摘要
随着分子生物学的研究进展,动物新品种的培育技术也得到了快速的发展,寻找有价值的分子标记或主效基因作为分子标记辅助选择的基础,结合传统的表型性状进行选择是目前猪遗传改良工程所采用的主要方法之一。同时,科技的发展和人们生活水平的提高也使猪育种的目标发生了变化,育种的重点也已由原来的降低背膘厚、提高生长速度等转变为提高瘦肉组织的生长效率、改良肉质等各项指标的改善。肌内脂肪含量能直接影响猪肉的嫩度和风味,脂肪性状作为影响猪肉质的主要指标之一越来越引起科学家们的关注,2009年启动的国家转基因新品种培育重大专项就把培育高肌内脂肪肉质新品种猪种列为其中一个主要目标。
在人体中,脂滴(lipid droplets)积累过量就会导致肥胖,而在所有的真核细胞生命中,从最简单的酵母到最复杂的人类,细胞内脂滴储存脂肪的能力都是最基本的过程,之前科学家已经确定了很多负责合成脂肪的基因,但是控制将脂肪“打包”成脂滴的基因一直未被发现。07年12月17日出版的《PNAS》上,美国艾伯特·爱因斯坦医学院的David L Silver和同事发现了两个将脂肪打包成脂滴的关键基因,分别命名为FIT1和FIT2 (fat-inducing transcript,诱导脂肪转录基因)。文献报道通过多种卖验方法均证明FIT基因在人、小鼠和斑马鱼等生物中有重要功能,鉴于在猪中脂滴方面研究的不足,很有必要在猪中进行该基因的研究,进而了解脂滴的生物合成机制,探讨瘦肉型猪和脂肪型猪差异形成的分子机理,为人类肥胖提供更细致的解释,为从分子水平上选育高瘦肉率且肉质较好的猪种提供理论依据。
本试验对FIT1和FIT2基因主要进行了基因克隆及其遗传效应的研究。初步结果如下:
1.充分利用NCBI数据库和猪基因组计划公布的数据,克隆鉴定了猪FIT1和FIT2基因的cDNA序列,并进行生物信息学预测和分析,发现两基因均只由两个外显子和一个内含子构成,猪FIT1基因编码290个氨基酸,位于7号染色体,包含一个肌糖磷脂合成蛋白超家族结构域,物种间同源性比较发现猪FIT1编码的氨基酸序列与人、牛、大鼠和小鼠有很高的相似性(>93%),且遗传进化关系和人的距离最近。FIT2基因编码262个氨基酸,位于17号染色体。
2.猪FIT2基因第二外显子的559bp处存在T/C突变,引起内切酶BcnI酶切位点的改变,利用PCR-BcnI-RFLP进行基因型分析,发现在梅山猪中A等位基因占优势,大白猪中B等位基因占优势,但在170头“大白×梅山”F2资源群体中进行与胴体性状的关联分析,结果发现该突变位点仅与肌肉pH值(SC)呈显著相关(P<0.05),与肌肉pH值(BF)接近显著相关,但是与其它性状关联不大。
3.猪FIT1基因第一外显子72bp处G/A突变,引起内切酶FspBI酶切位点的改变,利用PCR-FspBI-RFLP进行基因型分析,发现在梅山猪中A等位基因占优势,大白猪中B等位基因占优势,在279头“大白×梅山”F2资源群体中进行性状关联分析表明,该突变位点对骨率、肥肉率、臀部平均膘厚、肋骨数、板油重的效应达到极显著水平(P<0.01);对皮率、平均背膘厚的效应达到显著水平(P<0.05)。
4.在猪FIT1基因第二外显子590-595bp处存在CACTCC的插入和缺失,在梅山猪中为插入型,在大白猪中为缺失型,而且在该突变前9个碱基,即581bp处存在C/A突变,共引起3个氨基酸的改变,利用直接测序和PCR-SSCP方法证明两位点连锁,用PCR-SSCP进行基因型分析发现梅山猪中C等位基因占优势,大白猪中D等位基因占优势,在314头“大白×梅山”F2资源群体中进行性状关联分析,表明该位点与肥肉率、6-7腰椎间膘厚、臀部平均膘厚、板油重、内脂合计、内脂率(%)等脂肪沉积性状的相关性达到极显著水平(P<0.01)。
5.获得FIT1基因的启动子序列,利用CPGPLOT, NNPP, SignalScan MotifFinder及TFSEARCH等在线软件预测启动子的顺式元件和转录因子结合位点,预测仅发现一处可能的转录起始位点,即位于ATG上游-310bp处的C,且分值较高,为0.91。同时该段序列还具备启动子的一些特征元件,如帽子信号,TATA box, CCAAT box等,另外还有促使脂肪细胞分化的转录因子C/EBP和与调控成肌细胞分化相关的转录因子MyoD的结合位点。
6.利用RT-PCR技术对FIT1和FIT2基因进行了不同组织的表达谱分析。表达谱显示两基因在肌肉和心脏中均高表达,总体趋势是FIT1基因只在特异组织中表达,而FIT2基因在多数组织中均有表达。
7.由于大白和梅山猪的FIT1基因存在氨基酸的不同,我们分别构建了不同DNA序列的融合蛋白表达载体,转染猪成纤维细胞,在细胞水平初步研究其功能的差异。
With the fast development in molecular biology, the technology of cultivate new animal breeds are making great progress. To determine valuale molecular markers and efficiency genes are the first step to do molecular marker-assisted selection(MAS). The combination of MAS and traditional breeding methods is one and the most popular means in the swine genetic improvement engineering. At the same time, with the progress made in science and improvement of people's living standand, the focus of breeding is changing from reducing the fat thickness, increasing the grow speed to enhancing the grow efficience of lean meat, improve the meat taste, and homogeneous and better other indicators. Intramuscular fat (IMF) can effect tenderness and flavor of the meat, while fatty trait, one of the major index to judge pork quality, is being given more and more focus. In 2009, our state started an importantly special-purpose project, one of its objectives is to improve the IMF.
