphate pathway were signi?cantly upregulated when tilapia were fed with LP diet(Table8).
In our study,the resultant map(Fig.3)revealed5remarkably up-regulated genes(GCK:Glucokinase,FASN:fatty acid synthase, MAP2K1:mitogen-activated protein kinase1,PTPRF:receptor-type tyrosine-protein phosphatase F,ACAC:acetyl-CoA carboxylase) involved in insulin signaling pathway after fed with LP diet.Insulin signaling pathway triggers the uptake of glucose,fatty acids and amino acids into adipose tissue,muscle and liver and promotes the storage of these nutrients in the form of lipids,protein and glycogen respectively(Lizcano and Alessi,2002).Glucokinase catalyze the phosphorylation of glucose to glucose-6-phosphate,which be used in glycogenesis and the pentose-phosphate pathway.Gilthead sea bream fed with high carbohydrate/low protein diets showed higher expres-sion level of hepatic glucokinase(Caseras et al.,2002;Metón et al., 2004),which is in accordance with our results in tilapia.Azaza et al. (2013)found that carcass lipid accumulation of tilapia fed with high carbohydrate diet could be explained by de novo synthesis of lipid
Table7
GO terms enriched in signi?cant differentially expressed gene.
GO category GO term name FDR P-value Relative genes
Biological processes
GO:0006091Generation of precursor metabolites and energy 6.6E?3 2.6E?5PDHB,ME1,IDH1,GCK,ACLY,CYC,PDHA
GO:0009058Biosynthetic process 5.0E?1 5.8E?3SERC,SCD,CPT1B,LIPE,PDHB,CMPK1,FASN,ACAC,GCK,GLNA,SLC25A10,PDHA GO:0005975Carbohydrate metabolic process 5.0E?1 6.8E?3PDHB,PGD,GCK,GLNA,ACLY,PDHA
GO:0044710Single-organism metabolic process 5.0E?17.8E?3SCD,PGD,CPT1B,LIPE,CYP24A1,FASN,ACAC
GO:0006629Lipid metabolic process 6.1E?1 1.4E?2SCD,CPT1B,LIPE,CYP24A1,FASN,ACAC
GO:0009056Catabolic process7.8E?1 2.1E?2PDHB,PGD,LIPE,PRSS35,ME1,IDH1,OLA.20807,GCK,ACLY,PDHA
Cellular component
GO:0005811Lipid particle 6.1E?1 1.4E?2LIPE
GO:0005856Cytoskeleton 1.0E0 4.8E?2DES,OLA.20807,XLOC_005293,ZGC:101560,ACTB_G1
Molecular function
GO:0003824Catalytic activity9.7E?1 3.0E?2ADAMTS9,SERC,PDHB,MAP2K1,ACSL,CMPK1,SCD,PGD,CPT1B,XLOC_013177,LIPE,
PRSS35,CYP24A1,FASN,ACAC,MOGAT2,OTC,ME1,IDH1,OLA.20807,GCK,DHRS11A,
GLNA,PTPRF,ACLY,ZGC:112484,PDHA
Table8
Pathway with more than3differentially-expressed genes.
Pathway Genes
Metabolic pathways ME1,IDH1,PGD,PDHA,PDHB,CYP7A1,
OTC,FASN,serC,GCH1,ACLY,ACSL,glnA,
CYP24A1,CYC,ACAC,GCK,CMPK1,ACSBG
Microbial metabolism in diverse environments ME1,IDH1,PGD,PDHA,PDHB,serC,ACLY, glnA,CYC,GCK
Carbon metabolism ME1,IDH1,PGD,PDHA,PDHB,serC,GCK Biosynthesis of secondary
metabolites
IDH1,PGD,PDHA,PDHB,OTC,ACLY Fatty acid metabolism SCD,FASN,ACSL,CPT1,ACAC,ACSBG PPAR signaling pathway CYP7A1,SCD,ACSL,CPT1,ACSBG Insulin signaling pathway FASN,MAP2K1,PTPRF,ACAC,GCK,HSL Biosynthesis of amino acids IDH1,OTC,serC,glnA
Citrate cycle(TCA cycle)IDH1,PDHA,PDHB,ACLY
Pyruvate metabolism ME1,PDHA,PDHB,ACAC
Pathogenic Escherichia coli infection CDHE,ACTB_G1,TUBB
Glycolysis/gluconeogenesis PDHA,PDHB,GCK
HIF-1signaling pathway PDHA,PDHB,MAP2K1
Adherens junction CDHE,ACTB_G1,PTPRF
Fatty acid degradation ACSL,CPT1,ACSBG
Adipocytokine signaling pathway ACSL,CPT1,ACSBG
Phagosome ACTB_G1,TUBB,CALR
Rap1signaling pathway MAP2K1,CDHE,
ACTB_G1Fig.2.Expression analyses of IGF2,GCK,FASN and HSL genes by relative quantitative real-time PCR when tilapia were fed with LP diet.
