摘要
猪作为目前世界上饲养最广泛的肉用家畜，其产肉性能一直是研究者们关注的焦点。本研究室在利用LongSAGE技术对不同品种和不同发育阶段的猪胚胎骨骼肌进行全基因组转录谱研究的过程中得到6个差异表达基因，其中5个基因均与线粒体NADH呼吸链的功能有关。骨骼肌是一种代谢旺盛的器官，其肌纤维中含有丰富的线粒体，而线粒体是细胞中产生能量的主要部位，与线粒体功能相关的基因必然与肌肉的生长和品质有关。但是，目前对线粒体相关基因的研究集中于大肠杆菌、牛和人，在猪上还没有进行相关研究的报道。因此，本研究选择NADH呼吸链相关基因，以及与人类心血管疾病相关的SMTN基因，以猪的骨骼肌为研究对象，用人×仓鼠体细胞杂种克隆板进行6个基因的染色体定位、CDS克隆和序列分析、用半定量RT-PCR进行时空表达谱分析、进行突变位点的筛选、并利用3个猪群的产肉性状资料进行基因型与性状的关联分析，以期初步了解这些基因的结构和功能，并为改进猪产肉性能的标记辅助选择提供资料。同时，利用3种小型猪群体的资料进行的性状关联分析结果可为其已经作为研究人类疾病的动物模型增添宝贵的种质特性与遗传学资料。
本研究的结果如下：
1．利用猪的EST信息分离了猪骨骼肌中差异表达的6个新基因，它们分别为NDUFAB1、NDUFS2、NDUFS3、NDUPS5、NDUFS7和SMTN，获得了基因的基因组DNA片段，并对其序列进行了分析；
2．用猪×仓鼠体细胞辐射杂种板(IMpRH)，对6个新基因进行了染色定位，分别将NDUFAB1基因定位于SSC3p，与标记S0174连锁，LOD值10.46；NDUFS2基因定位于SSC4q，与标记SW589连锁，LOD值8.52；NDUFS3定位于SSC2p，与标记SW1450连锁，LOD值13.46；NDUFS5定位于SSC6q，与标记SW1473连锁，LOD值11.14；NDUFS7定位于SSC2q21-q24，与标记SW395连锁，LOD值7.43；SMTN基因定位于SSC14q21-q22，与标记SW2612连锁，LOD值12.56。并与人的相应基因定位结果进行了符合性比较。
3．用猪的EST信息设计引物扩增，获得了6个基因的CDS，根据CDS序列对基因编码产物的结构和功能进行了预测，并对猪、人、牛和小鼠的相应基因进行了物种间进化树分析。NDUFAB1的CDS长471bp，NDUFS2的CDS长1392bp，NDUFS3的
CDS长795bp，NDUFS5的CDS长321bp，NDUFS7的CDS长648bp，SMTN的CDS长2475bp。
4．利用半定量RT-PCR技术研究了6个新基因在猪的骨骼肌、心、肝、脑、脾、淋巴结、肺、肾、大肠、小肠和脂肪等11种组织中的表达情况，以及在33d、65d、90d胚胎和成年猪骨骼肌中的表达情况。发现5个NADH相关基因在心、肝、脑、肾中的表达量都很高，而在骨骼肌中的表达量都较低。SMTN基因在脾中的表达量很高，而在骨骼肌、心、脑、肾中的表达量则较低。在不同发育阶段骨骼肌中的表达趋势则一致：6个基因在65d胚胎的骨骼肌中表达量最高，90d趋于降低，在33d和成年猪的骨骼肌中表达量很低，这证实65d左右的胚胎是肌纤维分化的最旺盛阶段，基因的表达水平较高，随后会逐渐降低。
5．利用DHPLC检测、EST拼接策略，对NDUFAB1、NDUFS3、NDUFS5和SMTN基因进行了多态位点的筛查，在NDUFAB1中发现一个G／A突变，可引起限制性内切酶MspAlI的切割位点改变；用DHPLC技术在NDUFS3中检测到1个Exon突变A／G，可引起氨基酸改变，有2个BFaI的酶切位点；在NDUFS5中检测到一个C／G突变；在SMTN中检测到一个C／T突变，该突变位于Exon，可引起氨基酸改变。
6．用DHPLC和酶切法对4个SNP在通城、大×长×通、长×大×通、大白、长白、贵州香猪、五指山猪和巴马香猪群体中的基因型频率和基因频率分布进行了检测，NDUFAB1基因的GG基因型频率0.526，GA基因型频率0.428，从基因型频率0.046；对于基因频率，大白猪群的A等位基因频率高，而通城猪和长白猪的A等位基因频率极低。NNDUFS3基因的从基因型频率0.699，AG基因型频率0.275，GG基因型频率0.026，A等位基因在各个群体中呈现优势分布；NDUFS5基因的CC基因型频率0.650，CG基因型频率0.312，GG基因型频率0.038，C等位基因显示出优势分布。可以得出结论：这3个基因的基因型呈严重的偏态分布。对于SMTN基因，TT基因型频率0.547，TC基因型频率0.267，CC基因型频率0.186，大×长×通群体中无TT基因型个体，五指山群体中无CC基因型个体，且CT基因型个体只有1个，贵州香猪群体中CC基因型个体只有3个，而巴马香猪群体中全部为TT基因型个体，因此对于基因频率，在地方品种中T等位基因分布占优势。
7．利用SPSS的GLM多因素方差分析法，对通城群体的15个生产性状指标进行了基因型和性状的关联分析。对于NDUFAB1基因，基因型与腿臀比率(Ham)极显著相关，其中GG基因型与GA基因型、GG基因型与AA基因型之间差异极显著，而GA与
