Mol Biol Rep (2010) 37:3487–3493 DOI 10.1007/s11033-009-9941-4
Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds Qinghua Nie • Yongsheng Hu • Liang Xie • Chengguang Zhang • Xu Shen • Xiquan Zhang
Received: 7 August 2009 / Accepted: 19 November 2009 / Published online: 29 November 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (‘‘GASAG’’ for ‘‘GXSXG’’) and the Yongsheng Hu, Liang Xie, Chengguang Zhang contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-009-9941-4) contains supplementary material, which is available to authorized users. Q. Nie Y. Hu L. Xie C. Zhang X. Shen X. Zhang (&) Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, 510642 Guangzhou, Guangdong, China e-mail:
[email protected]
glycine rich motif (‘‘GCGFLG’’ for ‘‘GXGXXG’’) were completely conservative among 14 species. Association analyses showed that c.950?24C[A, c.950?45C[G, c.950? 73G[A, c.950?83C[T and c.950?128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P \ 0.05 or P \ 0.01), and c.777-26C[A, c.950?45C[G, c.950?73G[A and c.950? 118C[T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P \ 0.05). Keywords The adipose triglyceride lipase (ATGL) gene Parrot Quail Duck Association analyses
Introduction The adipose triglyceride lipase (ATGL) gene, also named desnutrin or calcium-independent phospholipase A2 f (iPLA2f) gene, was firstly reported in mouse and human [1–3]. The identification of ATGL gene proved that hormone-sensitive lipase (HSL) was not the only lipase responsible for triglyceride hydrolysis [4], as suggested by former studies since the obese phenotype was not observed in HSL-knockout mice [5, 6]. The ATGL contained the patatin region, the ‘‘GXSXG’’ (‘‘Gly-X-Ser-X-Gly’’) and ‘‘GXGXXG’’ (‘‘Gly-X-Gly-X-X-Gly’’) motif and a/b hydrolase folds, which were conservative structures of lipase. As the second member of patatin-like phospholipase domain containing protein (PNPLA2), ATGL gene was activated by a protein termed ‘‘comparative gene identification 58 (CGI-58 or ABHD5)’’ [7–9]. Recently, it was reported that FoxO1 controlled the ATGL gene expression and regulated lipolysis in adipocytes [10]. In mammals, it was known that ATGL was a key lipase for triglyceride degradation and produce diglyceride,
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whereas HSL played roles in further hydrolysis of diglyceride. The human and mouse ATGL gene encode a 504- and a 486-amino acid precursor (ATGL) with their identities of 86%, and both express predominantly in adipose tissue [1]. With higher expression in fat and muscle tissue, the pig ATGL gene at chromosome 11p15.5 translated a 486-amino acid protein with 83 and 78% homology to mouse and human counterpart [11–13]. The differences of ATGL gene expression were found between Jinhua pig and Landrace pig, and ATGL-siRNA significantly decreased the expression of ATGL and HSL in cultured porcine adipocytes [14]. Additionally, the ATGL genes of rat, dog, sheep, goose and Russian Dwarf Hamster is now available from NCBI. Until now, it was reported that many variations of ATGL gene were related to plasma free fatty acid concentrations, risk of type 2 diabetes, glucose levels, metabolic syndrome, neutral lipid storage disease with myopathy, as well as ChanarinDorfman syndrome [15–19]. As a meat type species after long history of domestication and many generations of breeding, chicken is notable for its outstanding growth characteristics and fat accumulation. The chicken ATGL gene, meanwhile, exhibited much conservation with mammalian counterparts. It encodes a 483amino acid peptide with identities of 67% to human ATGL, and expresses predominantly in adipose tissue including subcutaneous fat and abdominal fat [8, 20, 21]. The chicken ATGL gene spans over 30 kb at GGA5, and is tightly linked to CD151 (CD151 antigen, Raph blood group) and RPLP2 (ribosomal protein, large, P2), which is consistent with that of human, mouse and dog [21]. The chicken CGI-58 gene that encoded activation protein for ATGL lipase was also found to express predominantly in chicken adipose tissue, and then in muscle tissue [7, 8]. A recent study finished by us indicated that a missense SNP (G833A or S261N) of ATGL gene and its haplotype block were related to chicken fatty traits (data submitted). Until now, no study of ATGL gene in wild birds was reported. It is known to us that very little fat is accumulated in wild birds, which is distinguished with mammals and chicken. We wonder that if there are any differences of ATGL gene structure or its expression pattern between wild birds and mammals (or chicken)? Therefore, two species of parrot and quail were used for cDNA cloning and mRNA real time analyses by this study, in order to characterize the ATGL gene in birds with compared to mammals and chicken.