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from carbohydrate.High carbohydrate diets induce insulin secretion,which stimulate hepatic glucose uptake,thus enhancing hepatic glyco-lytic enzyme activities and leading to an increase in hepatic glycogen content to maintain glucose homeostasis.Glucose,as one of the precur-sors of acetyl-CoA,is the primary substrate for fatty acid synthesis in the liver.Acetyl-CoA carboxylase catalyzes the ATP and bicarbonate-dependent carboxylation of acetyl-CoA into malonyl-CoA.This reaction is generally considered as the rate-limiting step of the fatty acid synthe-sis (Iritani et al.,1984).Hepatic acetyl-CoA carboxylase expression of tilapia fed with low protein diet was signi ?cantly elevated by 2.437fold,which is in accordance with that of Rollin et al.(2003)which found that liver acetyl-CoA carboxylase activity of rainbow trout is increased by a high starch feed.
Expression of hepatic fatty acid synthase (FASN)was increased by 2.537fold by low dietary protein to starch ratio.FASN plays a pivotal role in the process of de novo lipogenesis by catalyzing acetyl coenzyme A and malonyl coenzyme A into the ?nal end product,palmitate,which is subsequently esteri ?ed and stored in adipose tissue (Smith et al.,2003).It is demonstrated that increased expression of FASN can mark-edly induce triglyceride deposition in body (Ohlrogge and Jaworski,1997).The expression of FASN and its activity directly control the synthesis of body fat.
Another signaling pathway affected by dietary protein and starch ratio in the tilapia was the PPAR signaling pathway (Fig.4).This path-way has been demonstrated to regulate the expression of genes involved in the lipid metabolism (Barger and Kelly,2000).Some key PPAR signaling pathway-related genes were identi ?ed in our tran-scriptome,including CYP7A1(cholesterol 7α-hydroxylase),SCD (stearoyl-CoA desaturase),ACSL (long-chain acyl-coenzyme A synthe-tase),ACSBG (acyl-coenzyme A synthetase bubblegum)and CPT-1(carnitine palmitoyltransferases-1).Long-chain-fatty-aid coenzyme A ligase catalyze the bio-activation of fatty acids forming acyl-coenzyme A thioesters that are substrates for anabolic and catabolic pathways.Acyl-coenzyme A thioesterases catalyze the hydrolysis of the thioester bond present within acyl-CoA ester molecules to generate coenzyme A and the corresponding non-esteri ?ed fatty acid,which play important cellular roles in animal fatty acid metabolism through modulation of cellular concentrations of activated fatty acyl-CoAs (Hunt and Alexson,2008).Stearoyl-CoA desaturase,as one of the key enzymes in the forma-tion of unsaturated fatty acids (Hsieh et al.,2007),catalyzes the initial oxidation reaction for the desaturation of long-chain saturated fatty acids into monounsaturated fatty acids.These monounsaturated fatty acids are important substrates for the formation of complex lipids such as cholesterol esters,triglycerides and diacylglycerols.Results showed that tilapia fed with LP diet had signi ?cant high expression of long-chain-fatty-aid CoA ligases and stearoyl-CoA desaturase.Some researchers had demonstrated that stearoyl-CoA desaturase levels were elevated by dietary carbohydrate (Miyazaki et al.,2004,2007).High levels of stearoyl-CoA desaturase may be a signi ?cant contributor to the carcass lipid accumulation induced by low dietary protein to starch ratio.
Carnitine palmitoyltransferases (CPT)play a key role in the regula-tion of mitochondrial β-oxidation of fatty acid in ?sh (McGarry and Brown,1997).CPT-1catalyzes the carnitine-dependent esteri ?cation of palmitoyl-CoA to form palmitoyl-carnitine,which is the main regula-tory step of fatty acid oxidation (Kerner and Hoppel,2000).Some research suggested high carbohydrate diet will induce an increase of acetyl-CoA and malonyl-CoA in the mammals'liver and muscle,the increased malonyl-CoA content will inhibit CPT-1(Saha et al.,1995;Sidossis et al.,1996).Our study indicated that the hepatic CPT-1mRNA levels of tilapia fed with low protein diet with high carbohydrate was higher than that fed with high protein diet,which is not in accordance with reports in mammals.Elevation of CPT-1gene ex-pression also was observed when rainbow trout were fed with a
high
Fig.3.Gene list involved in insulin signaling pathway generated by KEGG when tilapia were fed with LP diet.Black,signi ?cantly increased expression;gray,unchanged expression.White denotes genes that were not identi ?ed in the expression pro ?le analysis.
304Y.Xiong et al./Aquaculture 433(2014)299–306
carbohydrate/low protein diet (Seiliez et al.,2011).These results suggest that low protein diet triggered a switch from the use of as amino acids fuel to the use of fatty acids.
