抑制消减杂交法筛选中外猪种基因组差异研究
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摘要
抑制消减杂交技术(Suppression Subtractive Hybridizition，SSH)是以抑制PCR为基础的一种高效寻找差异表达基因的新方法，而通城猪和大白猪是各具优点，具有代表性的中外猪种，本文以SSH筛选两品种猪基因组差异，旨在寻找导致这两个品种猪性状表现差异不同的分子遗传基础，从而为猪的育种提供一条新的途径和方法。
本实验共得到如下结果：
1．建立了在猪中运用SSH进行分子生物学研究的方法。本课题率先在国内将SSH运用于猪的分子生物学研究，并得到了经初步鉴定的差异片段。
2．扩增得到基因组差异条带。通过对基因组酶切产物加接头，两次杂交后，进行两次扩增，得到两品种猪基因组差异条带。
3．克隆扩增产物建立了消减文库。将第二次PCR产物插入pGE-T Easy Vector得到消减文库。
4．初步鉴定了一些插入片段：通过EcoR Ⅰ酶切，得到400~600bp的插入片段，对酶切产物进行PCR扩增。得到与酶切的插入片段大小相近的扩增片段。
5．点杂交鉴定了一些差异片段。
6．本研究认为SSH可以在猪的分子生物学研究中得到很好应用。本课题所得到的结果，还有待进一步鉴定。得到差异片段后寻找差异基因、差异基因的功能及其在生产中的应用还有待进一步研究。
Suppression Subtractive Hybridization (SSH) Based on Suppression PCR is a highly effective method for finding differently expressed genes in the genome of animals. Tongcheng and Laige White pigs are famous indigenous and foreign pig breeds with different excellent traits, so were used as experimental materials. In these study SSH was used to investigate the genome differcce, i.e molecular basis for the different traits between the two breeds, thereby to provide a new method for swine breeding.
The results are as follow:
1. A method for using SSH to study swine molecular biology was established. Using this method establish differently expressed fragments were obtained.
2. Genome different bands were gained by PCR amplification. After adding adaptor and two times of hybridization, genome different bands between the two breeds were obtained by two times of PCR amplification.
3. A subtractive library was built by cloning the amplified products. The library was built by inserting the second PCR products into pGEM-T Easy Vector.
4. Some inserted fragments were initially identified After Eco R I digestion some fragments between 400 to 600bp were obtained, some fragments similar with those of the inserted fragments were gained by PCR amplification using the digested products as template.
5. Some different fragments was identified by dot blot
6. We came to a conclusion that SSH could be effectively used in the studies of swine molecular biology. The fragments obtained in this studies awaited further identification, meanwhile the jobs of finding new genes through differently expressed fragment, the function of the differently expressed genes and their application in animal production also awaited deepening and broadening.
本实验共得到如下结果：
1．建立了在猪中运用SSH进行分子生物学研究的方法。本课题率先在国内将SSH运用于猪的分子生物学研究，并得到了经初步鉴定的差异片段。
2．扩增得到基因组差异条带。通过对基因组酶切产物加接头，两次杂交后，进行两次扩增，得到两品种猪基因组差异条带。
3．克隆扩增产物建立了消减文库。将第二次PCR产物插入pGE-T Easy Vector得到消减文库。
4．初步鉴定了一些插入片段：通过EcoR Ⅰ酶切，得到400~600bp的插入片段，对酶切产物进行PCR扩增。得到与酶切的插入片段大小相近的扩增片段。
5．点杂交鉴定了一些差异片段。
6．本研究认为SSH可以在猪的分子生物学研究中得到很好应用。本课题所得到的结果，还有待进一步鉴定。得到差异片段后寻找差异基因、差异基因的功能及其在生产中的应用还有待进一步研究。
Suppression Subtractive Hybridization (SSH) Based on Suppression PCR is a highly effective method for finding differently expressed genes in the genome of animals. Tongcheng and Laige White pigs are famous indigenous and foreign pig breeds with different excellent traits, so were used as experimental materials. In these study SSH was used to investigate the genome differcce, i.e molecular basis for the different traits between the two breeds, thereby to provide a new method for swine breeding.
