超饲养对鹅肝脏差异表达基因转化生长因子β_2基因表达的影响
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摘要
超饲养(Overfeeding)引起鹅肝脏代谢发生改变,使得大量脂肪沉积在肝脏中形成肥肝(fatty liver)。但目前鹅肥肝形成的分子机理仍然不清楚。
本研究以朗德鹅和溆浦鹅为试验材料,使用了90对引物进行mRNA差异显示分析,总共分析了大约800条cDNA片段,最后,经过检验,共确定了超饲养后的6个差异表达基因,其中,基因9105的表达水平上调了。在mRNA差异显示分析的基础上,并对基因9105进行了预测,预测结果表明基因9105的cDNA序列含有一个1239bp的开放读码框架(ORF),与鸡和其它物种的转化生长因子β_2基因(transforming growth factor-beta 2,TGF-β_2)同源。编码一个412个氨基酸的蛋白。蛋白质序列存在2个保守的功能域,即一个引导序列(TGF-β前肽),一个是功能结构域(TGF-β)。该蛋白质与鸡、非洲爪蛙、短尾猊、人、猪、牛、绵羊、小鼠、大鼠的TGF-β_2蛋白分别具有99%、96%、91%、90%、90%、89%、89%、88%、87%的同源性。相对定量RT-PCR结果表明鹅基因9105在多种组织中表达,其表达量在肌胃中最高,其次是肝脏、脾脏和卵巢,然后是子宫、肺和肌肉,最低的是肾脏和腹脂。
The liver metabolize of goose was dramatically changed induced by overfeeding, and causing large fats deposited in liver. At present, the molecular mechanism of fatty liver forming is still remaining un-clear.
In this study, mRNA differential display technique was applied to isolate the differentially expressed gene between fatty liver and normal liver of Landes and Xupu goose. Ninety primer pairs were used and approximately eight hundred pieces of cDNA segments were analyzed in this study. Eventually six differentially expressed genes were confirmed by using real-time quantitative PCR technique, among which gene 9105 was confirmed as up regulated in both fatty livers. And besides, gene 9105 was predicted by the bio-informatics technique. The result indicated that cDNA fragments of gene 9105 which was obtained though cDNA cloning contained an open reading frame of 1239 bp which encoded a protein of 412 amino acids, and the sequence of this fragment showed homologous with the mRNA transforming growth factor- beta 2 (TGF-β_2) of chicken and other species, and the protein of 412 amino acids contained two putative conserved domains of TGF-β-propeptide and TGF-β, and has high homology with its homologues-chicken 99%, Xenopus laevis 96%, Monodelphis domestica 91%, human 90%, pig 90%, cattle 89%, Ovis aries 89%, mouse 88%, rat 87%. And by semi-quantitative RT-PCR technique the result showed gene 9105 was expressed in manifold tissues. The expressed level is the highest in muscular stomach, secondly in liver, spleen and ovary, thirdly in uterus, lung and muscle, the lowest in abdominal fat and kidney.
本研究以朗德鹅和溆浦鹅为试验材料,使用了90对引物进行mRNA差异显示分析,总共分析了大约800条cDNA片段,最后,经过检验,共确定了超饲养后的6个差异表达基因,其中,基因9105的表达水平上调了。在mRNA差异显示分析的基础上,并对基因9105进行了预测,预测结果表明基因9105的cDNA序列含有一个1239bp的开放读码框架(ORF),与鸡和其它物种的转化生长因子β_2基因(transforming growth factor-beta 2,TGF-β_2)同源。编码一个412个氨基酸的蛋白。蛋白质序列存在2个保守的功能域,即一个引导序列(TGF-β前肽),一个是功能结构域(TGF-β)。该蛋白质与鸡、非洲爪蛙、短尾猊、人、猪、牛、绵羊、小鼠、大鼠的TGF-β_2蛋白分别具有99%、96%、91%、90%、90%、89%、89%、88%、87%的同源性。相对定量RT-PCR结果表明鹅基因9105在多种组织中表达,其表达量在肌胃中最高,其次是肝脏、脾脏和卵巢,然后是子宫、肺和肌肉,最低的是肾脏和腹脂。
The liver metabolize of goose was dramatically changed induced by overfeeding, and causing large fats deposited in liver. At present, the molecular mechanism of fatty liver forming is still remaining un-clear.