Materials and methods Samples and tissue collection For quail (Coturnix coturnix), parrot (Budgerigar: Melopsittacus undulatus), and duck (Anas platyrhynchos), three
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adult females and three males were bought from Guangzhou Fangcun Huadiwan Flower Bird Fish & Insect market (Guangzhou, China). Each bird was performed for blood extraction and slaughtered for tissue collection. Additionally, a F2 designed chicken resource population described by Lei et al. [22] was used for association analyses of single nucleotide polymorphism (SNP) of ATGL gene with fatness traits. In parrot, quail and duck, a total of 18 kinds of tissues were collected, and they were abdominal fat, breast muscle, cerebellum, cerebrum, heart, hypothalamus, kidney, leg muscle, liver, lung, ovary, pituitary, gizzard, subcutaneous fat, small intestine, spleen, stomachus glandularis, as well as testis. In parrot, quail and duck, the breast muscle tissue was used for ATGL gene cloning, and all tissues available were used for mRNA real time analysis. Primers As described by on-line supplementary Table 1, a total of 18 primer pairs were used by this study. Based on reported cDNA of chicken ATGL gene (NCBI accession number: EU240627), primers of pPM1, pPM2, qPM1 and qPM2 were designed considering the results of ATGL gene homology analysis [21]. pPM1 and pPM2 were used to amplify partial cDNA of parrot ATGL (pATGL) gene by RT–PCR, and qPM1 and qPM2 were for quail ATGL (qATGL) gene. The obtained partial sequences were then used to design the rest primers of pPM3*pPM5 and qPM3*qPM5. 50 rapid amplification of cDNA ends (50 RACE) and 30 RACE was performed with pPM3 and pPM4, qPM3 and qPM4 for pATGL gene and qATGL gene each. For mRNA real time analysis, pPM5 and qPM5 were used to amplify target genes of pATGL and qATGL, whereas pPM7 and qPM6 were for housekeeping gene of beta actin (b-actin) as control in parrot and quail. qPM6 was designed based on reported quail b-actin gene sequences (NCBI accession number: AB199913). As the nucleotide sequences of parrot b-actin gene were unavailable, which were therefore amplified by this study with pPM6 that was designed based on quail b-actin gene sequences (NCBI accession number: AB199913). The obtained partial sequences of parrot b-actin gene were then used to design pPM7 primer for real time analyses. dPM1 and dPM2 were used for cDNA cloning and mRNA real time analyses of duck ATGL (dATGL) gene. dPM3 was designed based on reported duck b-actin gene sequences (NCBI accession number: EF6673 45). To amplify partial intron 5, exon 6 and partial intron 6 of chicken ATGL (cATGL) gene, cPM1 was used for SNP identification, genotyping and association analysis (on-line supplementary Table 1). All primers were designed with GeneTool Lite software (http://www.BioTools.com/), and sent for synthesis by Biosune Co. Ltd (Shanghai, China) for commercial service.
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DNA and RNA preparation
followed by 35 cycles of 30 s at 94°C, 45 s at X°C (AT of Table 1), 1 min at 72°C, and a final extension of 5 min at 72°C in a Mastercycler gradient (Eppendorf Limited, Hamburg, Germany). The amplified fragments were cloned into pGEM-T Easy plasmid vector (Promega, Tokyo, Japan), and then sequenced by Biosune Co. Ltd (Shanghai, China) with commercial service. Furthermore, SNP identification and genotyping of cATGL gene were performed by directly sequencing of PCR products amplified by cPM1. Sequencing service was also provided by Biosune Co. Ltd (Shanghai, China).
Genomic DNA was extracted from EDTA-anticoagulated blood with Phenol–Chloroform. Each tissue were frozen immediately with liquid nitrogen and stored at -80°C before RNA extraction. An improved single step RNA isolation method (Invitrogen, California, USA) was used for total RNA isolation following the manufacture’s instructions. Before reverse transcription, total RNA were treated with RNase-free DNase I (Roche, Mannheim, Germany) for 15 min at 37°C to eliminate genomic DNA contamination. Synthesis of cDNA was performed with 2 lg total RNA, 1 lg oligo dT primer and 20 nmol deoxynucleotide triphosphates using M-MLV Reverse Transcriptase (Promega, Madison, WI).