The results are as follow:
1. A method for using SSH to study swine molecular biology was established. Using this method establish differently expressed fragments were obtained.
2. Genome different bands were gained by PCR amplification. After adding adaptor and two times of hybridization, genome different bands between the two breeds were obtained by two times of PCR amplification.
3. A subtractive library was built by cloning the amplified products. The library was built by inserting the second PCR products into pGEM-T Easy Vector.
4. Some inserted fragments were initially identified After Eco R I digestion some fragments between 400 to 600bp were obtained, some fragments similar with those of the inserted fragments were gained by PCR amplification using the digested products as template.
5. Some different fragments was identified by dot blot
6. We came to a conclusion that SSH could be effectively used in the studies of swine molecular biology. The fragments obtained in this studies awaited further identification, meanwhile the jobs of finding new genes through differently expressed fragment, the function of the differently expressed genes and their application in animal production also awaited deepening and broadening.
引文
1．川井田博著．猪肉肌纤维粗细与肉质的关系。郁明确译．国外畜牧学——猪与禽，1983，3：51
2．大石孝雄．豚血液型上蛋白质多型上应用(6)。畜产研究，1981，35：339-343
3．王亚鸣．江西玉山猪肌肉组织学特征与肉质的关系。江西农业大学学报．1994，10(3)：284-287
4．王楚端．长白猪北京黑猪民猪肌肉组织学特征研究。中国畜牧杂志．1996．32(4)：33-34
5．刘建忠．转基因小鼠乳腺表达人瘦蛋白的研究。[博士学位论文]。武汉，华中农业大学，1999
6．沈元新．金华猪及其杂种肌肉组织学特征与肉质的关系。浙江农业大学学报，1984，10(3)：265-271
7．吴桢方．HSL和LPL作为猪脂肪沉积性状候选基因的研究。[博士学位论文]。武汉，华中农业大学，1998
8．陈润生．关于改善猪肉品质研究的进展。国外畜牧科学，1981，3：32-35
9．邹峄，陈世荃，傅伟龙．江西玉山黑猪、赣州白猪和台湾本地猪血清转铁蛋白类型的比较。畜牧兽医学报，1984，15(1)：63-66
10．杨在清，马志科，孙超，张恩平，汪邦李，杨静萍，郭泽坤．猪脂肪沉积的品系差异及其cAMP的调控作用。畜牧兽医学报，1996，27：489-494
11．易国华，柳小春，成廷水，李文平，施启顺．猪的血液生化指导和蛋白位点基因平均杂合度与断奶重关系的研究。养猪，1998，2：28-29
12．周俊玲，彭中镇，李奎．赵书红，龚炎长，熊统安，刘平华，陈永久．张亚平，罗明，黄路生．二花脸猪和杜洛克猪的RPPD分析。畜牧兽医学报．1998，29(5)，392-396
13．黄路生，邹峄．中国部分猪种同种异名问题的研究。畜牧兽医学报，1989，20(2)：117-122
14．陶钧，邹峄．湖南地方猪种群亲缘关系的生化遗传学研究。畜牧兽医学报，1992，23(1)：13-21
15．简运华．异种器官移植供体的遗传纯度研究。[硕士学位论文]。武汉，华中农业大学，1998
16．熊远著等，猪生化及分子遗传导论。北京，中国农业出版社，1999
17. Ayala M, Balint P F, Fernai dez-de-Cossio M E. New primer strategy improves precision of differential display. Biotechniques,1995, 8(5):840-848
18. Bogush ML, Velikodvorskaya TV, Lebedev YB, Nikolaev LG, Lukyanov SA, Fradkov AF, Pliyer BK,Boichenko MN, Usatova GN, Vorobien AA, Anderson GL,Sverdlov ED. Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization. Mol.Gen. Genet, 1999, 262(4-5): 721-9
19. Cross HR, et al. Palatability of individual muscles from ovine leg steaks as related to chemical and histological trait. J. food sci. 1972, 37:282
20. Cui D X., Yah X J, Su C Z. Differential expressed geres in GC7901. Or GES-I were isolated by optimized differential display RT-PCR and their primary clinical significance. Journal of Fourth Military University, 1998, 18(6): 601-605