The present study indicates that tilapia fed with LP diet signi ?cantly reduce the growth and feed utilization.Our results concur with other reports of tilapia (Abdel-Tawwab et al.,2010).After analyzing the data of RNA-seq,we found that insulin-like growth factor 2(IGF-2)expres-sion of tilapia fed with LP diet was signi ?cantly reduced by 2.586fold.Insulin-like growth factors play a key role in regulating somatic growth in ?sh via the modulation of growth hormone,IGF-2has a similar growth stimulating function to IGF-1in ?shes (Wilkinson et al.,2006).Chen et al.(2000)demonstrated administration of either recombinant native IGF-1or IGF-2markedly stimulated growth of tilapia.
Present study showed that nitrogen retention of tilapia fed with LP diet was markedly elevated than that fed with HP diet.Glutamine synthetase,isocitrate dehydrogenase,ornithine carbamoyltransferase and phosphoserine aminotransferase,which participated in amino acid biosynthesis,were respectively elevated by 5.997,3.706,3.813and 3.728fold when tilapia were fed with LP diet.Glutamine synthetase is an essential enzyme in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to synthesize glutamine,glutamine can serve as a precursor for protein synthesis and donates ni-trogen for the formation of nucleotides,pyrimidines,purines and amino
sugars (Li et al.,2007),and also can be utilized as energy source for cell growth (Bols et al.,1994).Our results indicated the ability of juvenile Nile tilapia to effective utilize protein by dietary carbohydrate.5.Conclusion
In conclusion,this study,the ?rst attempt to apply the RNA-seq tech-nique to evaluation of the effects of dietary protein to starch ratio on the liver gene expression pro ?le of tilapia,has revealed that a variety of genes are affected by dietary protein and starch content.Key pathways modulated by low dietary protein to starch ratio include those involved with glycolysis,gluconeogenesis,biosynthesis of amino acids,fatty acid biosynthesis and fatty acid metabolism etc.The present results should make an important contribution to better understand protein and carbohydrate utilization mechanisms of ?sh.
Supplementary data to this article can be found online at https://www.doczj.com/doc/595204064.html,/10.1016/j.aquaculture.2014.06.009.Acknowledgment
The work was ?nancially supported by the National Natural Science Foundation of China (Grant No.
31202007).
Fig.4.Gene list involved in PPAR signaling pathway generated by KEGG when tilapia were fed with LP diet.Black,signi ?cantly increased expression;gray,unchanged expression,and white denotes genes that were not identi ?ed in the expression pro ?le analysis.
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路漫漫其修远兮，吾将上下而求索- 百度文库Rolling In The Deep There's a fire starting in my heart 胸中燃起怒火 Reaching a fever pitch and it's bringing me out the dark 狂热救赎我于黑暗 Finally, I can see you crystal clear 终于看清本性 Go ahead and sell me out and I'll lay your sheet bare. 继续背叛而我亦将不再留恋 See how I leave, with every piece of you 看我如何将你撕碎 Don't underestimate the things that I will do 请别低估我的能耐 There's a fire starting in my heart 我胸中升起的怒火 Reaching a fever pitch and it's bringing me out the dark 熊熊燃烧驱走黑暗 The scars of your love, remind me of us 爱之伤疤疼痛于心 They keep me thinking that we almost had it all 让我回想曾经的拥有 The scars of your love, they leave me breathless 爱之伤疤令人窒息 I can't help feeling 思绪万千不能自已 We could have had it all 我们本应幸福Rolling in the deep 如今却在深渊中翻滚 You had my heart inside of your hands 你将我的心捏在手里 