AA基因型之间差异不显著。基因型与肌内脂肪含量(IMF)显著相关，其中GG基因型与GA基因型、GG基因型与AA基因型之间差异极显著，而GA与从基因型之间差异显著。基因型与其它性状无显著相关，但LSD比较发现平均日增重(ADG)、眼肌面积(LMA)、平均背膘厚(BF)、大理石纹评分(MS)、剪切力(Shear force)的基因型间两两比较有差异。对于NDUFS3基因：基因型与平均背膘厚趋于显著相关，其中AA与GG基因型差异极显著，AA与AG、AG与GG基因型差异显著。基因型与其它性状无显著相关，但LSD比较发现板油率(LFP)、平均背膘厚、肌内脂肪含量、屠宰率(DP)、腿臀比率和眼肌面积的基因型间两两比较有差异。对于NDUFS5基因：基因型与腿臀比率极显著相关，但基因型之间的差异不显著；基因型与达90kg日龄(Age)趋于显著相关，各基因型之间差异不显著。对于SMTN基因：基因型与腿臀比率极显著相关。基因型与平均日增重、肌内脂肪含量、平均背膘厚、屠宰率、眼肌面积无显著相关，但各基因型之间有差异。T检验法显示，血液生理生化指标甘油三酯(TG)、总胆固醇(CHOL)、高密度脂蛋白胆固醇(HDL—C)、低密度脂蛋白胆固醇(LDL—C)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、谷氨酰转肽酶(GGT)、C反应蛋白(CRP)、平均红细胞血红蛋白浓度(MCHC)的基因型间差异极显著，而载脂蛋白B(APO-B)、白细胞数(WBC)、大未染色细胞(LVC)、淋巴细胞百分数(LYMPH％)指标的基因型间差异显著，肌酐(CREA)的基因型间差异趋于显著，其它指标的基因型间差异不显著。
Swine is the most dominating domestic animals feeding for meat production in the world. The meat production performance is the investigative emphasis of investigators including our lab at all times. We found 6 genes differential expression in the skeletal muscle of porcine fetus when we study the porcine genome transcription profile in different breeds and different developmental stages using LongSAGE. 5 among the 6 genes are related to the function of NADH respiratory chain of mitochondrion. As a kind of active organ of metabolism, skeletal muscle contains abundant mitochondrion. Since mitochondrion is the primary organelle productting energy in cells, the genes related to the function of mitochondrion would be associated with the growth and quality of skeletal muscle. However, the study of genes related to mitochondrion is focused on E.coli, cattle and human, and there has no report on swine. Therefore, we selected the 5 genes, as well as SMTN gene related to human cardiovascular diseases, for chromosomal mapping, CDS cloning and analysis, spatio-temporal distribution, mutation riddling, and association analysis with production traits of 3 porcine populations. We expect to know the structure and function of these 6 genes primarily, and supply data for porcine marker assistant selection of improving meat production. Meanwhile, the results of association analysis of 3 pint-sized swine may supply valuable genetics data of germ plasm characteristics for the study of animal model of human disease.
