版纳微型猪近交系GOT1基因克隆、序列分析及表达分析
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摘要
【目的】细胞质型天冬氨酸氨基转移酶又称谷草转氨酶,由细胞核GOT1基因编码,催化天冬氨酸的氨基转移到α-酮戊二酸形成草酰乙酸和谷氨酸,在物质代谢调控中起重要作用。本研究以版纳微型猪近交系(Banna mini-pig inbred line, BMI)为材料,克隆GOT1基因,并对其进行生物信息学及组织mRNA表达分析。【方法】利用RT-PCR技术扩增猪GOT1基因,并进行生物信息学分析,同时应用半定量RT-PCR技术明确其mRNA的组织表达特征。【结果】获得了BMI GOT1基因全长1 484 bp的cDNA序列(GenBank登录号:KU705636.1),包含1 242 bp的开放阅读框。生物信息学分析表明:猪GOT1基因位于14号染色体,包含9个外显子和8个内含子;猪GOT1蛋白无信号肽,无跨膜螺旋,有36个磷酸化位点和1个O连接的糖基化位点,定位于细胞质的概率是94.1%,与水牛、黄牛、黑猩猩、人、食蟹猴、大鼠和小鼠的GOT1蛋白依次有95.6%、95.4%、93.0%、92.7%、92.5%、90.3%和90.1%的同源性;二级结构中以α螺旋为主,无规则卷曲和延伸链含量中等,β转角只在局部出现;三级结构同源建模成功。组织表达分析表明:GOT1 mRNA在检测的15个组织中差异表达,肌肉中表达量最高。【结论】为进一步了解该基因的生理功能及调控机制积累了资料。
[Purposes] Aspartate aminotransferase, cytoplasmic, also called glutamic-oxaloacetic transaminase 1, is an enzyme that is encoded by the nuclear GOT1 gene. It catalyzes the reversible transamination reaction from L-aspartate and α-ketoglutarate to L-glutamate and oxaloacetate, and plays a crucial role in the metabolic regulation. The aim of this study was to amplify GOT1 gene from Banna mini-pig inbred line(BMI) and to obtain its expression pattern.[Methods]The GOT1 gene was cloned using RT-PCR method from BMI. Nucleotide and protein sequences of GOT1 were utilized to carry out bioinformatics analysis. The semi-quantitive RT-PCR technology was applied to analysis the mRNA expression profiles of tissues.[Results]A cDNA of 1 484 bp(GenBank accession No. KU705636.1) of BMI GOT1 was obtained, which contained 1 242 bp opening reading frame.Bioinformatics analysis showed GOT1 gene localized to Sus scrofa chromosomes 14 and consisted of nine exons and eight introns without signal peptide sequences and transmembrane helix. GOT1 protein contained 36 potential phosphorylation sites and 1 O-GlcNAc glycosylation site and to be located in the cytoplasm with 94.1% certainty. The predicted amino acid sequence of the gene has high homology with the water buffalo(95.6%), cattle(95.4%), chimpanzee(93.0%), human(92.7%), cynomolgus monkey(92.5%), rat(90.3%) and mouse(90.1%). The secondary structure of BMI GOT1 was mainly consisted of α-helices, random coils and and extended strands were medium; whereas beta turns were locally present. Its tertiary structure was also constructed by homology modeling. GOT1 mRNA was widely expressed in the 15 tissues of BMI with highest expression level in muscle.[ Conclusion] The above results will lay a foundation for further study of the gene about its physiological function and regulatory mechanisms in BMI.