21. Devel D L, et al. Variation in composition and palatability traits and relationships between muscle characteristics and palatability in a random samole of pork carcasses. J.Anim. Sci, 1988,66:385
22. G.J.Hausman and G.B.Thomas. The development of the inner layer of backfat in fetal and young pigs. Journal of Animal Science, 1984, 58:1550-1559
23. M.Hubank and D.G.Schatz.Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Research, 1994, 22(25): 5640-5648
24. Ito, T., K.Kito, N.Adati, Y. Mitsui, H.Hagiwara and Y.Sakaki. Fluorescent differential display: arbitrarily primed RT-PCR finger printing on an automated DNA sequencer. FEBS Lett., 1994, 351: 231-236
25. Fang D C, LaFrance R, Su Z Z, et al. Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences. Pro Natl Acad Sci USA, 1998, 95(23):13788-13793
26..Killefer, J. And Hu, C. Y.. Production of a noveol monoclonal antibody to porcine adipocyte plasma membrane. Proc. Soc. Exp. Bio. And Med. 1990b, 194:172-176
27. Kim JY, Chung YS, Pack KH, Park YI, Kim JK, Yu SN, Oh BJ, Shin JS. Isolation and
Characterization of a cDNA encoding the cysteine proteinase inhibitor, induced upon flower maturation in carnation using suppression subtractiue hybridization. Mol. Cells, 1999, 31:9(4):392-397
28. Peng Liang and Arthur B. Pardee. Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction. Science, 1992, 257(14):967-970
29. Liang P, Zhu W. Zhang X,et al. Differential tlisplay using one-base anchored oligo-dT primer. Nucleic Acids Res, 1994, 22(25):5763-5764
30. Liang P, Factors ensuring successful use ofdifferential display. Methods, 1998, 16(4): 42-45
31. Mersmann, H.J. Adipose tissue lipolyticrate in genetically obese and lean swine, J.Anim. Sci., 1985, 60:131
32. Mark Schena, Dari Shalon, Ronald W. Davis, Patrick O.Brown. Quantiative monitoring of gene expression Patterns with a complementary DNA microrray. Science, 1995,270(20): 467-470
33. Martin Hadman, Bao-Ling Adam, George L.Wright, Jr., and Timothy J. Bos. Modifications to the differential display technique reduce background and increase sensitivity. Analytical biochemistry, 1995, 226:383-386
34. Massimo Romani, Jula V.M. Marchi, Barbara Banelli, and Ida Casciano. Analytical Biochemistry. 1999, 271:204-207
35. Nikolai lisitsyn, Natalya lisilsyn, Michael wigler. Cloning the differenles between two complex genomes. Science, 1993 259(12): 946-j950
36. Nemeth E, Millar LK, Bryant-Cneenwood G. Fetal membrane. Distention: IJ. Differentially expressed genes regulated by a cute distention in vitro. Am J Obstet Gyuecol., 2000, 182:60-67
37. Oishi et al. Blood groups and serum protein polymnorphisms in the pitman-moore and Ohmini strains of miniature pigs. Anim. Bld.Grps. Bioch. Genet., 1976, 6(7):27-32
38. Oliver D.Von Stein, Wolf-Gerolf Thies and Martin Hofmann. A high throughput screening for rarely transcribed differentially expressed geues Nucleic Acids Researth, 1997, 25(B):2598-2602
39. Olga D. Ermolacva, Eugene D. Sverdlov. Subtractine hybridization, a technique for extraction of DNA sequences distinguishing two closely related genomes: critical analysis.