The results of this study as follows:
1. The 6 genes are isolated using porcine EST information, namely NDUFAB1, NDUFS2、NDUFS3、NDUFS5、NDUFS7 and SMTN gene, the genome DNA segments of 6 genes are cloned and the sequence analysis are completed.
2. The 6 genes are mapped on porcine chromosome using IMpRH respectively, NDUFAB1 mapped on SSC3p, linked with marker S0174, the LOD value 10.46; NDUFS2 mapped on SSC4q, linked with marker SW589, the LOD value 8.52; NDUFS3 mapped on SSC2p, linked with marker SW1450, the LOD value 13.46; NDUFS5 mapped on SSC6q, linked with marker SW1473, the LOD value 11.14; NDUFS7 mapped on SSC2q21-q24, linked with marker SW395, the LOD value 7.43; SMTN mapped on SSC14q21-q22, linked with marker SW2612, the LOD value 12.56. The results of mapping are consistent with the results of human corresponding genes.
3. The CDS of 6 porcine genes are obtained. The structure and function of coding products of 6 genes are predicted according to the CDS, and the phylogenetic relationship of 6 genes are analysed among swine, human, cow and rat. The results show that the CDS length of NDUFAB1 is 471bp, NDUFS2 1392bp, NDUFS3 795bp, NDUFS5 321bp, NDUFS7 648bp, and SMTN 2475bp respectively.
4. By semi-quantitative RT-PCR, the expressive level of 6 porcine genes are studied in skeletal muscle, heart, liver, brain, spleen, lymph node, lung, kidney, large intestine, small intestine, fat tissue of grown pigs, and the skeletal muscle of 33d fetuses, 65d fetuses, 90d fetuses, and grown pigs. The results indicate that the expressive levels of 5 mitochondrion genes are higher in heart, liver, brain and kidney, while lower in skeletal muscle. The expressive level of SMTN gene is higher in spleen, whereas lower in skeletal muscle, heart, brain and kidney. The changing trend of expressive level of 6 genes is similar in different stages skeletal muscle, that is, the expressive level is higher in the skeletal muscle of 65d fetuses, lower in 90d fetuses, very low in 33d fetuses and grown pigs. This improves that 65d post coitus is the most active stage of differentiation of muscle fibre, with higher expressive level of genes, and then the expressive level tends to lower.
5. The single nucleotide polymorphism of NDUFAB1、NDUFS3、NDUFS5 and SMTN gene are detected with DHPLC, EST aligning and sequencing. A G/A nucleotide mutation of NDUFAB1was found, which changed the cutting site of MspA1I. A G/A nucleotide mutation of NDUFS3 was detected with DHPLC in an exon region, which leads to the change of amino acid. A G/A nucleotide mutation of NDUFS5 was found in the 5'-UTR, and a C/T mutation of SMTN in an exon.
6. The genotype frequency of 4 genes are detected in the pig populations of Tongcheng, White×Landrance×Tong, Large White, Landrance, Guizhou, Wuzhishan and Bama with DHPLC and enzyme cutting. The GG genotype frequency of NDUFAB1 is 0.526, GA 0.428, and AA 0.046 respectively. The frequency of allele A is higher in Large White pig population, whereas very low in Tongcheng and Landrance pig populations. The AA genotype frequency of NDUFS3 is 0.699, AG 0.275, and GG 0.026 respectively. The frequency of A allele is higher among every populations. The CC genotype of NDUFS5 is 0.650, CG 0.312, and GG 0.038 respectively. The C allele show preponderant distribution among every population. The conclusion can be drawed subsequently that the genotype of 3 genes shows serious leaning distribution. The TT genotype of SMTN gene is 0.547, TC 0.267, and CC 0.186. There has no TT genotype in White×Landrance×Tong population, no CC genotype in Wuzhishan population, and no CC and CT genotype in Bama population. There are 1 CT genotype sample in Wuzhishan and 3 CC genotype samples. Therefore, the T allele shows preponderant distribution among local breeds.