In this study, mRNA differential display technique was applied to isolate the differentially expressed gene between fatty liver and normal liver of Landes and Xupu goose. Ninety primer pairs were used and approximately eight hundred pieces of cDNA segments were analyzed in this study. Eventually six differentially expressed genes were confirmed by using real-time quantitative PCR technique, among which gene 9105 was confirmed as up regulated in both fatty livers. And besides, gene 9105 was predicted by the bio-informatics technique. The result indicated that cDNA fragments of gene 9105 which was obtained though cDNA cloning contained an open reading frame of 1239 bp which encoded a protein of 412 amino acids, and the sequence of this fragment showed homologous with the mRNA transforming growth factor- beta 2 (TGF-β_2) of chicken and other species, and the protein of 412 amino acids contained two putative conserved domains of TGF-β-propeptide and TGF-β, and has high homology with its homologues-chicken 99%, Xenopus laevis 96%, Monodelphis domestica 91%, human 90%, pig 90%, cattle 89%, Ovis aries 89%, mouse 88%, rat 87%. And by semi-quantitative RT-PCR technique the result showed gene 9105 was expressed in manifold tissues. The expressed level is the highest in muscular stomach, secondly in liver, spleen and ovary, thirdly in uterus, lung and muscle, the lowest in abdominal fat and kidney.
引文
1.鲍同柱.转化生长因子β超家族与肿瘤的研究进展.国外医学物理医学与康复学分册,2005,2(25):87-90
2.董明,李会成.转化生长因子β的研究进展.诊断学理论与实践,2003,1(3):45-47
3.金凤媚,薛俊,郏艳红,刘仲齐.半定量RT-PCR技术的研究及应用.天津农业科学,2008,14(1):10-13
4.李文雍,陈清轩.筛选差异表达基因的方法进展.阜阳师范学院学报(自然科学版),2004,21(1):1-5
5.李玉京,李自银,李振声.真核生物mRNA差显技术(Differential Display)的研究进展.生物技术通报,1998,5:23-30
6.李翠玲,崔正言.转化生长因子β分子生物学研究进展.国外医学免疫学分册,1995,3:124-127
7.刘国君.不同品种鹅填饲肥肝效果对比实验.黑龙江畜牧兽医,2006,8:49-51
8.倪虹,陈瑞阳.基因表达系列分析(SAGE)的研究进展.生命科学研究,2002,1(6):18-20
9.潘美辉,金虎林,袁建刚.基因差异表达的研究方法.基础医学与临床,1997,17(5):1-16
10.史春梦,粟永萍.全基因组基因表达频谱研究的新方法—SAGE和IPGI.生物工程进展,2001,21(2):205-207
11.孙玉鹏,胡蕴玉.β转化生长因子研究进展.中华骨科杂志,1994,8(14):505-507
12.王梁燕,洪奇华.实时定量PCR技术及其应用.细胞生物学杂志,2004,26(1):62-64
13.王涛,陆应麟.抑制消减杂交技术的原理及应用.国外医学分子生物学分册,1998,20(3):271-275
14.徐立新,袁潜华,罗越华.DNA微阵列方法与应用.贵州科学,2004,4(22):61-64
15.杨菲菲.基因差异表达的研究方法.现代农业科技,2007,17:232-234
16.张云燕,石雪蓉.转化生长因子β的研究进展.四川解剖学杂志,2006,14(3):30-32
17.瞿浩,王继文.鹅肥肝形成的分子机理研究进展.四川畜牧兽医,2003,5(30):33-34
18.Arya M,Shergill I S,Williamson M.Basic principles of real-time quantitative PCR.Expert Rev Mol Diagn,2005,5(2):209
19.Berger H,Feng X H,Jun Y.Resistance to transforming growth factor beta occurs in the presence of normal Smad activation Surgery,2002,132(2):310-316
20.Derynck R,Feng X H.TGF2beta receptor signaling.Biochim Biophys Acta,1997,1333(2):F105-150
21.Fournier E,Peresson R,Guy G,Hermier D.Relationships between storage and secretion of hepatic lipids in two breeds of geese with different susceptibility to liver steatosis.Poult Sci,1997,76(4):599-607
22.Hermier D,Rousselot-Pailley D,Peresson R,Sellier N.Influence of orotic acid and estrogen on hepatic lipid storage and secretion in the goose susceptible to liver steatosis.Biochim Biophys Acta,1994,1211(1):97-106
23.Hermier D,Saadoun A,Salichon M R,Sellier N,Rousselot-Paillet D,Champman M J.Plasma lipoproteins and liver lipids in two breeds of geese with different susceptibility to hepatic steatosis:changes induced by development and force-feeding.Lipids,1991,26(5):331-339
24.Hermier D,Salichon M R,Guy G,Peresson P.Differential channeling of liver lipids in relation to hepatic steatosis in the geese.Poultry-science,1999,78(10):1398-1406
25.Ito M,Kodama H,Komamine A.Expression of extensin genes is dependenton the stage of the cell cycle and cell proliferation in suspension-cultured catharhnthus roseus cells. Plant Mol Biol, 1998, 36 (3): 343-351
26. Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the ploymerase China reaction. Science, 1992,257: 967-971
27. Liu C, Lustiq A J. Genetic analtsis of Raplp/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae. Genetics, 1996, 143(1): 81-93
28. Livak K J, Flood S J, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 1995, 4: 357-362
29. Massague J. How cells read TGF-beta signals. EMBO J, 2000, 1 (3): 169-178
30. Pearce J. some differences between avian and mammalian biochemistry. Int.j.Bioche.1997, (8):269-279
31. Mou L, Miiler H, Li J, Wang E, Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun, 1994, 199 (2): 564-569
32. Persson U, Izumi H, Souchelnytskyi S.TheL45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. FEBS Lett, 1998, 14(34):83-87
33. Pilo B, George J C. Diurmal and seasonal variations in liver glycogen and fat in relation to metabolic status of liver and metabolic pectoralis in the migratory starling, Sturnus roseus, wintering in India. Comp Biochem Physiol A. 1983;74(3):601-604
34. Shim K S, Kim K H, Han W S. Elevated serumlevels of t ransforming growth factor2betal in patient swith colorectal carcinoma. Cancer, 1999, 85(3): 554-561
35. Sompaysac L, Jane S, Bum T C. Overcoming limitations of the mRNA differential display technique. Nucleic Acids Res, 1995, 23: 4738-4739
36. Taipale J, Saharinen J, Keski O J. Extracellular matrix-associated transforming growth factor-beta, rolein cancer cell growth and invasion. Adv CancerRes, 1998, 75: 87-134
37. Tajima Y, Tashiro K, Camerini D. Cloning of human chromosome 172specific cDNAs using representational difference analysis and human2mouse hyObrid cells. Genomics, 1997, 42(2): 353 -355
38. Twisk J, Gillian-Daniel D L, Tebon A, Wang L, Barrett P H, Artie A D. The role of the LDL receptor in apolipoprotein B secretion. J Clin Invest, 2000, 105(4): 521-532
39. Van Stein O D. Nucleic Acids Research. 1997, 25(13): 598-602
40. Wittwer C T, Ririe K M, Andrew R V, David D A, Gundry R A, Balis U J. The Light Cycler: A microvolume multi-sample fluorimeter with rapid temperature control. Bio Techniques, 1997, 22: 176-181
41. Yasuhara M, Ohama T, Matsuki N, Saito H, Shiga J, Inoue K, Kurokawa k, Teramoto T. Induction of fatty liver by fasting in suncus. Journal of Lipid Reseach. 1991,18: 887-891
42. Zegzouti H, Marty C, Jones B, Bouquin T, Latche A, Pech J C, Bouzayen M. Improved screening of cDNAs generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol Biol Rep, 1997, 15: 236-245
43. Zhang C K, Lang P, Dane F, Ebel R C, Singh N K, Locy R D, Dozier W A. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliate). Plant Cell Rep, 2005, 23 (10-11): 764-769
2.董明,李会成.转化生长因子β的研究进展.诊断学理论与实践,2003,1(3):45-47
3.金凤媚,薛俊,郏艳红,刘仲齐.半定量RT-PCR技术的研究及应用.天津农业科学,2008,14(1):10-13
4.李文雍,陈清轩.筛选差异表达基因的方法进展.阜阳师范学院学报(自然科学版),2004,21(1):1-5
5.李玉京,李自银,李振声.真核生物mRNA差显技术(Differential Display)的研究进展.生物技术通报,1998,5:23-30
6.李翠玲,崔正言.转化生长因子β分子生物学研究进展.国外医学免疫学分册,1995,3:124-127
7.刘国君.不同品种鹅填饲肥肝效果对比实验.黑龙江畜牧兽医,2006,8:49-51
8.倪虹,陈瑞阳.基因表达系列分析(SAGE)的研究进展.生命科学研究,2002,1(6):18-20
9.潘美辉,金虎林,袁建刚.基因差异表达的研究方法.基础医学与临床,1997,17(5):1-16
10.史春梦,粟永萍.全基因组基因表达频谱研究的新方法—SAGE和IPGI.生物工程进展,2001,21(2):205-207
11.孙玉鹏,胡蕴玉.β转化生长因子研究进展.中华骨科杂志,1994,8(14):505-507
12.王梁燕,洪奇华.实时定量PCR技术及其应用.细胞生物学杂志,2004,26(1):62-64
13.王涛,陆应麟.抑制消减杂交技术的原理及应用.国外医学分子生物学分册,1998,20(3):271-275
14.徐立新,袁潜华,罗越华.DNA微阵列方法与应用.贵州科学,2004,4(22):61-64
15.杨菲菲.基因差异表达的研究方法.现代农业科技,2007,17:232-234
16.张云燕,石雪蓉.转化生长因子β的研究进展.四川解剖学杂志,2006,14(3):30-32
17.瞿浩,王继文.鹅肥肝形成的分子机理研究进展.四川畜牧兽医,2003,5(30):33-34
18.Arya M,Shergill I S,Williamson M.Basic principles of real-time quantitative PCR.Expert Rev Mol Diagn,2005,5(2):209
19.Berger H,Feng X H,Jun Y.Resistance to transforming growth factor beta occurs in the presence of normal Smad activation Surgery,2002,132(2):310-316