[Purposes] Aspartate aminotransferase, cytoplasmic, also called glutamic-oxaloacetic transaminase 1, is an enzyme that is encoded by the nuclear GOT1 gene. It catalyzes the reversible transamination reaction from L-aspartate and α-ketoglutarate to L-glutamate and oxaloacetate, and plays a crucial role in the metabolic regulation. The aim of this study was to amplify GOT1 gene from Banna mini-pig inbred line(BMI) and to obtain its expression pattern.[Methods]The GOT1 gene was cloned using RT-PCR method from BMI. Nucleotide and protein sequences of GOT1 were utilized to carry out bioinformatics analysis. The semi-quantitive RT-PCR technology was applied to analysis the mRNA expression profiles of tissues.[Results]A cDNA of 1 484 bp(GenBank accession No. KU705636.1) of BMI GOT1 was obtained, which contained 1 242 bp opening reading frame.Bioinformatics analysis showed GOT1 gene localized to Sus scrofa chromosomes 14 and consisted of nine exons and eight introns without signal peptide sequences and transmembrane helix. GOT1 protein contained 36 potential phosphorylation sites and 1 O-GlcNAc glycosylation site and to be located in the cytoplasm with 94.1% certainty. The predicted amino acid sequence of the gene has high homology with the water buffalo(95.6%), cattle(95.4%), chimpanzee(93.0%), human(92.7%), cynomolgus monkey(92.5%), rat(90.3%) and mouse(90.1%). The secondary structure of BMI GOT1 was mainly consisted of α-helices, random coils and and extended strands were medium; whereas beta turns were locally present. Its tertiary structure was also constructed by homology modeling. GOT1 mRNA was widely expressed in the 15 tissues of BMI with highest expression level in muscle.[ Conclusion] The above results will lay a foundation for further study of the gene about its physiological function and regulatory mechanisms in BMI.
引文
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[11]曾养志.版纳微型猪近交系研究进展[C]//2010广州-东莞首届国际小型猪学术论坛论文集,2010.
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[14]MACI?GA M,SZKOP M,PASZKOWSKI A.Chromosomal localization and contribution of three homoeologous genes to biosynthesis of cytosolic aspartate aminotransferase in common wheat[J].Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2016,86(4):945.DOI:10.1007/s40011-015-0536-7.
[15]WU H J,YANG Y,WANG S,et al.Cloning,expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3[J].The FEBS Journal,2011,278(8):1345.DOI:10.1111/j.1742-4658.2011.08054.x.
[16]SON H F,KIM K J.Structural insights into a novel class of aspartate aminotransferase from corynebacterium glutamicum[J].PLoS One,2016,11(6):e0158402.DOI:10.1371/journal.pone.0158402.
[17]李秀,张立江,汤玉清,等.血清天门冬氨酸转氨酶对急性心肌梗塞的诊断价值[J].吉林医学,1996,17(6):320.
[18]唐洪彦,周季英,钟亮伊,等.血清天门冬氨酸转氨酶极度升高的病因和死亡率研究[J].现代临床医学生物工程学杂志,2000,6(2):37.DOI:10.3760/cma.j.issn.1674-1927.2000.02.019.
[19]WEI H J,QING Y B,PAN W Y,et al.Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells[J].PLoSOne,2013,8(2):e57728.DOI:10.1371/journal.pone.0057728.
[20]袁进,顾为望.小型猪作为人类疾病动物模型在生物医学研究中的应用[J].动物医学进展,2011,32(2):108.DOI:10.3969/j.issn.1007-5038.2011.02.026.
[21]朱星红.版纳微型猪近交系解剖组织学[M].北京:高等教育出版社,2004.
[22]OBARU K,NOMIYAMA H,SHIMADA K,et al.Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes[J].Journal of Biological Chemistry,1986,261(36):16976.
[2]TORRE F DE LA,SANTIS L D,SUáREZ M F,et al Identification and functional analysis of a prokaryotic type aspartate aminotransferase:implications for plan amino acid metabolism[J].The Plant Journal,200646(3):414.DOI:10.1111/j.1365-313X.2006.02713.x.
[3]TORRE F DE LA,SUáREZ M F,SANTIS L D,et al The aspartate aminotransferase family in conifers:bio chemical analysis of a prokaryotic-type enzyme from maritime pine[J].Tree Physiology,2007,27(9):1283DOI:10.1093/treephys/27.9.1283.