Genetic analysis, 1996, 13:49-58
40. Patricia A, et al.Eating quality of pork in Denmark. Pig Farming Supplement. 1985, 10:56-57
41. Pitzer C. Stassar M, Zoller M. Identifcation of renal-cell-carcinoma-related cDNA clones by suppression subtractive hybridization. J. Cancer Res.Clin. Oncol, 1999, 125(8-9)487-92
42. RONALD K.TUME, STEPHENR.LEE and ANTHONY CRYER. A Comparison of the polypeptide composition of plasma membranes prepared from the white adipose tissue and adipocytes of the mouse, rat,rabbit, ox and chicken by a percoll self-forming gradient procedure. Comp. Bio chem. physiol,1985, 80B:127-134
43. Sakamoto K, Yamasaki Y, Kaneto H, Fujitani Y, Matsuoda T, Yoshioka R, Tagawa T, Matsuhisa M, Kajimoto Y, Hori M. Identification of oxidative stress-regulated genes in rat aortic smooth muscle cells by suppression subtractive hybridization. FEBS Lett, 1999, 461(1-2):47-51
44. Scott, R.A., S.G.Comelius and H.J.Mersmann, Effects of age on lipogenesis and lipolysis in lean and obese swine. J.Anim. Sci., 1981, 52:505
45. Steele, N.C., L.T.Frobish and M.Keeney. Lipogenesis and cellularity of adipose tissue from genetically lean and obese swine, J. Anim. Sci.., 1974, 39:712
46. Steele, N.C. and L.T.Frobish. Selected lipogenic enzyme activities of swine adipose tissue as influenced by genetic phenotgpe,age, feeding frequency and dietary energy source. Grouth, 1976, 40:369
47. Victor E. Velculesce, Lin Zhang, Bert vogelstein, Kenneth W. Kinzler. Serial analysis of gene expression. Science, 1995, 270:484-487
48. Wang X, Lix, Yaish-Ohead S, Sarau HM, Barone FC, Feuerstein GZ. Molecular eloning and expression of the rat monocyte protein-3 gene: a possible role in stroke. Brain Res. Mol. Brain Res, 1999,25;27(2):304-312
49. Wayne W. Kuang, DevonA. Thompson, Renee V. Hoch and Ronald J. Weigel. Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma crll line. Nucleic Acids Research, 1998,26(4):1116-1123
50. Wu WX, Zhang Q, Unno N, Derks JB, Nathanielsz PW. Characterization of decorin mRNA in pregnant in trauterine tissues of the ewe and regulation by steroids. Am J Physiol Cell Physiol., 2000, 278(1): C199-206
51. Zylka MJ, Reppert SM. Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches. Brain Res Mol Brain Res, 1999, 74(1-2):175-181
2．大石孝雄．豚血液型上蛋白质多型上应用(6)。畜产研究，1981，35：339-343
3．王亚鸣．江西玉山猪肌肉组织学特征与肉质的关系。江西农业大学学报．1994，10(3)：284-287
4．王楚端．长白猪北京黑猪民猪肌肉组织学特征研究。中国畜牧杂志．1996．32(4)：33-34
5．刘建忠．转基因小鼠乳腺表达人瘦蛋白的研究。[博士学位论文]。武汉，华中农业大学，1999
6．沈元新．金华猪及其杂种肌肉组织学特征与肉质的关系。浙江农业大学学报，1984，10(3)：265-271
7．吴桢方．HSL和LPL作为猪脂肪沉积性状候选基因的研究。[博士学位论文]。武汉，华中农业大学，1998
8．陈润生．关于改善猪肉品质研究的进展。国外畜牧科学，1981，3：32-35
9．邹峄，陈世荃，傅伟龙．江西玉山黑猪、赣州白猪和台湾本地猪血清转铁蛋白类型的比较。畜牧兽医学报，1984，15(1)：63-66
10．杨在清，马志科，孙超，张恩平，汪邦李，杨静萍，郭泽坤．猪脂肪沉积的品系差异及其cAMP的调控作用。畜牧兽医学报，1996，27：489-494
11．易国华，柳小春，成廷水，李文平，施启顺．猪的血液生化指导和蛋白位点基因平均杂合度与断奶重关系的研究。养猪，1998，2：28-29
12．周俊玲，彭中镇，李奎．赵书红，龚炎长，熊统安，刘平华，陈永久．张亚平，罗明，黄路生．二花脸猪和杜洛克猪的RPPD分析。畜牧兽医学报．1998，29(5)，392-396
13．黄路生，邹峄．中国部分猪种同种异名问题的研究。畜牧兽医学报，1989，20(2)：117-122
14．陶钧，邹峄．湖南地方猪种群亲缘关系的生化遗传学研究。畜牧兽医学报，1992，23(1)：13-21
15．简运华．异种器官移植供体的遗传纯度研究。[硕士学位论文]。武汉，华中农业大学，1998
16．熊远著等，猪生化及分子遗传导论。北京，中国农业出版社，1999