7. The correlative analysis of 4 genes was carried out with General Linear Model of SPSS software. The genotype of NDUFAB1 gene is highly significantly correlative with percentage of ham (Ham), while significantly correlative with intramuscular fat (IMF). The differences are highly significant between GG genotype and the other two genotypes for the two traits, while no significant difference been found between GA and AA for the percentage of ham, and significant difference between GA and AA for IMF. There have no significant correlation between genotype and the other traits, while there have difference between genotypes for average daily gain from birth to market (ADG), backfat thickness (BF), loin-muscle area (LMA), marbling score (MS) and shear force.
The genotype of NDUFS3 gene tends to be significantly correlative with BF. There are highly significant difference between AA and GG genotype, while significant difference between AG and the other two genotypes. The genotype has no significant correlation with other traits, but the results of LSD analysis show that there have differences between genotypes for percentage of leaf fat (LFP), percentage of intramuscular fat (IMF), dressing percent (DP), Ham and LMA.
There are highly significant correlation between genotype of NDUFS5 gene and Ham, while no significant difference exists between genotypes. The genotype tends to be significantly correlative with days of age at an ideal market weight (Age).
The genotype of SMTN gene is highly significant correlative with Ham, while there have differences between genotypes for ADG, IMF, BF, DP and LMA. The results of T-test show that the difference is very significant between 2 genotypes for triglyceride (TG)、cholesterol (CHOL)、high density lipoprotein cholesterol (HDL-C)、low density lipoprotein cholesterol (LDL-C)、alanine amiotransferase (ALT)、alkaline phosphatase (ALP)、γ-glutamyl transpeptadase (GGT)、c-reactive protein (CRP) and mean erythrocytes hemoglobin concentration (CHCM), while there have significant difference between 2 genotypes for apolipoprotein-B (APO-B)、white blood cell count (WBC)、LVC and lymphocyte percent (LYMPH%). Creatinine (CREA) tends to be significant difference. There have no defference between genotypes for the other traits.
引文
1．蔡诚忠，许洪卫，谭龙益．分时同步循环半定量RT-PCR检测胃癌细胞E-钙粘附素表达的方法学建立．中国肿瘤临床，2006，33(8)：424—426
2．陈启龙．RACE技术的研究进展及其应用．黄山学院学报，2006，8(3)：95—98
3．戴茹娟，吴常信．DNA标记及其在家禽遗传育种中的应用．中国畜牧杂志，1996，32(5)：55—57
4．邓亚军，童维，陈雁炯，胡松年，李生斌．猪脂肪组织表达序列标签(ESTs)大规模测序及分析．遗传学报，2004，31(11)：1211—1217
5．甘世祥．贵州小型猪实验用小香猪[M]．贵阳：贵州科技出版社，1997，1—17
6．贺福初，鱼咏涛，魏汉东，翟云，瞿祥虎．一种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究．遗传，2001，6：503—510
7．黄中波，牛荣，魏泓，甘世祥．STR-PCR方法检测贵州小型香猪遗传变异性的研究．第三军医大学学报，2000，22(7)：642—644
8．姜玉武，黄奕辉，秦炯，袁云，戚豫，肖江喜，杨艳玲，吴希如．儿童Leigh综合征的临床、神经病理及分子遗传学研究．中华儿科杂志，2001，39(6)：330—334