50 RACE and 30 RACE For pATGL gene, 50 RACE and 30 RACE was performed with pPM3 and pPM4 to amplify its 50 UTR and 30 UTR each. For qATGL gene, qPM3 and qPM4 was used for 50 UTR and 30 UTR amplification by 50 RACE and 30 RACE each (Table 1). RACE PCR was performed with RACE kit (Roche, Mannheim, Germany) by following its manufacture’s instructions step by step. For both 50 RACE and 30 RACE reaction, nested PCR was conducted with two specific primers and one random primer (Table 1). The PCR condition was as follows: 94°C for 2 min, 30 cycles of 94°C for 30 s, X°C for 30 s (AT of Table 1) and 72°C for 3 min, and a final extension of 72°C for 10 min. PCR products were cloned into pGEM-T Easy plasmid vector (Promega, Tokyo, Japan), and sent for sequencing service by Biosune Co. Ltd (Shanghai, China). The obtained
RT–PCR, cloning and sequencing Using cDNA transcribed from parrot breast muscle as template, RT–PCR was performed to obtain two fragments of pATGL gene by pPM1 and pPM2 primers. Similar experiment was also performed to amplify qATGL gene partial sequences with qPM1 and qPM2 primers. Additionally, pPM6 was used to amplify partial cDNA sequences of parrot b-actin gene. PCR was performed in 25 ll reaction mixtures containing 1 ll cDNA, 1.25 U of ExTaq polymerase (Takara, Osaka, Japan), 5 ll of 59 buffer supplied by the manufacturer and 200 lM each of dNTP. The PCR conditions were 3 min at 94°C,
Table 1 Quantification of ATGL mRNA in different tissues of parrot, quail and duck
Note: Abd abdominal fat, Brm breast muscle, Ceb cerebellum, Cer cerebrum, Hea heart, Hyp hypothalamus, Kid kidney, Lem leg muscle, Liv liver, Lun lung, Ova ovary, Pit pituitary, Giz gizzard, Sbf subcutaneous fat, Smi small intestine, Spl spleen, Stg stomachus glandularis, Tes testis
Tissues
pATGL mRNA Average
qATGL mRNA SD
Average
dATGL mRNA SD
Average
SD
Abd
0.0634
0.3258
Failed
0.0412
0.0333
Brm
0.2892
0.1419
10.4228
0.8551
0.0188
0.0207
Ceb
0.0042
0.1206
0.0168
0.8481
0.0019
0.0008
Cer Hea
0.0016 0.0988
0.2485 0.4028
0.0133 0.8526
1.0146 1.2903
0.0064 0.0225
0.0095 0.0063
Hyp
0.0024
0.1389
0.0253
1.1651
0.0035
0.0037
Kid
0.0048
0.2050
0.0614
0.6714
0.0010
0.0004
Lem
0.1312
0.3894
4.5002
1.1851
0.0181
0.0116
Liv
0.0112
0.3035
0.0986
1.3407
0.0056
0.0037
Lun
0.0051
0.1153
0.0456
0.5132
0.0056
0.0031
Ova
0.0023
0.1493
0.0387
0.2615
Failed
Pit
0.0118
0.1242
0.0403
0.6373
0.0021
Giz
0.0015
0.3394
0.0299
0.3173
Failed
0.0008
Sbf
0.0616
0.2610
Failed
0.0553
0.0299
Smi
0.0034
0.2252
0.0133
0.7091
0.0010
0.0006
Spl
0.0019
0.2910
0.0086
0.7307
0.0223
0.0318
Stg
0.0008
0.1563
0.0071
1.5051
0.0084
0.0056
Tes
0.0008
0.3848
0.0057
0.4400
Failed
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sequences were assembled to obtain the full-length cDNA sequences of pATGL and qATGL gene.