And you played it to the beat 玩弄于股掌之间 Baby I have no story to be told 宝贝我已无话可说 But I've heard one of you and I'm gonna make your head burn 可我亦知你愁肠百结 Think of me in the depths of your despair 在绝望深处想着我 Making a home down there, as mine sure won't be shared 纠结着吧，老娘不再与你同甘共苦 The scars of your love, remind me of us 爱之伤疤疼痛于心 They keep me thinking that we almost had it all 让我回想曾经的拥有 The scars of your love, they leave me breathless 爱之伤疤令人窒息 I can't help feeling 思绪万千不能自已 We could have had it all 我们本应幸福 Rolling in the deep 如今却在深渊中翻滚 You had my heart inside of your hands
Rolling in the deep肉铃音则地谱 There's a fire starting in my heart, 贼而子额FAI而司大婷音买哈特 Reaching a fever pitch and it's bringing me out the dark 瑞驰英额飞唔儿皮尺案的一次布瑞英米奥特则大可 Finally, I can see you crystal clear. Fai呢里，爱看细有克瑞斯头科利尔 Go ahead and sell me out and I'll lay your ship bare. 勾阿?的案的赛哦米奥特案的爱哦累幼儿谁谱?耳 See how I leave, with every piece of you 细好唉礼物，维斯爱吴瑞劈死奥夫有 Don't underestimate the things that I will do. 懂特安德尔瑞斯四蹄梅特则丝印死在特唉唯有读 There's a fire starting in my heart, 贼而子额FAI而司大婷音买哈特 Reaching a fever pitch and it's bringing me out the dark 瑞驰英额飞唔儿皮尺案的一次布瑞英米奥特则大可 The scars of your love, remind me of us. 则死卡死奥夫幼儿辣舞，瑞慢的米奥夫阿斯 They keep me thinking that we almost had it all 贼?谱米丝印刻印在特为哦某斯特?得特傲 The scars of your love, they leave me breathless 则死卡死奥夫幼儿辣舞，贼礼物米布瑞斯里斯 I can't help feeling... 唉康特还奥普非零 We could have had it all... (you're gonna wish you, never had met me)... 为苦的还无?得特傲（有啊够那为石油，乃武还的麦特米） Rolling in the Deep (Tears are gonna fall, rolling in the deep) 肉铃音则地谱（提而死啊够那佛，肉铃音则地谱） Y ou had my heart... (you're gonna wish you)... Inside of your hand (Never had met me) 有还得买哈特（有啊够那为石油）因赛的奥夫幼儿??（乃武还的麦特米）And you played it... (Tears are gonna fall)... To the beat (Rolling in the deep) 案的有普雷的伊特（提而死啊够那佛）图则比特（肉铃音则地谱） Baby I have no story to be told, 北鼻唉还无no 四道瑞图比投的 But I've heard one of you and I'm gonna make your head burn. 巴特爱屋赫尔德午安奥夫有案的爱慕够那没课幼儿?得伯儿恩 Think of me in the depths of your despair.
rolling in the deep谐音歌词 There's a fire starting in my heart, 贼而死啊 FAI而司大婷音买哈特 Reaching a fever pitch and it's bringing me out the dark 瑞驰Ing 额飞我皮尺案的一次布瑞ING 米奥特则大可 Finally, I can see you crystal clear. 烦NOU里，爱看 sei 有克瑞斯头科利尔 Go ahead and sell me out and I'll lay your ship bare. 勾阿海的案的赛哦米奥特案的爱哦来幼儿谁谱拜耳 See how I leave, with every piece of you SEI 好唉礼物，维斯爱吴瑞劈死奥夫有 Don't underestimate the things that I will do. 懂特安德尔RUAI四蹄梅特则丝印死在特唉唯有读 There's a fire starting in my heart, 贼而死啊 FAI而司大婷音买哈特 Reaching a fever pitch and it's bringing me out the dark 瑞驰Ing 啊飞我皮尺案的一次布瑞ING 米奥特则大可 The scars of your love, remind me of us. 则死盖尔死奥夫幼儿辣舞，瑞慢的米奥夫阿斯 They keep me thinking that we almost had it all 贼 KEI谱米丝印刻印在特为哦某斯特还得伊特傲 The scars of your love, they leave me breathless 则死盖尔死奥夫幼儿辣舞，贼礼物米布瑞斯里斯 I can't help feeling... 唉康特还奥普非零 We could have had it all... (you're gonna wish you, never had met me)... 为苦的还无还得伊特傲（有啊够那为石油，乃武还的麦特米） Rolling in the Deep (Tears are gonna fall, rolling in the deep) 揉林音则地谱（提而死啊够那佛，揉林音则地谱） You had my heart... (you're gonna wish you)... Inside of your hand (Never had met me) 有还得买哈特（有啊够那为石油）因赛的奥夫幼儿汗的（乃武还的麦特米）And you played it... (Tears are gonna fall)... To the beat (Rolling in the deep) 案的有普雷的伊特（提而死啊够那佛）图则比特（揉林音则地谱） Baby I have no story to be told, 北鼻唉还无 nou 四道瑞图比投的 But I've heard one of you and I'm gonna make your head burn. 巴特爱屋赫尔德午安奥夫有案的爱慕够那没课幼儿还得伯儿恩 Think of me in the depths of your despair. SIN可奥夫米音则带普斯奥夫幼儿第四百二 Making a home down there, as mine sure won't be shared. 没刻印啊后母当贼而，爱死迈恩说儿翁特比晒儿的 The scars of your love, remind me of us.