9．李重生，陈瑶生，王翀，李加琪．猪肌肉组织差异表达ESTs的克隆与分析．中国科学C辑，2006，36(2)：139—144
10．李刚，胡迎春，张开泰，吴德昌．“电子”cDNA文库筛选指导基因的全长cDNA克隆．生物技术通讯，2000，11(1)：1—4
11．李宁，赵志辉，刘兆良，赵兴波，连正兴，吴常信．猪肝脏组织表达序列标签(ESTs)的初步分析．中国农业科学，2002，12：1525—1528
12．Livak K J，Schmittgen T D．利用实时定量PCR技术通过2-△△CT方法分析相对基因表达差异．METHODS，2001，25：402—408
13．李祥龙，巩元芳，刘铮铸，张建文，Valentini A．利用D H PLC研究我国几个地方绵羊品种黑素细胞刺激素受体基因单核苷酸多态性．遗传，2004，26(6)：841—844
14．李振有，贾允英．动脉粥样硬化机理新说．武警医学，2001，12(2)：112—113
15．刘润幸，邝建．医学统计方法与应用(上册，基础统计)．广州：广东人民出版社，2001，139—160
16．刘祖洞．遗传学(下册)．北京：高等教育出版社，1979
17．卢大学，万文君．力量素质及其发展．西安体育学院学报，2002，19(3)：50-60
18．闾宏伟，杨向东，何淑雅，杨永宗．cDNA文库基础上运用热启动聚合酶链反应末端延伸快速分离全长cDNA序列．中国动脉硬化杂志，2002，10(5)：396—399
19．孟斌，陈风．C-反应蛋白对心血管病的预测作用．中西医结合心脑血管病杂志，2006，4(9)：797—798
20．单体中，汪以真，李民．猪脂蛋白脂酶基因片段的克隆及不同体重的表达差异．农业生物技术学报，2006，14(2)：151—155
21．宋元宗，王自能，柳国胜，刘新．Leigh综合征肌肉超微结构观察．暨南大学学报(自然科学与医学版)，2004，25(2)：235—236，239
22．唐中林，万平，李小平，李勇，赵书红，刘榜，李奎．猪胚胎骨骼肌LongSAGE文库的构建．农业生物技术学报，2007，15(1)：171-172
23．王翀，陈瑶生，李重生，黄志宏，田兴国．DHPLC对猪肌肉组织差异表达EST的鉴定．遗传学报，2003，30(12)：1085—1089
24．王忠华，孙玉民，谢幼梅，夏广德，李同树，曾勇庆．新浦东、AA肉鸡肉用性能的比较．山东农业大学学报，1992，23(4)：368-374
25．王昕，陈宏，曹红鹤．中国10个地方猪种的群体近交程度分析．遗传，2006，28(10)：1229—1232
26．王彦芳．猪PA28和PA700基因家族相关基因的分离、定位、SNPs检测及其与性状的关联分析．[博士学位论文]．武汉：华中农业大学图书馆，2004
27．王延云，徐亚欧，胡强，於建国，袁列，杨德孝．SNP研究技术在畜牧业中的应用．畜禽业，2005，5：16—17
28．无．脂肪肝可能是心血管病的独立危险因子．高血压杂志，2006，14(7)：587
29．吴开平，吴丰春，魏泓，王爱德，甘世祥，曾养志．五种品系猪亲缘关系的RAPD分析．遗传，2000，22(4)：217—220
30．杨金娥．猪12号染色体上10个新基因的分离、定位及其与部分性状的关联分析．[博士学位论文]．武汉：华中农业大学图书馆，2004
31．张曼，安成，郝林，许华林，张敏，张秀敏．细胞分裂周期蛋白基因6半定量检测方法的条件建立．中华检验医学杂志，2003，26(4)：197—199
32．赵志辉，刘兆良．鸡下丘脑cDNA文库的构建及部分克隆ESTs序列初步分析．遗传学报，2001，28(4)：301—305
33．周顺伍．动物生物化学．北京：农业出版社，1999
34．朱正茂，赵书红，陈浩，余梅，刘榜，樊斌，朱猛进，李奎．基于cDNA宏阵列的系统聚类分析猪发育阶段的基因表达谱．武汉大学学报(理学版)，2004，50(4)：482—486
35．左波，熊远著，苏玉虹，邓昌彦，余雳，郑嵘．猪4号和7号染色体部分微卫星位点的遗传图谱构建．农业生物技术学报，2003，11(1)：70—74
36. Ahlers P M, Garofano A, Kerscher S J, Brandt U. Application of the obligate aerobic yeast Yarrowia lipolytica as a eucaryotic model to analyse Leigh syndrome mutations in the complex Ⅰ core subunits PSST and TYKY. Biochim Biophys Acta Bioenergetics, 2000, 1459(2): 258-265
37. Asanumaa M, Ogawa N, Hirata H, Choua H, Kondo Y, Mori A. Ischemia induced changes in a-tubulin and β-actin mRNA in the gerbil brain and effects of bifemelane hydrochloride. Brain Res, 1993, 600(2): 243-248
38. Augenlicht L H, Bordonaro M, Heerdt B G, Mariadason J, Velcich A. Cellular mechanisms of risk and transformation. Ann N Y Acad Sci, 1999, 889: 20-31
39. Bai Y, Shakeley R M, Attardi G. Tight control of respiration by NADH dehydrogenase ND5 subunit gene expression in mouse mitochondria. Mol Cell Biol, 2000, 20(3): 805-815
40. Belogrudov G, Hatefi Y. Catalytic sector of complex Ⅰ(NADH: ubiquinone oxidoreductase): subunit stoichiometry and substrate-induced conformation changes. Biochemistry, 1994, 33: 4571-4576