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Results The pATGL cDNA sequences
mRNA real time analyses The b-actin was used as internal control in quantitative real time RT–PCR with SYBR green dye to quantify mRNA expression of pATGL and qATGL gene. The 15-ll mixture contained 19 Light Cycler-DNA Master SYBR Green I (Roche), 4.5 mM MgCl2, and 0.4 lM primers (pPM5 for pATGL gene, qPM5 for qATGL gene, pPM7 for parrot b-actin gene and qPM6 for quail b-actin gene). Real time PCR was performed at 94°C for 3 min, followed by 40 cycles of 30 s at 94°C, 30 s at 62°C, 40 s at 72°C in ABI7500 (Applied Biosystems, Foster City, CA, USA). The PCR products of PM3 and PM4 were both sequenced by Biosune Co. Ltd (Shanghai, China) for product confirmation. Varying lengths of oligonucleotides produce dissociation peaks at different melting temperatures. Following 40 cycles, the PCR products were consequently analyzed using the heat dissociation protocol to confirm that one single PCR product was detected by SYBR Green dye. Each data point was repeated four times. Quantitative values were obtained from the threshold PCR cycle number (Ct) at which the increase in signal associated with an exponential growth for PCR product starts to be detected. The relative mRNA levels in each sample were normalized to its content. The relative expression levels of ATGL gene was indicated by 2-DCt, for which DCt = Cttarget gene - Ctb-actin.
Methods Datasets, ATGL homology and bioinformatic analyses The protein and CDs data sets of ATGL for 13 species were downloaded from NCBI website (on-line supplementary Table 2). The prediction of relative molecular weight of ATGL and their identities within each other were performed with DNASTAR soft package (www.dnastar.com). The amino acid comparison of ATGL patatin domain was performed with MEGA 4.1 Beta 2 program (http://www.mega software.net/mega41.html). This program was also used for blast of both CDs and ATGL sequences to construct a phylogenetic tree among 13 species. Marker trait association analyses Association analyses of cATGL gene SNP and chicken fatness traits were performed with GLM procedure of SAS 8.0, as completely described by Lei et al. [22]. All identified SNP of cATGL gene were named with putative standards (http://www.hgvs.org/mutnomen/examplesDNA.html).
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The obtained pATGL cDNA was 1,651 bp in length, which comprised 212 bp 50 UTR, 1446 open reading frame (ORF) and 3 bp 30 UTR. These cDNA sequences were submitted to NCBI database and now released with accession number of GQ221784. The encoded pATGL was a 481-amino acid peptide (ACT09362) with a predicted relative molecular weight of 53,701.53 Da. Its N-terminal region contained a 169-amino acid ‘‘patatin’’ domain and a ‘‘GXSXG’’ motif with a putative active serine (on-line supplementary Figure 1). The pATGL had identities of 90.0, 89.8, 90.8, 92.2 and 92.5% with that of turkey, chicken, quail, finch and duck, respectively. However, its identities with mammalian counterparts were lower than 70% (on-line supplementary Figure 2). The qATGL cDNA sequences The obtained qATGL cDNA was 1,557 bp in length, which comprised 1,449 bp open reading frame (ORF) and 108 bp 30 UTR. These cDNA sequences were submitted to NCBI database and now released with accession number of GQ221783. The encoded qATGL was a 482-amino acid peptide (ACT09361) with a predicted relative molecular weight of 53,649.54 Da. Its N-terminal region contained a 169-amino acid ‘‘patatin’’ domain and a ‘‘GXSXG’’ motif with a putative active serine (on-line supplementary Figure 3). The qATGL had identities of 90.8, 93.6, 97.1, 97.1 and 95.2% with that of parrot, finch, turkey, chicken and duck each, which were much higher than that with mammals (on-line supplementary Figure 2). The dATGL cDNA sequences The obtained dATGL cDNA was 1,440 bp in length, only comprised the open reading frame (ORF) and encoded a 479-amino acid dATGL peptide with a predicted relative molecular weight of 53,197.02 Da. Its N-terminal region contained a 169-amino acid ‘‘patatin’’ domain and a ‘‘GXSXG’’ motif with a putative active serine (on-line supplementary Figure 4). The dATGL had identities of 92.5, 93.6, 95.2, 94.8 and 95.2% with that of parrot, finch, turkey, chicken and quail each, which were much higher than that with mammals (on-line supplementary Figure 2). ATGL homology and phylogenetic tree among 13 species The patatin region of ATGL was much conservative among 14 species. The cATGL patatin region was the same as that