[4]MCKENNA M C,HOPKINS I B,LINDAUER S L,e al.Role of lipids in modulating the activity of mitochon drial aspartate aminotransferase,a key enzyme in meta bolism[J].Journal of Neurochemistry,2008,104(S1):7.
[5]OTTO-?LUSARCZYK D,GRABO?W,MIELCZAREK-PUTA M.Aspartate aminotransferase-key enzyme in the human systemic metabolism[J].Postepy Higieny Medycyny Doswiadczalnej,2016,70:219.DOI:10.560417322693.1197373.
[6]宫传丽,孙敏.心肝疾病患者血清线粒体天门冬氨酸转氨酶变化及临床意义[J].山东医药,2001,41(11):30DOI:10.3969/j.issn.1002-266X.2001.11.018.
[7]GAZE D C.The role of existing and novel cardiac bio markers for cardioprotection[J].Current Opinion in In vestigational Drugs,2007,8(9):711.
[8]ZHANG L,LI Y,LIU J,et al.Activation of human co agulation system by liver-derived clotting factors o Banna minipig inbred line[J].Transplantation proceedin gs,2004,36(8):2490.DOI:10.1016/j.transproceed.200407.064.
[9]WANG P,HUO H L,WANG S Y,et al.Cloning,se quence characterization,and expression patterns of mem bers of the porcine TSSK family[J].Genetics and molecular research,2015,14(4):14908.DOI:10.4238/2015.October.18.56.
[10]XIN J G,YANG H Q,FAN N N,et al.Highly efficient generation of GGTA1 biallelic knockout inbred minipigs with TALENs[J].PLoS One,2013,8(12):e84250.DOI:10.1371/journal.pone.0084250.
[11]曾养志.版纳微型猪近交系研究进展[C]//2010广州-东莞首届国际小型猪学术论坛论文集,2010.
[12]FLOREA L,HARTZELL G,ZHANG Z,et al.A computer program for aligning a cDNA sequence with a genomic DNA sequence[J].Genome Research,1998,8(9):967.
[13]郭安源,朱其慧,陈新,等.GSDS:基因结构显示系统[J].遗传,2007,29(8):1023.DOI:10.1360/yc-007-1023.
[14]MACI?GA M,SZKOP M,PASZKOWSKI A.Chromosomal localization and contribution of three homoeologous genes to biosynthesis of cytosolic aspartate aminotransferase in common wheat[J].Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2016,86(4):945.DOI:10.1007/s40011-015-0536-7.
[15]WU H J,YANG Y,WANG S,et al.Cloning,expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3[J].The FEBS Journal,2011,278(8):1345.DOI:10.1111/j.1742-4658.2011.08054.x.
[16]SON H F,KIM K J.Structural insights into a novel class of aspartate aminotransferase from corynebacterium glutamicum[J].PLoS One,2016,11(6):e0158402.DOI:10.1371/journal.pone.0158402.
[17]李秀,张立江,汤玉清,等.血清天门冬氨酸转氨酶对急性心肌梗塞的诊断价值[J].吉林医学,1996,17(6):320.
[18]唐洪彦,周季英,钟亮伊,等.血清天门冬氨酸转氨酶极度升高的病因和死亡率研究[J].现代临床医学生物工程学杂志,2000,6(2):37.DOI:10.3760/cma.j.issn.1674-1927.2000.02.019.
[19]WEI H J,QING Y B,PAN W Y,et al.Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells[J].PLoSOne,2013,8(2):e57728.DOI:10.1371/journal.pone.0057728.
[20]袁进,顾为望.小型猪作为人类疾病动物模型在生物医学研究中的应用[J].动物医学进展,2011,32(2):108.DOI:10.3969/j.issn.1007-5038.2011.02.026.
[21]朱星红.版纳微型猪近交系解剖组织学[M].北京:高等教育出版社,2004.
[22]OBARU K,NOMIYAMA H,SHIMADA K,et al.Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes[J].Journal of Biological Chemistry,1986,261(36):16976.