roling in the deep There's a fire starting in my heart 胸中燃起怒火 Reaching a fever pitch and it's bringing me out the dark 狂热救赎我于黑暗 Finally, I can see you crystal clear 终于看清本性 Go ahead and sell me out and I'll lay your sheet bare. 继续背叛而我亦将不再留恋 See how I leave, with every piece of you 看我如何将你撕碎 Don't underestimate the things that I will do 请别低估我的能耐 There's a fire starting in my heart 我胸中升起的怒火 Reaching a fever pitch and it's bringing me out the dark 熊熊燃烧驱走黑暗 The scars of your love, remind me of us 爱之伤疤疼痛于心 They keep me thinking that we almost had it all 让我回想曾经的拥有 The scars of your love, they leave me breathless 爱之伤疤令人窒息 I can't help feeling 思绪万千不能自已 We could have had it all 我们本应幸福 Rolling in the deep 如今却在深渊中翻滚 You had my heart inside of your hands 你将我的心捏在手里 And you played it to the beat 玩弄于股掌之间 Baby I have no story to be told
There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bringing me out the dark 熊熊烈焰带我走出黑暗 Finally, I can see you crystal clear 最终我将你看得一清二楚 Go ahead and sell me out and I'll lay your ship bare 去吧出卖我我会让你一无所有See how I'll leave with every piece of you 看我怎么离你而去带走你的一切 Don't underestimate the things that I will do 不要低估我将来的所作所为 There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bring me out the dark 熊熊烈焰带我走出黑暗 The scars of your love remind me of us 你的爱情伤痕让我想起了我们曾经的甜蜜They keep me thinking that we almost had it all 它们总在提醒我我们几乎拥有了一切
The scars of your love, they leave me breathless 你的爱情伤痕让我窒息 I can't help feeling 我不禁心生感触 We could have had it all 我们本该拥有一切 (You're gonna wish you never had met me) （你会祈祷要是从未遇见我该有多好）Rolling in the deep 内心深处爱恨交织 (Tears are gonna fall, rolling in the deep) （眼泪快要掉下来，内心深处爱恨交织）You had my heart inside your hand 你俘虏了我的芳心 (You're gonna wish you never had met me) （你会祈祷要是从未遇见我该有多好）And you played it to the beat 但是你玩弄它伴着每一次心跳 (Tears are gonna fall, rolling in the deep) （眼泪快要掉下来，内心深处爱恨交织）Baby, I have no story to be told 宝贝我没有故事可讲
《Rolling，in，The，Deep》中文歌词 There'safirestartinginmyheart 我怒火中烧 Reachingafeverpitchandit'sbringingmeoutthedark 熊熊烈焰带我走出黑暗 Finally,Icanseeyoucrystalclear 最终我将你看得一清二楚 GoaheadandsellmeoutandI'lllayyourshipbare 去吧出卖我我会让你一无所有 SeehowI'llleavewitheverypieceofyou 看我怎么离你而去带走你的一切 Don'tunderestimatethethingsthatIwilldo 不要低估我将来的所作所为 There'safirestartinginmyheart
我怒火中烧 Reachingafeverpitchandit'sbringmeoutthedark 熊熊烈焰带我走出黑暗 Thescarsofyourloveremindmeofus 你的爱情伤痕让我想起了我们曾经的甜蜜 Theykeepmethinkingthatwealmosthaditall 它们总在提醒我我们几乎拥有了一切 Thescarsofyourlove,theyleavemebreathless 你的爱情伤痕让我窒息 Ican'thelpfeeling 我不禁心生感触 Wecouldhavehaditall 我们本该拥有一切 (You'regonnawishyouneverhadmetme) (你会祈祷要是从未遇见我该有多好)
Rollinginthedeep 内心深处爱恨交织 (Tearsaregonnafall,rollinginthedeep) (眼泪快要掉下来，内心深处爱恨交织) Youhadmyheartinsideyourhand 你俘虏了我的芳心 (You'regonnawishyouneverhadmetme) (你会祈祷要是从未遇见我该有多好) Andyouplayedittothebeat 但是你玩弄它伴着每一次心跳 (Tearsaregonnafall,rollinginthedeep) (眼泪快要掉下来，内心深处爱恨交织) Baby,Ihavenostorytobetold 宝贝我没有故事可讲 ButI'veheardoneonyouandI'mgonnamakeyourheadburn
Roll in the deep There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bringing me out the dark 熊熊烈焰带我走出黑暗 Finally, I can see you crystal clear 最终我将你看得一清二楚 Go ahead and sell me out and I'll lay your ship bare 去吧出卖我我会让你一无所有 See how I'll leave with every piece of you 看我怎么离你而去带走你的一切Don't underestimate the things that I will do 不要低估我将来的所作所为 There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bring me out the dark 熊熊烈焰带我走出黑暗 The scars of your love remind me of us 你的爱情伤痕让我想起了我们曾经的甜蜜 They keep me thinking that we almost had it all 它们总在提醒我我们几乎拥有了一切 The scars of