20.Derynck R,Feng X H.TGF2beta receptor signaling.Biochim Biophys Acta,1997,1333(2):F105-150
21.Fournier E,Peresson R,Guy G,Hermier D.Relationships between storage and secretion of hepatic lipids in two breeds of geese with different susceptibility to liver steatosis.Poult Sci,1997,76(4):599-607
22.Hermier D,Rousselot-Pailley D,Peresson R,Sellier N.Influence of orotic acid and estrogen on hepatic lipid storage and secretion in the goose susceptible to liver steatosis.Biochim Biophys Acta,1994,1211(1):97-106
23.Hermier D,Saadoun A,Salichon M R,Sellier N,Rousselot-Paillet D,Champman M J.Plasma lipoproteins and liver lipids in two breeds of geese with different susceptibility to hepatic steatosis:changes induced by development and force-feeding.Lipids,1991,26(5):331-339
24.Hermier D,Salichon M R,Guy G,Peresson P.Differential channeling of liver lipids in relation to hepatic steatosis in the geese.Poultry-science,1999,78(10):1398-1406
25.Ito M,Kodama H,Komamine A.Expression of extensin genes is dependenton the stage of the cell cycle and cell proliferation in suspension-cultured catharhnthus roseus cells. Plant Mol Biol, 1998, 36 (3): 343-351
26. Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the ploymerase China reaction. Science, 1992,257: 967-971
27. Liu C, Lustiq A J. Genetic analtsis of Raplp/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae. Genetics, 1996, 143(1): 81-93
28. Livak K J, Flood S J, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 1995, 4: 357-362
29. Massague J. How cells read TGF-beta signals. EMBO J, 2000, 1 (3): 169-178
30. Pearce J. some differences between avian and mammalian biochemistry. Int.j.Bioche.1997, (8):269-279
31. Mou L, Miiler H, Li J, Wang E, Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun, 1994, 199 (2): 564-569
32. Persson U, Izumi H, Souchelnytskyi S.TheL45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. FEBS Lett, 1998, 14(34):83-87
33. Pilo B, George J C. Diurmal and seasonal variations in liver glycogen and fat in relation to metabolic status of liver and metabolic pectoralis in the migratory starling, Sturnus roseus, wintering in India. Comp Biochem Physiol A. 1983;74(3):601-604
34. Shim K S, Kim K H, Han W S. Elevated serumlevels of t ransforming growth factor2betal in patient swith colorectal carcinoma. Cancer, 1999, 85(3): 554-561
35. Sompaysac L, Jane S, Bum T C. Overcoming limitations of the mRNA differential display technique. Nucleic Acids Res, 1995, 23: 4738-4739
36. Taipale J, Saharinen J, Keski O J. Extracellular matrix-associated transforming growth factor-beta, rolein cancer cell growth and invasion. Adv CancerRes, 1998, 75: 87-134
37. Tajima Y, Tashiro K, Camerini D. Cloning of human chromosome 172specific cDNAs using representational difference analysis and human2mouse hyObrid cells. Genomics, 1997, 42(2): 353 -355
38. Twisk J, Gillian-Daniel D L, Tebon A, Wang L, Barrett P H, Artie A D. The role of the LDL receptor in apolipoprotein B secretion. J Clin Invest, 2000, 105(4): 521-532
39. Van Stein O D. Nucleic Acids Research. 1997, 25(13): 598-602
40. Wittwer C T, Ririe K M, Andrew R V, David D A, Gundry R A, Balis U J. The Light Cycler: A microvolume multi-sample fluorimeter with rapid temperature control. Bio Techniques, 1997, 22: 176-181
41. Yasuhara M, Ohama T, Matsuki N, Saito H, Shiga J, Inoue K, Kurokawa k, Teramoto T. Induction of fatty liver by fasting in suncus. Journal of Lipid Reseach. 1991,18: 887-891
42. Zegzouti H, Marty C, Jones B, Bouquin T, Latche A, Pech J C, Bouzayen M. Improved screening of cDNAs generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol Biol Rep, 1997, 15: 236-245
43. Zhang C K, Lang P, Dane F, Ebel R C, Singh N K, Locy R D, Dozier W A. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliate). Plant Cell Rep, 2005, 23 (10-11): 764-769