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Fig. 1 Expression of pATGL, qATGL and dATGL gene in different tissues. Note: a Expression of pATGL gene in different tissues. b Expression of qATGL gene in different tissues. c Expression of dATGL gene in different tissues. The horizontal axis and vert axis indicate different tissues and 2-DCt value (mean ± SD) each. Abd abdominal fat, Brm breast muscle, Ceb cerebellum, Cer cerebrum, Hea heart, Hyp hypothalamus, Kid kidney, Lem leg muscle, Liv liver, Lun lung, Ova ovary, Pit pituitary, Giz gizzard, Sbf subcutaneous fat, Smi small intestine, Spl spleen, Stg stomachus glandularis, Tes testis. Abd, Sbf , Ova, Giz and Tes indicated these tissues were failed in mRNA real time analyses
of turkey, and very little difference was found within mammals or poultries (on-line supplementary Figure 5). The active serine hydrolase motif (‘‘GASAG’’ for ‘‘GXSXG’’) and the glycine rich motif (‘‘GCGFLG’’ for ‘‘GXGXXG’’) were completely the same among 14 species, which were regarded as conservative motifs for lipase (on-line supplementary Figure 5). Phylogenetic tree based on ATGL gene sequences indicated that 14 species clearly included into three groups: mammals, birds and frog (on-line supplementary Figure 2). The ATGL identities among 14 species varied from 65.8% (pig and frog) to 98.3% (mouse and rat) (on-line supplementary Figure 2).
In quail, very higher ATGL mRNA was also found in breast muscle (10.4228 ± 0.8551) and leg muscle (4.5002 ± 1.1851). Except for breast muscle, leg muscle and heart (0.8526 ± 1.2903), very lower ATGL mRNA was found in other 13 tissues (2-DCt \ 0.1) (Fig. 1; Table 1). In duck, the dATGL gene was found to predominantly express in subcutaneous fat (0.0553 ± 0.0299) and abdom inal fat (0.0412 ± 0.0333), quite higher ATGL mRNA was also found in heart (0.0225 ± 0.0063), spleen (0.0223 ± 0.0318), breast muscle (0.0188 ± 0.0207) and leg muscle (0.0181 ± 0.0116) (Fig. 1; Table 1).
Tissue-specific expression of pATGL and qATGL gene
Associations of cATGL gene SNP with chicken fatness traits
In parrot, the pATGL gene was found to predominantly express in breast muscle (0.2892 ± 0.1419) and leg muscle (0.1312 ± 0.3894), and quite higher ATGL mRNA was also found in heart (0.0988 ± 0.4028), abdominal fat (0.0634 ± 0.3258) and subcutaneous fat (0.0616 ± 0.2610) (Fig. 1; Table 1).
A total of 10 SNP were identified in intron 5 (c.777-86T[C and c.777-26C[A) and intron 6 (c.950?24C[A, c.950? 34T[C, c.950?45C[G, c.950?49T[A, c.950?73G[A, c.950?83C[T, c.950?118C[T and c.950?128delA) of cATGL gene. Association analyses showed that c.77726C[A was significantly associated with pH value of breast
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muscle (BMPH) (P = 0.0221) and cross-sectional fiber area of breast muscle (CFBM) (P = 0.0379) (on-line supplementary Table 3). c.950?24C[A was highly significantly associated with cingulated fat width (CFW) (P = 0.0019) (on-line supplementary Table 4), and c.950?45C[G, was also significantly associated with CFW (P = 0.021) and BMPH (P = 0.0455) (on-line supplementary Table 5). c.950?73G[A was significantly associated with CFW (P = 0.0172), BMPH (P = 0.0152) and crude protein content of breast muscle (CPBM) (P = 0.026) (on-line supplementary Table 6). c.950?83C[T and c.950?128delA were both highly associated with CFW (P = 0.0014, P = 0.003) (on-line supplementary Tables 7 and 8), Moreover, c.950?118C[T was significantly associated with BMPH (P = 0.0186) and crude fat (ether extract) content of breast muscle (EEBM) (P = 0.0327) (on-line supplementary Table 9).