your love, they leave me breathless 你的爱情伤痕让我窒息 I can't help feeling 我不禁心生感触 We could have had it all 我们本该拥有一切 (You're gonna wish you never had met me) （你会祈祷要是从未遇见我该有多好） Rolling in the deep 内心深处爱恨交织 (Tears are gonna fall, rolling in the deep) （眼泪快要掉下来，内心深处爱恨交织） You had my heart inside your hand 你俘虏了我的芳心 (You're gonna wish you never had met me) （你会祈祷要是从未遇见我该有多好） And you played it to the beat 但是你玩弄它伴着每一次心跳 (Tears are gonna fall, rolling in the deep) （眼泪快要掉下来，内心深处爱恨交织） Baby, I have no story to be told 宝贝我没有故事可讲 But I've heard one on you and I'm gonna make your head burn 但是我听说了一件有关你的事情我会让你焦头烂额 Think of me in the depths of your despair 在绝望的深渊中想起我
Roling in the deep -Adele 歌词及翻译 Rolling In the Deep -- Adele 滑向深处 There's a fire starting in my heart, 我心中燃起了火焰 Reaching a fever pitch and it's bringing me out the dark 那温度驱走了黑暗 Finally, I can see you crystal clear. 我终于看得清你 Go ahead and sell me out and I'll lay your ship bare. 放弃自己的全部赤裸的留在你的心中 See how I leave, with every piece of you 我会一片一片把你剥离我的记忆 Don't underestimate the things that I will do. 不要以为我真的不会这么做 There's a fire starting in my heart, 心中燃起了火焰 Reaching a fever pitch and it's bringing me out the dark 那温度驱走了黑暗 The scars of your love, remind me of us. 害怕爱你让我更清晰的了解你我 They keep me thinking that we almost had it all 让我觉得总是有一步之遥 The scars of your love, they leave me breath less 害怕爱你让我无法呼吸 I can't help feeling... 我无法阻止自己的思绪 We could have had it all... (you're gonna wish you, never had met me)... 我们本应幸福（你会祈祷你从未遇见我） Rolling in the Deep (Tears are gonna fall, rolling in the deep) 在内心的深处辗转（流下的泪水在心中反侧） Your had my heart... (you're gonna wish you)... Inside of your hand (Never had met me) 你拥有我的心（你会希望）在你的手掌上（从未遇见我） And you played it... (Tears are gonna fall)... To the beat (Rolling in the deep) 你却没有珍惜（泪水滑下）没有留恋（滑向内心深处） Baby I have no story to be told,
ROLLING IN THE DEEP by, Adele CAPO 3 Am(Palm Mute) / / / / / / / / / / / / / / / / Am E There's a fire starting in my heart, G E G Reaching a fever pitch and it's bringing me out the dark Am E Finally, I can see you crystal clear. G E G Go ahead and sell me out and I'll lay your ship bare. Am E See how I leave, with every piece of you G E G Don't underestimate the things that I will do. Am E There's a fire starting in my heart, G E G Reaching a fever pitch and it's bringing me out the dark F G The scars of your love, remind me of us. Em F They keep me thinking that we almost had it all F G The scars of your love, they leave me breathless Em E I can't help feeling... E Am G We could have had it all... (I wish you, never had met me)... F(hold) G Rolling in the Deep (Tears are gonna fall, rolling in the deep) Am G Your had my heart... (I wish you)... Inside of your hand (Never had met me) F(hold) G
rollinginthedeep的歌词There's a fire starting in my heart 胸中燃起怒火 Reaching a fever pitch and it's bringing me out the dark 狂热救赎我于黑暗 Finally, I can see you crystal clear 终于看清本性 Go ahead and sell me out and I'll lay your sheet bare. 继续背叛而我亦将不再留恋 See how I leave, with every piece of you 看我如何将你撕碎 Don't underestimate the things that I will do 请别低估我的能耐 There's a fire starting in my heart 我胸中升起的怒火 Reaching a fever pitch and it's bringing me out the dark 熊熊燃烧驱走黑暗 The scars of your love, remind me of us 爱之伤疤疼痛于心 They keep me thinking that we almost had it all 让我回想曾经的拥有 The scars of your love, they leave me breathless 爱之伤疤令人窒息 I can't help feeling 思绪万千不能自已 We could have had it all 我们本应幸福 Rolling in the deep 如今却在深渊中翻滚 You had my heart inside of your hands 你将我的心捏在手里 And you played it to the beat 玩弄于股掌之间