In human, if excessive lipid droplets are cumulated, it may results in obesity. While in all eukaryotes, from the simplest yeast to the most complicated human, it is a basic course that fat is depoted as lipid droplets in cells. Scientists have determined many genes regard with fat synthesis, but a controling gene to package fat into lipid droplets is yet not found. David L Silver and his colleagues from Albert Einstein Medicine Institute detected two key genes that can package fat into lipid droplets, named FIT1and FIT2(fat-inducing transcript)respectively; and their reseach result was published in PNAS in December 17th 2007. Important functions of these two genes were proved in Homo sapien, Mus musculus and Zebra fish by multiple experimental methods. In view of their importance and little knowledge about lipid droplet, it's necessary to research deeply in porcine to get the message about biosynthesis of lipid droplet, and the molecure difference among pork type pigs and fatty type ones. To some extent, we want to give more detailed explaination to human obesity, and give some theoretical support to select superordinary pigs possessing higher lean meat percentage and better tasted meat quality.
Based on these, we cloned FIT1 and FIT2, and studied their genetic effects, the main results are as follows:
1. Isolation and characterization of cDNA sequence of FIT1 and FIT2, with full use of database published in NCBI and swine genome project, and bioinformatical prediction and analysis the cloned sequences. It's found that both of FIT1 and FIT2 contain two exones and one intron, respectively. Porcine FIT1 located at SSC7, codes 290 amino acids, including Inositol phospholipid synthesis protein Scs3p superfamily. Porcine FIT1 protein has high homology with corresponding protein in homo sapien, cattle, rat and mus musculus(>93%). As for FIT2, it is located at SSC17, codes 262 amino acid.
2. The T559C mutation in exon 2 of FIT2 gene was detected, and PCR-BcnI-RFLP was used to detect the polymorphism of the genotypes among Large White×MeiShan F2 resource population. The result shows that Meishan breed mainly possess A allele, while Large white possess B allele, but T/C polymorphism is not significant associated with traits except Meat PH (m.Semispinalis Capitis) (P<0.05).
3. An A/G mutation in exon one of FIT1 gene was detected, and PCR-FspBI-RFLP was developed to detect the A/G polymorphism. Results show that Meishan breed mainly possess A allele, while Large white possess B allele. And FspBI-RFLP polymorphism was extremely significantly associated with Bone percentage(BP), Fat percentage (FW), Buttock fat thickness(BFT) and Leaf fat weight(LFW)(P<0.01),while significantly correlated with Skin percentage(SP) and Average bakefat thickness(ABF) (P<0.05).