17. Ayala M, Balint P F, Fernai dez-de-Cossio M E. New primer strategy improves precision of differential display. Biotechniques,1995, 8(5):840-848
18. Bogush ML, Velikodvorskaya TV, Lebedev YB, Nikolaev LG, Lukyanov SA, Fradkov AF, Pliyer BK,Boichenko MN, Usatova GN, Vorobien AA, Anderson GL,Sverdlov ED. Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization. Mol.Gen. Genet, 1999, 262(4-5): 721-9
19. Cross HR, et al. Palatability of individual muscles from ovine leg steaks as related to chemical and histological trait. J. food sci. 1972, 37:282
20. Cui D X., Yah X J, Su C Z. Differential expressed geres in GC7901. Or GES-I were isolated by optimized differential display RT-PCR and their primary clinical significance. Journal of Fourth Military University, 1998, 18(6): 601-605
21. Devel D L, et al. Variation in composition and palatability traits and relationships between muscle characteristics and palatability in a random samole of pork carcasses. J.Anim. Sci, 1988,66:385
22. G.J.Hausman and G.B.Thomas. The development of the inner layer of backfat in fetal and young pigs. Journal of Animal Science, 1984, 58:1550-1559
23. M.Hubank and D.G.Schatz.Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Research, 1994, 22(25): 5640-5648
24. Ito, T., K.Kito, N.Adati, Y. Mitsui, H.Hagiwara and Y.Sakaki. Fluorescent differential display: arbitrarily primed RT-PCR finger printing on an automated DNA sequencer. FEBS Lett., 1994, 351: 231-236
25. Fang D C, LaFrance R, Su Z Z, et al. Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences. Pro Natl Acad Sci USA, 1998, 95(23):13788-13793
26..Killefer, J. And Hu, C. Y.. Production of a noveol monoclonal antibody to porcine adipocyte plasma membrane. Proc. Soc. Exp. Bio. And Med. 1990b, 194:172-176
27. Kim JY, Chung YS, Pack KH, Park YI, Kim JK, Yu SN, Oh BJ, Shin JS. Isolation and
Characterization of a cDNA encoding the cysteine proteinase inhibitor, induced upon flower maturation in carnation using suppression subtractiue hybridization. Mol. Cells, 1999, 31:9(4):392-397
28. Peng Liang and Arthur B. Pardee. Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction. Science, 1992, 257(14):967-970
29. Liang P, Zhu W. Zhang X,et al. Differential tlisplay using one-base anchored oligo-dT primer. Nucleic Acids Res, 1994, 22(25):5763-5764
30. Liang P, Factors ensuring successful use ofdifferential display. Methods, 1998, 16(4): 42-45
31. Mersmann, H.J. Adipose tissue lipolyticrate in genetically obese and lean swine, J.Anim. Sci., 1985, 60:131
32. Mark Schena, Dari Shalon, Ronald W. Davis, Patrick O.Brown. Quantiative monitoring of gene expression Patterns with a complementary DNA microrray. Science, 1995,270(20): 467-470
33. Martin Hadman, Bao-Ling Adam, George L.Wright, Jr., and Timothy J. Bos. Modifications to the differential display technique reduce background and increase sensitivity. Analytical biochemistry, 1995, 226:383-386
34. Massimo Romani, Jula V.M. Marchi, Barbara Banelli, and Ida Casciano. Analytical Biochemistry. 1999, 271:204-207
35. Nikolai lisitsyn, Natalya lisilsyn, Michael wigler. Cloning the differenles between two complex genomes. Science, 1993 259(12): 946-j950