Rolling In the Deep – Adele There's a fire starting in my heart, 我心中燃起了火焰 Reaching a fever pitch and it's bringing me out the dark 达到狂热的程度，将我带出了黑暗。 Finally, I can see you crystal clear. 最后，我终于看清了你。 Go ahead and sell me out and I'll lay your ship bare. (你)出卖了我，离开我。而我也将彻底把你忘记。 See how I leave, with every piece of you 看我会如何离开你的点点滴滴 Don't underestimate the things that I will do. 不要看轻我将要做的事情。 There's a fire starting in my heart, 心中燃起了火焰 Reaching a fever pitch and it's bringing me out the dark 那温度驱走了黑暗 The scars of your love remind me of us. 你的爱留下的伤痕，提醒着我我们的曾经 They keep me thinking that we almost had it all 它一直让我认为我们几乎拥有了那一切幸福。 The scars of your love, they leave me breathless 你的爱留下的伤痕，让我无法呼吸 I can't help feeling... 我无法阻止我的感觉 We could have had it all... 我们本应幸福的 Rolling in the Deep 思绪翻滚 Your had my heart Inside of your hand 曾经我的心放在你的手心， And you played it. To the beat 你玩弄它，让它伤痕累累。 Baby I have no story to be told, 宝贝，我没有什么故事要说 But I've heard one of you and I'm gonna make your head burn. 可我听到一个你的故事——我将让你的头脑崩溃 Think of me in the depths of your despair. 在绝望的深处想着我
Rolling in the Deep 在灵魂深处翻滚 There's a fire starting in my heart. Reaching a fever pitch it's bringing me out the dark 一团火焰在我心中熊熊燃烧，狂热的温度带我走出黑暗 Finally I can see you crystal clear. Go head and sell me out and I'll lay your shit bare 我终于看透你了，继续出卖我吧，小心我揭你老底 See how I'll leave with every piece of you. Don't underestimate the things that I will do 等着瞧我离去后让你体无完肤，别低估我将来的所作所为 There's a fire starting in my heart. Reaching a fever pitch and it's bring me out the dark 一团火焰在我心中熊熊燃烧，狂热的温度让我远离黑暗 The scars of your love remind me of us. They keep me thinking that we almost had it all 你爱情的伤疤让我回忆起了你我的当初，让我想起我们几乎可以拥有一切 The scars of your love they leave me breathless. I can't help feeling we could have had it all.Rolling in the deep 你爱情的伤疤让我无法呼吸，让我不禁心生感触，我们本来可以拥有一切，内心深处爱恨交织 You had my heart inside of your hand and you played it to the beat 你把我的真心拿在手上，你百般玩弄，我无法忍受 Baby I have no story to be told. But I've heard one of you and I'm gonna make your head burn 我已无话可说，但我听说你倒有一个，我要让你焦头烂额 Think of me in the depths of your despair. Making a home down there as mine sure won't be shared 让你想起我，当你深陷绝望时，在那建立自己的家园与世隔绝 The scars of your love remind me of us. They keep me thinking that we almost had it all 你爱情的伤疤让我回忆起了当初，它一直提醒着我我们曾几乎拥有一切 The scars of your love they leave me breathless. I can't help feeling we could have had it all.Rolling in the deep 你爱情的伤疤让我无法呼吸，让我不禁感到我们本来可以拥有一切，内心深处爱恨交织You had my heart inside of your hand and you played it to the beat 你把我的真心拿在手上，你百般玩弄，令我心碎 We could have had it all. Rolling in the deep 我们本来可以拥有一切，泪水只能流淌在心底 You had my heart inside of your hand but you played it with the beat 你把我的真心拿在手上，但你百般玩弄，伴随着每次心跳
Roll the Dice（掷骰子) Sometimes you wonder 有时候你会期望 what if you could rewrite the moment 如果重新来过会怎么样 Sometimes you figure out 有时候去发现其实 there's another luck in imperfection 不完美才是完美的真相 Sometimes you've been hurt 有时候难免要受伤 then you learn to think about and feel more 却收获了感悟的力量 Sometimes you've been bored 有时候你也厌倦了 with the life you hate 生活本来的模样 always be amazed 要一直好奇啊 always take a risk 要不断冒险啊 always keep the pace in a lifetime game 要在生活这场游戏里前进的自如漂亮 1,2,3,4,5,6, life is rolling the dice,babe 人生的骰子不停地转啊 you can not imagine what would happen in a second 谁能猜得透下一秒是哪一面朝上 1,2,3,4,5,6 life is rolling the dice,babe 人生的骰子不停地转啊 while you're lost in the dark,close your eyes, 迷失在黑暗里就把眼睛闭上 you'll find the lights shine in the other side 我们总会发现彼岸之光 （重复） Sometimes you wonder 有时候你会期望 what if you could rewrite the moment 如果重新来过会怎么样 Sometimes you figure out 有时候去发现其实 there's another luck in imperfection 不完美才是完美的真相