41. Benit P, Slama A, Cartault F, Giurgea I, Chretien D, Lebon S, Marsac C, Munnich A, Rotig A, Rustin P. Mutant NDUFS3 subunit of mitochondrial complex Ⅰ causes Lei-gh syndrome. J Med Genet, 2004, 41(1): 14-17
42. Boneh A. Regulation of mitochondrial oxidative phosphorylation by second messenger-mediated signal transduction mechanisms. Cell Mol Life Sci, 2006, 63(11): 1236-1248
43. Bramham J, Hodgkinson J L, Smith B O, Uhrin D, Barlow P N, Winder S J. Solution structure of the calponin CH domain and fitting to the 3D-helical reconstruction of F-actin: calponin. Structure, 2002, 10(2): 249-258
44. Brandt U. Energy Converting NADH: Quinone Oxidoreductase (compound Ⅰ). Annu Rev Biochem, 2006, 75: 69-92
45. Chen J, Kitchen CM, Streb J W, Miano J M. Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol, 2002,34 (10): 1345-1356
46. Chol M, Lebon S, Benit P, Chretien D, de Lonlay P, Goldenberg A, Odent S, Hertz-Pannier L, Vincent-Delorme C, Cormier-Daire V, Rustin P, Rotig A, Mun-nich A. The mitochondrial DNA G13513A MELAS mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh-like syndrome with isolated complex I deficiency. J Med Genet, 2003,40 (3): 188-191
47. Ciobanu D C, Bastiaansen J W, Lonergan S M, Thomsen H, Dekkers J C, Plastow G S, Rothschild M F. New alleles in calpastatin gene are associated with meat quality traits in pigs. J Anim Sci, 2004, 82 (10): 2829-2839
48. Clark M A, Baumann L, Baumann P. Buchnera aphidicola (endosymbiont of aphids) contains nuoC (D) genes that encode subunits of NADH dehydrogenase. Curr Micro-biol, 1997, 35 (2):122-123
49. Crimi M, Bordoni A, Menozzi G, Riva L, Fortunato F, Galbiati S, Del Bo R, Pozzoli U, Bresolin N, Comi G P. Skeletal muscle gene expression profiling in mitochondrial disorders. FASEBJ, 2005,19 (7): 866-868
50. Cronan J E, Fearnley I M, Walker J E. Mammalian mitochondria contain a soluble acyl carrier protein. FEES Lett, 2005,579 (21): 4892-4896
51. Darrouzet E, Issartel J P, Lunardi J, Dupuis A. The 49-kDa subunit of NADH-ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone-related inhibitors. FEBS Lett, 1998, 431 (1): 34-38
52. de Koning D J, Janss L L G, Rattink A P, van Oers P A M, de Vries B J, Groenen M A M, van der Poel J J, de Groot P N, Brascamp E W, van Arendonk J A M. Detection of Quantitative Trait Loci for Backfat Thickness and Intramuscular Fat Content in Pigs (Sus scrofa). Genetics, 1999,152: 1679-1690
53. de Koning D J, Harlizius B, Rattink A P, Groenen M A M, Brascamp E W, van Arendonk J A M. Detection and characterization of quantitative trait loci for meat quality traits in pigs. J Anim Sci, 2001, 79: 2812-2819
54. Duborjal H, Beugnot R, De Camaret B M, Issartel J P. Large functional range of steady-state levels of nuclear and mitochondrial transcripts coding for the subunits of the human mitochondrial OXPHOS system. Genome Res, 2002,12 (12): 1901-1909