Discussion In this study, we cloned the full-length coding sequences of parrot, quail and dATGL gene cDNA. As a newly discovered and endogenous gene, the ATGL gene is extensively studied in human, mouse and chicken due to its catalysis effects on lipid hydrolysis [1, 4, 8, 20, 21]. However, this is the first report on ATGL gene in wild birds. The obtained pATGL gene cDNA included the complete 50 UTR, partial 30 UTR and the full-length open reading frame (ORF), as released by NCBI with accession number of GQ221784. There was a deletion of six nucleotides (TTCAGC) or two amino acids (L–Q) in pATGL cDNA compared with that of chicken. The obtained qATGL gene cDNA included the full-length open reading frame (ORF) and the complete 30 UTR, as released by NCBI with accession number of GQ221783. There was a deletion of three nucleotides (CTG) or one amino acid (AA) in qATGL cDNA compared with that of chicken, which was similar with that of turkey [20]. As far as coding sequences of qATGL gene were concerned, there were seven nucleotide substitutions between our sequence (GQ22178) and the formerly reported one (EU852336), and these substitutions led to changes of three amino acids (L51H, P103A and P360L). The recently obtained dATGL cDNA contained only ORF, and encoded a 479-AA ATGL peptide with a predicted relative molecular weight of 53,197.02 Da. The ATGL in birds seemed to be shorter than that in mammals, whereas higher conservation was found between birds and mammals. The reported ATGL precursor of parrot, turkey, quail, chicken and duck contained 481, 481, 482, 483 and 479 amino acids, which were shorter that that of human (504 amino acids), mouse (486), pig (486) and
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cattle (486). Despite of the ATGL precursor length, much conservation still existed between birds and mammals based on homology analyses by this study. Among 13 species, much conservation was found in ATGL patatin region, and in which both the active serine hydrolase motif (‘‘GASAG’’ for ‘‘GXSXG’’) and the glycine rich motif (‘‘GCGFLG’’ for ‘‘GXGXXG’’) are completely the same. The constructed phylogenetic tree based on the ATGL sequences was consistent with the putative relationship among 14 species [23, 24]. A recent study indicated that the patatin domain and the hydrophobic domain of ATGL were both conserved in avian, in which the patatin domain contained lipase activity and the hydrophobic domain exhibited lipid droplet binding [20]. For another lipase gene of lipoprotein lipase (LPL), its high conservation among four mammalian species and chicken was also reported [25]. As proved in mammals, the conservation of the ATGL gene probably indicated that it would also catalyze the first step of triglyceride degradation in birds. Even though little difference was found among three birds of parrot, quail and duck, ATGL gene was predominantly expressed in either fat or muscle tissue. In wild birds of parrot and duck, the ATGL gene was found to express predominantly in muscle tissue (breast muscle [ leg muscle) and fat tissue (subcutaneous fat and abdominal fat), which was much higher than other tissues. In duck, the ATGL gene was found to predominantly express in subcutaneous fat and abdominal fat, and then in heart, spleen, breast muscle and leg muscle. In chicken, three independent studies proved that the highest ATGL mRNA was found in adipose tissues (subcutaneous fat [ abdominal fat), and then muscle tissue (leg or thigh muscle [ breast or pectoralis muscle) [8, 20, 21]. The human and murine ATGL gene was found to predominantly express in white and brown adipose tissue, and then in cardiac and skeletal muscle as well as the testis [1, 4]. The porcine ATGL gene was also reported to highly express in adipose tissue, to a lesser degree in muscle tissue [13]. As in mammals, the ATGL gene was also related to fat metabolism in birds. Most SNP identified in intron 5 and 6 of cATGL gene were significantly or highly significantly associated with some fatness traits like with cingulated fat width (CFW) and pH value of breast muscle (BMPH). It indicated that ATGL gene was probably involved in fat metabolism in birds, this seemed to be further proved by the fact that the highest mRNA expression of ATGL gene was found in either fat or muscle tissues. Many closer SNP were significantly associated with CFW and BMPH also suggested us that a potential SNP for chicken fatness traits probably existed nearby. In conclusion, we firstly cloned the ATGL gene cDNA of parrot, quail and duck by this study, and investigated their characterization in birds by mRNA real time, homology
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and association analyses. It was suggested that ATGL gene was a candidate gene for lipid metabolism in birds. Acknowledgments This work was funded by projects under the Major State Basic Research Development Program, China, project no. 2006CB102100, and the National Natural Science Foundation of China (project number: 30600429).
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