4. An CACTCC insertion/deletion mutation exists at 590-595bp of exon 2 in FIT1 gene, and at 581bp, namely,9bp before the insertion/deletion mutation exists C/A mutation, and the two mutations changed three amino acid in all. We carried out PCR-SSCP analysis followed by association analysis in F2 "Large white×Meishan" resource family. The results are as follow:in all tested individuals, all Meishan pigs harbor the insertion, which was designated type C. All Large white pigs harbor the deletion and was named as type D. Association analysis in F2 resource family showed that this site was highly significantly associated with Fat percentage (FP),6-7rib fat thickness(RFT), Buttock average fat thickness(BAFT), Leaf fat weigh(LFW), Total internal fat weigh(TFW) and Internal fat rate(FP)(P<0.01).
5. FIT1 promoter was isolated and NNPP, CPGPLOT, SignalScan, MotiFinder and TFSEARCH softwares were used for prediction of initial position of transcription, distribution of CpG island and transfactors banding sites. It is predicted to contain a sole transcription startpoint at the site of-310bp, with score 0.91. Also, some potential promoter characteristic elements exist in this sequence predictively, eg:Cap signal, TATA box, CCAAT box, et al. Interestingly, some transfactors banding locations were predicted that some C/EBP transfactors related with adipose cell differentiation, and some MyoD transfactor related with muscle differentiation were exist.
6. The semi-quantitative RT-PCR was performed to detect the expression of porcine FITI and FIT2 in different tissues. It's found that these two genes are mainly expressed in the longest back muscle and heart. And in total view, FIT1 is only expressed in special tissue, while FIT2 in most tissues.
7. In addition, recombined plasmids were constructed, considering the mutations existing between Meishan and Large White breeds. And then transfected transiently into fibroblast to study its function difference in cellular level.
引文
1.陈俊峰.猪脂肪细胞分化、脂肪沉积相关候选基因的分离、定位及遗传效应分析.[博士学位论文].武汉:华中农业大学,2006
2. 陈炜,张戈,张思仲.基于生物信息学的SNP侯选位点搜寻方法.遗传,2001,23(2):153-156
3.戴丽荷.猪脂肪沉积三个候选基因的分子生物学基础研究.[博士学位论文].武汉:华中农业大学,2008
4.余文敏.猪四个肌肉相关基因分离、SNPs检测及遗传效应分析.[硕士学位论文].武汉:华中农业大学,2008
5.程蕾.猪PNLIPRP2基因的克隆、原核表达、启动子调控及其与生产性状的关联分析.[硕士学位论文].武汉:华中农业大学,2008
6. 戴茹娟,李宁,吴常信.猪肥胖基因部分序列的克隆与多态性分析.遗传,1997,19(增刊):29-31
7.丁艳,鲁绍雄,连林生,李明丽.影响猪肌内脂肪含量的QTL研究进展.家畜生态学报,2004,(04)
8.侯娅丽,周倩,刘文忠.单核苷酸多态性及其在猪育种中的应用.动物科学与动物医学,2004,21(6):46-47
9.李国华,张沅,李宁.奶牛乳铁蛋白基因部分序列的PCR-SSCP分析.农业生物技术学报,2001,9(2):139—141
10.刘梅芳,徐国恒.PAT家族蛋白在细胞内脂滴代谢过程中的作用.生理科学进展,2006,37(2):103
11.马海明,柳小春,施启顺.猪肌内脂肪酸候选基因研究进展.国外畜牧学猪与禽,2004,(06).