55. Duehlmeier R, Sammet K, Widdel A, von Engelhardt W, Wernery U, Kinne J, Sal-lmann HP. Distribution patterns of the glucose transporters GLUT4 and GLUT1 in skeletal muscles of rats (Rattus norvegicus), pigs (Sus scrofa), cows (Bos taurus), adult goats, goat kids (Capra hircus), and camels (Camelus dromedarius). Comp Biochem PhysiolA Mol Integr Physiol, 2007,146 (2):274-282
56. Emahazion T, Beskow A, Gyllensten U, Brookes A J. Intron based radiation hybrid mapping of 15 complex I genes of the human electron transport chain. Cytogenet Cell Genet, 1998, 82 (1-2): 115-119
57. Engelen J J, Esterling L E, Albrechts J C, Detera-Wadleigh S D, van Eys G J. Assignment of the human gene for smoothelin (SMTN) to chromosome 22ql2 by fluorescence in situ hybridization and radiation hybrid mapping. Genomics, 1997, 43 (2): 245-247
58. Ernst C W, Robic A, Yerle M, Wang L, Rothschild M F. Mapping of calpastatin and three microsatellites to porcine chromosome 2q2.1-q2A. Anim Genet, 1998, 29 (3): 212-215
59. Estrade M, Ayoub S, Talmant A, Monin G. Enzyme activities of glycogen metabolism and mitochondfial characteristics in muscles of RN carrier pigs(Sus scrofa domesticus). Comparative Biochemistry and Physiology Biochemistry and Molecular Biology, 1994,108 (3): 295-301
60. Flemming D, Schlitt A, Spehr V, Bischof T, Friedrich T. Iron-sulfur cluster N2 of the Escherichia coli NADH: ubiquinone oxidoreductase (complex I) is located on subunit NuoB. J Biological Chemical, 2003a, 278 (48): 47602-47609
61. Flemming D, Hellwig P, Friedrich T. Involvement of tyrosines 114 and 139 of subunit NuoB in the proton pathway around cluster N2 in Escherichia coli NADH: ubiquinone oxidoreductase. J Biological Chemical, 2003b, 278 (5): 3055- 3062
62. Flemming D, Stolpe S, Schneider D, Hellwiq P, Friedrich T. A possible role for iron-sulfur cluster N2 in proton translocation by the NADH: ubiquinone oxidoreductase (complex I). J Molecular Microbiological Biotechnology, 2005, 10 (2-4): 208-222
63. Flemming D, Hellwig P, Lepper S, Kloer D P, Friedrich T. Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH: ubiquinone oxidoreductase (complex I). J Biological Chemical, 2006, 281 (34): 24781- 24789
64. Galante Y M, Hatefi Y. Purification and molecular and enzymic properties of mit-ochondrial NADH dehydrogenase. Arch Biochem Biophys, 1979,192: 559-568
65. Georges M, Andersson G, Braunschweig M, Buys N, Collette C, Moreau L, Nezer C, Nguyen M, Van Laere A S, Andersson L. Genetic dissection of an imprinted QTL mapping to proximal SSC2. Plant & Animal Genomes XI Conference, 2003, San Diego, CA
66. Gerald Karp. Cell and Molecular Biology: concepts and experiments, 2nd Edition. 1999, NEW York: John Wiley & Sons Inc.