Rolling in the deep 爱情漩涡 There's a fire starting in my heart 心，躁动，如火Reaching a fever pitch and it's bringing me out the dark 让我狂，黑暗远去Finally, I can see you crystal clear 终于我看清Go ahead and sell me out and I'll lay your ship bare 你的背叛，就让它继续吧 我有我的做法，你将一无所有See how I'll leave with every piece of you 看我离你而去，不必挽留Don't underestimate the things that I will do 我有我的做法，永远不要低估There's a fire starting in my heart 我心如火Reaching a fever pitch and it's bring me out the dark 让我狂，逃离黑暗The scars of your love remind me of us 你给我的爱满是伤痕，不禁想起They keep me thinking that we almost had it all 一切都只是即将拥有The scars of your love, they leave me breathless 你给我的爱满是伤痕，不忍呼吸 I can't help feeling 不禁感触We could have had it all 我们本该拥有Rolling in the deep 爱情漩涡将我困住You had my heart inside your hand 我心已为你所俘And you played it to the beat 丢掉的珍惜，反被玩弄 一次，又一次…Baby, I have no story to be told Baby，我没有故事But I've heard one on you and I'm gonna make your head burn 你的故事， 我有听过，你的意志，由我掌握Think of me in the depths of your despair 绝望中忆起Making a home down there as mine sure won't be shared 我给的迁就，这次不会再有 The scars of your love remind me of us 你给我的爱满是伤痕，不禁想起They keep me thinking that we almost had it all 一切都只是即将拥有The scars of your love, they leave me breathless 你给我的爱满是伤痕，不忍呼吸 I can't help feeling 不禁感触We could have had it all 我们本该拥有Rolling in the deep 深深无奈，苦苦纠结You had my heart inside your hand 我的心已为你所俘And you played it to the beat 丢掉的珍惜，反被玩弄 一次，又一次…We could have had it all 我们本该拥有Rolling in the deep 深深无奈，苦苦纠结You had my heart inside your hand 我的心已为你所俘But you played it with a beating 丢掉的珍惜，反被玩弄 一次，又一次…Throw your soul through every open door 抛弃灵魂
Adele(歌手) 中英文对照歌词 声明;翻译参照网上,仅供学习交流，任何用于商业目的的行为，触犯原作者版权的，与本人无关！ 可用歌词制作工具直接制作双语歌词 歌词正文： Rolling In The Deep 爱恨交织 There's a fire starting in my heart 我满腔怒火 Reaching a fever pitch and it's bring me out of the dark 熊熊燃烧的烈火带我远离黑暗 Finally I can see you crystal clear 终于我看透了你 Go ahead and sell me out and I'll lay your ship bare 来吧背叛我吧我会令你一无所有 See how I'll leave with every piece of you 看我如何带走你所有的一切 Don't underestimate The things that I will do 千万不要低估我的能耐 There's a fire starting in my heart 我满腔怒火 Reaching a fever pitch and it's bring me out of the dark 熊熊燃烧的烈火带我远离黑暗 The scars of your love remind me of us 你的爱情疤痕让我想起我们 They keep me thinking that we almost had it all 往事历历在目我们似乎拥有一切 The scars of your love,they leave me breashless 你的爱情疤痕让我无法呼吸
遗憾- 许美静 别再说是谁的错 让一切成灰 除非放下心中的负累一切难以挽回 你总爱让往事跟随 怕过去白费 你总以为要体会人生就要多爱几回 与其让你在我怀中枯萎宁愿你犯错后悔 让你飞向梦中的世界留我独自伤悲 与其让你在我爱中憔悴宁愿你受伤流泪 莫非要你尝尽了苦悲才懂真情可贵 别再说是谁的错 让一切成灰 除非放下心中的负累一切难以挽回 你总爱让往事跟随 怕过去白费 你总以为要体会人生就要多爱几回 与其让你在我怀中枯萎宁愿你犯错后悔 让你飞向梦中的世界留我独自伤悲 与其让你在我爱中憔悴宁愿你受伤流泪 莫非要你尝尽了苦悲才懂真情可贵 与其让你在我怀中枯萎宁愿你犯错后悔 让你飞向梦中的世界留我独自伤悲 与其让你在我爱中憔悴宁愿你受伤流泪 莫非要你尝尽了苦悲
才懂真情可贵 莫非要你尝尽了苦悲 才懂真情可贵 才懂真情可贵 Apologize歌词 歌手：Timbaland 歌曲：Apologize I'm holding on your rope 我紧紧握住你给的希望 Got me ten feet off the ground 它让我双脚悬空 I'm hearing what you say but I just can't make a sound 听懂了你的言意我却只能绝望沉默 You tell me that you need me 爱我要我曾是你山盟海誓 Then you go and cut me down, but wait 伤心于你的离去我却只能空虚等待 You tell me that you're sorry 不说你的歉意是那么苍白无力 Didn't think I'd turn around, and say... 可你不会想到我要重新向你致意 It's too late to apologize, it's too late 你说的对不起已是太迟，真的太迟 I said it's too late to apologize, it's too late 我说过现在已是太迟，真的太迟 I'd take another chance, take a fall 我想我还会努力，宁愿再次被你伤害 Take a shot for you 也再给你一次让我接受你的机会 And I need you like a heart needs a beat 我需要你就像我的心要跳动得一次又一次 But it's nothing new