12.王爱国,李宁.猪基因定位及其标记辅助选择在育种中的应用.中国畜牧杂志,1998,134(3):45-48
13.魏彩虹.猪脂肪沉积调控酶的研究进展.甘肃畜牧兽医,1997,(04)
14.熊远著,邓昌彦.种猪测定原理与方法.北京:中国农业出版社,1999:57-118
15.熊远著.猪的生化及分子遗传实验导论.北京:中国农业出版社,1999
16.徐冲,何金汗,徐国恒.脂滴包被蛋白(perilipin)调控脂肪分解.生理科学进展,2006,37(3):221
17.杨公社,张浩卫,白亮,李惠侠,张立杰.猪—研究肥胖和糖尿病的理想模式动物.自然科学进展.2008,18(5):481-487
18.张沅.现代动物育种原理与方法.北京:北京农业大学出版社,1989,310-311
19.钟金城,陈智华分子遗传学与动物育种.成都:四川大学出版社,2001
20.周顺伍.动物生物化学.北京:中国农业出版社,1999
21. Andersson L, Haley C S, Ellegren H, et al. Genetic mapping of quantitative trait loci for growth and fatness in pigs.Science,1994,263:1771-1774
22. Beller M, Riedel D, Jansch L, Dieterich Q Wehland J, Jackle H, Kuhnlein RP. Characterization of the Drosophila lipid droplet subproteome. Mol Cell Proteomics. 2006 Jun,5(6):1082-94. Epub 2006 Mar 16
23. Belliger D A,Merricks E P,Nichols T C.Swine Models of DiabeteMellitus:Insulin Resistance,Glucose Tolerance and Cardiovascular Complications.2006,47(3):243-258
24. Bidanel JP, Milan D,Chevalet C,et al.,Mapping of quantitative trait loci(Qtl) in F2 crosses between Meishan and Large White pig breeds in France.Proc of Intern Conf on Pig Production,Beijing,1998:51-55
25. Blum JS,Temenoff JS,Park H,et al. Development and characterization of enhanced green fluorescent protein and luciferase expressing cell line for non-destructive evaluation of tissue engineering constructs. Biomaterials.2004;25(27):5809-5819.
26. B.P. Mallikarjuna Swamy, N. Sarla.Yield-enhancing quantitative trait loci (QTLs) from wild species.biotechnology advances,26(2008):106-120
27. Brasaemle DL, Rubin B, Harten IA, Gruia-Gray J, Kimmel AR, Londos C.Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem.2000 Dec 8;275(49):38486-93
28. Brasaemle DL, Dolios G, Shapiro L, Wang R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J Biol Chem.2004 Nov 5;279(45):46835-42. Epub 2004 Aug 27.
29. Brown DA. Lipid droplets:proteins floating on a pool of fat. Curr Biol,2001,11 (11):R446
30. Chang BH, Chan L. Regulation of Triglyceride Metabolism. Ⅲ. Emerging role of lipid droplet protein ADFP in health and disease. Am J Physiol Gastrointest Liver Physiol.2007 Jun;292(6):G1465-8. Epub 2006 Dec 28
31. Chen M,Hancock LC, Lopes JM.Transcriptional regulation of yeast phospholipid biosynthetic genes.Biochim Biophys Acta.2007 Mar; 1771(3):310-21.
32. Dalen KT, Ulven SM, Arntsen BM, Solaas K, Nebb HI..PPARalpha activators and fasting induce the expression of adipose differentiation-related protein in liver. J Lipid Res.2006 May;47(5):931-43. Epub 2006 Feb 17
33. Duncan RE, Ahmadian M, Jaworski K, Sarkadi-Nagy E, Sul HS. Regulation of lipolysis in adipocytes.Annu.Rev.Nutr,2007,27:79-101
34. Edvardsen H,Irene Grenaker Alnaes G,Tsalenko A, et al.Experimental validation of data mined single nucleotide Polymorphisms from several databases and consecutive dbSNP builds.Pharmacogenet Genomics.2006Mar,16(3):207-217
35. Fan W, Boston B A, Kesterson R A, Hruby V J, Cone R D. Role of melanocortin ergicneuronsin feeding and the agouti obesity syndrome. Nature,1997,385:165-168
36. Freking B A, Keele J w, Leymaster K A.Evaluation of the ovine caflipyge locus:I Relative chromosomal position and gene action.Anim Sei,1998 76(8):2062-2071
37. Gerbens F, Rettenberger G, LenstraJ A, VeerkamJ H,te Pas MF.chromosomal localization and genetic variation of the porcine heart fatty acid-binding protein gene. Mamm Genome,1997,8 (5):328-332
38. Goldspink G, Yang S P. Muscle Structure, Development and Growth. Poultry meat Science.1999,11
39. Gu F, Harbitz I, Chowdhary B P, Bosnes M, Gustavsson I. Chromosomal localizations of the hormone-sensitive lipase and insulin receptor genes in pig. Hereditas,1992,17:231-236
40. Harbitz I, Langset M, Ege A G, Hoyheim B,Davies W.The porcine hormone sensitive lipase gene:sequence, structure, polymorphism and linkage mapping. Animal Genetics,1999,30:10-15
41. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O'Shea EK.Global analysis of protein localization in budding yeast. Nature.2003 Oct 16;425(6959):686-91
42. Harbitz I, Chowdhary B, Thomsen P D, Davie s W, Kaufmann U, Kran S Gustavsson I, Chris tensen K, Hauge J G. Assignment of the porcine calcium release 1.1 2.1 channel gene a candidate for the malignant hyperthermial locus to the 6p-q segment of chromosome 6. Genomics,1990,8:243-248
43. Hawken R J, Murtaugh J, Flickinger G H. A firstgeneration porcine whole-genome radiation hybrid map. Mamm Genome,1999,10:824-830.