36. Nemeth E, Millar LK, Bryant-Cneenwood G. Fetal membrane. Distention: IJ. Differentially expressed genes regulated by a cute distention in vitro. Am J Obstet Gyuecol., 2000, 182:60-67
37. Oishi et al. Blood groups and serum protein polymnorphisms in the pitman-moore and Ohmini strains of miniature pigs. Anim. Bld.Grps. Bioch. Genet., 1976, 6(7):27-32
38. Oliver D.Von Stein, Wolf-Gerolf Thies and Martin Hofmann. A high throughput screening for rarely transcribed differentially expressed geues Nucleic Acids Researth, 1997, 25(B):2598-2602
39. Olga D. Ermolacva, Eugene D. Sverdlov. Subtractine hybridization, a technique for extraction of DNA sequences distinguishing two closely related genomes: critical analysis.
Genetic analysis, 1996, 13:49-58
40. Patricia A, et al.Eating quality of pork in Denmark. Pig Farming Supplement. 1985, 10:56-57
41. Pitzer C. Stassar M, Zoller M. Identifcation of renal-cell-carcinoma-related cDNA clones by suppression subtractive hybridization. J. Cancer Res.Clin. Oncol, 1999, 125(8-9)487-92
42. RONALD K.TUME, STEPHENR.LEE and ANTHONY CRYER. A Comparison of the polypeptide composition of plasma membranes prepared from the white adipose tissue and adipocytes of the mouse, rat,rabbit, ox and chicken by a percoll self-forming gradient procedure. Comp. Bio chem. physiol,1985, 80B:127-134
43. Sakamoto K, Yamasaki Y, Kaneto H, Fujitani Y, Matsuoda T, Yoshioka R, Tagawa T, Matsuhisa M, Kajimoto Y, Hori M. Identification of oxidative stress-regulated genes in rat aortic smooth muscle cells by suppression subtractive hybridization. FEBS Lett, 1999, 461(1-2):47-51
44. Scott, R.A., S.G.Comelius and H.J.Mersmann, Effects of age on lipogenesis and lipolysis in lean and obese swine. J.Anim. Sci., 1981, 52:505
45. Steele, N.C., L.T.Frobish and M.Keeney. Lipogenesis and cellularity of adipose tissue from genetically lean and obese swine, J. Anim. Sci.., 1974, 39:712
46. Steele, N.C. and L.T.Frobish. Selected lipogenic enzyme activities of swine adipose tissue as influenced by genetic phenotgpe,age, feeding frequency and dietary energy source. Grouth, 1976, 40:369
47. Victor E. Velculesce, Lin Zhang, Bert vogelstein, Kenneth W. Kinzler. Serial analysis of gene expression. Science, 1995, 270:484-487
48. Wang X, Lix, Yaish-Ohead S, Sarau HM, Barone FC, Feuerstein GZ. Molecular eloning and expression of the rat monocyte protein-3 gene: a possible role in stroke. Brain Res. Mol. Brain Res, 1999,25;27(2):304-312
49. Wayne W. Kuang, DevonA. Thompson, Renee V. Hoch and Ronald J. Weigel. Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma crll line. Nucleic Acids Research, 1998,26(4):1116-1123
50. Wu WX, Zhang Q, Unno N, Derks JB, Nathanielsz PW. Characterization of decorin mRNA in pregnant in trauterine tissues of the ewe and regulation by steroids. Am J Physiol Cell Physiol., 2000, 278(1): C199-206
51. Zylka MJ, Reppert SM. Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches. Brain Res Mol Brain Res, 1999, 74(1-2):175-181
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