44. Knight BL, Hebbachi A, Hauton D, Brown AM, Wiggins D, Patel DD, Gibbons GF.A role for PPARalpha in the control of SREBP activity and lipid synthesis in the liver. Biochem J.2005 Jul 15;389(Pt 2):413-21
45. Hu E, Liang P, Spiegelman B M. AdipoQ is a novel adipose specific gene are gulated in obesity. J Biol Chem,1996,271(18):10697-10703
46. Jiang Z h, Gibson J P. Genetic polymorphisms in the leptin gene and their association with fatness in four pig breeds. Mammalian Genome,1999 (10):191-193
47. Kadereit B, Kumar P, Wang WJ, Miranda D, Snapp EL, Severina N, Torregroza I, Evans T, Silver DL. Evolutionarily conserved gene family important for fat storage Proc Natl Acad Sci U S A.2008 Jan 8;105(1):94-9. Epub 2007 Dec 26
48. Kelsoe JR.Genomics and the Human Genome Project implications for psychiatry.Int Rev Psychiatry. 2004 Nov,16(4):294-300
49. Brookes AJ.The essence of SNPs.Gene,1999,234:177-186
50. Londos C, Brasaemle DL, Schultz CJ, et al. On the control of lipolysis in adipocytes. Ann N Y Acad Sci,1999,892 155-168.
51. Londos C, Brasaemle DL, Schultz CJ, et al.Perilipins, ADRP, and other proteins that associate with intracellular neutral lip id drop lets in animal cells. Semin Cell Dev Biol,1999,10(1):51
52. Londos C, Sztalryd C, Tansey JT, et al. Role of PAT proteins in lipid metabolism. Biochimie,2005,8745-49.
53. Londos C, Brasaemle DL, Schultz CJ, Segrest JP, Kimmel AR.Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin Cell Dev Biol.1999 Feb;10(1):51-8
54. Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara Kl. cDNA cloning and expression of a novel adipose specific collagen-like factor, APM1 (AdiPose Most abundant Gene transcript 1). Biochem BiophysRes Commun,1996, 221(2):286-289
55. Martin S, Parton RG. Lipid droplets:a unified view of a dynamic organelle. Nat Rev Mol Cell Biol.2006 May;7(5):373-8
56. McTernan C L, McTernan P G, Harte A L, Levick P L, Barnett A H, Kumar S.Resistin, central obesity, and type 2 diabetes. Lancet,2002,359:46-47
57. Miller E R,Ullrey D E.The pig as a model for human nutrition. Annu Rev N utr,1987,7:361-82
58. Mullner H, Daum G.Dynamics of neutral lipid storage in yeast.Acta Biochim Pol. 2004;51(2):323-47
59. NothnagelM, Rohde K.The effect of single-nucleotide polymorphism marker selection on pattems of haplotype blocks and haplotype frequency estimates.Am J Hum Genet.2005 Dec,77(6):988-98
60. Novikoff AB, Novikoff PM, Rosen OM, Rubin CS. Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol.1980 Oct;87(1):180-96
61. Obesity and the regulation of energy balance.Spiegelman BM, Flier JS. Cell.2001 Feb 23;104(4):531-43
62. Rapacz J and Hasler-Rapacz J.Animal Models-The Pig Genetic Factors in Atherosclerosis.Appro Mod Sys,1989,12:139-169
63. Remenyi A, Scholer HR, Wilmanns M. Combinatorial control of gene expression Nat Struct Mol Biol.2004 Sep;11(9):812-5
64. Robenek H, Hofnagel O, Buers I, Robenek MJ, Troyer D, Severs NJ.Adipophilin enriched domains in the ER membrane are sites of lipid droplet biogenesis. J Cell Sci. 2006 Oct 15;119(Pt 20):4215-24. Epub 2006 Sep 19
65. Rohrer G A, Alexander L J, Hu Z, et al. A comprehensive map of the porcine genome. Genome Res,1996,6:371-391
66. Sassaki S, Clutter A C, Pomp D. Assignment of the procine obese (leptin) gene to Chromosome 18 by linkage analysis of a new PCR-based polymorphism. Mammalian Genome,1996,7(4):471-472
67. Schiedner G,Bloch W,Hertel S,et al. A hemodynamic response to intravenous adenovirus vector particles is caused by systemic Kupffer cell-mediated activation of endothelial cells. Hum Genether.2003,14(17):1631-1641
68. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell.2001 Feb 23;104(4):531-43.
69. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC.Mechanism of action of fibrates on lipid and lipoprotein metabolism.Circulation. 1998 Nov 10;98(19):2088-93
70. Steppan C M, Bailey S T, Bhat S, Brown E J, Banerjee R R, Wright C M, Patel H R,Ahima R S, Lazar M A. The hormone resistin links obesity to diabetes. Nature, 2001,409(6818):307-312
71. Smyth DJ, Howson JM, Payne F, et al.Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune mediated diseases.BMC Med Genet.2006 Mar6,7:20
72. Sztalryd C, Xu G, Dorward H, et al. Perilip in A is essential for the translocation of hormone2sensitive lipase during lipolytic activation. J Cell Biol,2003,161 (6):1093
73. Tholpady SS, Katz AJ, Ogle RC. Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec A DiscovMol Cell Evol Biol, 2003,272 (1):398
74. Trinklein ND, Aldred SJ, Saldanha AJ, Myers RM. Identification and functional analysis of human transcriptional promoters Genome Res.2003 Feb;13(2):308-12
75. Tuggle C K, Green J A, Fitzsimmons C, et al. EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human. Mamm Genome, 2003,14(8):565-579
76. WallJD, Pritehard JK.Haplotype blocks and linkage disequilibriumin the human genome.Nat Rev Genet,2003,4(8):587-597
77. Whitworth KM, Agca C, Kim JG, Patel RV, Springer GK, Bivens NJ, Forrester LJ, Mathialagan N, Green JA, Prather RS. Transcriptional profiling of pig embryogenesis by using a 15-K member unigene set specific for pig reproductive tissues and embryos Biol Reprod.2005 Jun;72(6):1437-51. Epub 2005 Feb 9
78. Wolins NE, Brasaemle DL, Bickel PE.A proposed model of fat packaging by exchangeable lipid droplet proteins.FEBS Lett.2006 Oct 9;580(23):5484-91. Epub 2006 Sep 1
79. Xu G, Sztalryd C, Lu X,et al.Posttranslational regulation of adipose differerntiattion related protein by the ubiquitin/proteasome pathway. J Biol Chem,2005,280(52):42 841
80. Xu G, Sztalryd C, Londos C. Degradation of perilip in is mediated through ubiquitination proteasome pathway.Biochim Biophys Acta,2006,1761 (1):83
81. Yamaguchi T, Matsushita S, Motojima K, Hirose F, Osumi T.MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor alpha.J Biol Chem.2006,19:281(20): 14232-40.
82. Yerle M, Pinton P, Delcros C, et al. Generation of a 12 000 rad radiation hybrid panel for fine mapping in pigs.Cytogenet Genome Res,2002,97:219-228.
83. YUE Genhua, Petra Beeckmann, Gerhard Moser, Elisabeth Muller,Hans Bartenschlager, Stanislav Cepica, Jaroslav Schroffel,Antonin Stratil & Hermann Geldermann. QTL alleles on chromosome 7 from fatty Meishan pigs reduce fat deposition.science in china 2002,46(1):10-17
84. Zechner R, Strauss JG, Haemmerle G, Lass A, Zimmermann R..Lipolysis:pathway under construction. Curr Opin Lipidol.2005 Jun;16(3):333-40
85. Zimmermann K,Schogl D,Mannhalter JW. Digestion of terminal restriction endonuclease recognition sites on PCR products.Biotechniques.1998;24(4):582-584.
86. Zweytick D, Athenstaedt K, Daum G. Intracellular lipid particles of eukaryotic cells. Biochim Biophys Acta,2000,1469 (2):101