67. Gimona M, Djinovic-Carugo K, Kranewitter W J, Winder S J. Functional plasticity of CH domains. FEBS Let, .2002,513 (1): 98-106
68. Grand R J. Acylation of viral and eukaryotic proteins. J Biochemical, 1989, 258 (3): 625-638
69. Grgic L, Zwicker K, Kashani-Poor N, Kerscher S, Brandt U. Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I. J Biol Chem, 2004, 279 (20): 21193-21199
70. Grindflek E, Szyda J, Liu Z, Lien S. Detection of quantitative trait loci for meat quality in a commercial slaughter pig cross. Mammalian Genome, 2001,12: 299- 304
71. Gross E, Arnold N, Goette J,Schwarz-Boeger U, Kiechle M. A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC. Human Genetic, 1999,105 (1-2): 72-78
72. Gross E, Arnold N, Pfeifer K, Bandick K, Kiechle M. Identification of specific BRCAl and BRCA2 variants by DHPLC. Human Mutaton, 2000,16 (4): 345- 353
73. Harald Bar, Wende P, Watson L, Denger S, van Eys G, Kreuzer J, Jahn L. Smoot-helin is an indicator of reversible phenotype modulation of smooth muscle cells in balloon-injured rat carotid arteries. Basic Research in Cardiology, 2002, 97 (1): 9-16
74. Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem, 1985,54: 1015-1069
75. Hinttala R, Uusimaa J, Remes A M, Rantala H, Hassinen I E, Majamaa K. Sequence analysis of nuclear genes encoding functionally important compound I subunits in children with encephalomyopathy. Mol Med, 2005, 83 (10): 786-794
76. Hiraiwa H, Sawazaki T, Suzuki K, Fujishima-Kanaya N, Toki D, Ito Y, Uenishi H, Hayashi T, Awata T, Yasue H. Elucidation of correspondence between swine chromosome 4 and human chromosome 1 by assigning 27 genes to the ImpRH map, and development of microsatellites in the proximity of 14 genes. Cytogen etic and Genome Research, 2003, 101 (1): 84-89
77. Hu R M, Han ZG, Song H D, Peng Y D, Huang Q H, Ren S X, Gu Y J, Huang C H, Li Y B, Jiang C L, Fu G, Zhang Q H, Gu B W, Dai M, Mao Y F, Gao G F, Rong R, Ye M, Zhou J, Xu S H et al. Gene expression profiling in the human ypoth- alam- us-pituitary-adrenal axis and full-length cDNA cloning. Proc Natl Acad Sci U S A, 2000, 7 (17): 9543-9548
78. Huang G, Chen Y, Lu H, Cao X. Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death. Cell Death Differ, 2007,14 (2): 327-337
79. Hyslop S J, Duncan A M V, Pitkanen S, Robinson B H. Assignment of the PSST Subunit Gene of Human Mitochondrial Complex I to Chromosome 19pl3. Genomics, 1996, 37 (3): 375-380
80. Iuso A, Scacco S, Piccoli C, Bellomo F, Petruzzella V, Trentadue R, Minuto M, Ripoli M, Capitanio N, Zeviani M, Papa S. Dysfunctions of Cellular Oxidative Metabolism in Patients with Mutations in the NDUFS1 and NDUFS4 Genes of Complex I. J Biol Chem, 2006,281 (15): 10374-10380
81. Jorgenson J W, Lukacs K D. Zone electrophoresis in open tubular class capillary. Analycal Chemistry, 1981,1298-1302
82. Kavaler E M D, Landman J M D, Chang Y B A, Droller M J, Liu B C-S M D. Detecting human bladder carcinoma cells in voided urine samples by assaying for the presence of telomerase activity. Cancer, 1998, 82 (4): 708-714
83. Kawasaki H, Kretsinger R.H. Calcium-binding proteins 1: EF-hands. Protein Prof, 1995, 2: 305-490
84. Kelley D E, He J, Menshikova E V, Ritov V B. Dysfunction of Mitochondria in Human Skeletal Muscle in Type 2 Diabetes. Diabetes, 2002, 51: 2944-2950
85. Kempermann G, Chesler E J, Lu L, Williams R W, Gage F H. Natural variation and genetic cova riance in adult hippocampal neurogenesis. Proc Natl Acad Sci U S A, 2006,103 (3): 780-785
86. Kerscher S, Kashani-Poor N, Zwicker K, Zickermann V, Brandt U. Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics. J Bioenerg Biomembr, 2001, 33 (3): 187-196
87. Kim S H, Fountoulakis M, Dierssen M, Lubec G. Decreased protein levels of complex 130-kDa subunit in fetal Down syndrome brains. J Neural Transm Suppl. 2001, (61): 109-116
88. Kirby D M, Salemi R, Sugiana C, Ohtake A, Lee P, Bell K M, Kirk E P, Boneh A, Taylor R W, Dahl H H M, Ryan M T, Thorburn D R. NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J Clin Invest, 2004,114 (6): 837-845
89. Kisby G E, Olivas A, Standley M, Lu X, Pattee P, O'Malley J, Li X, Muniz J, Nagalla S R. Genotoxicants Target Distinct Molecular Networks in Neonatal Neurons. Environ Health Perspect, 2006,114 (11): 1703-1712
90. Krzecio E, Kury J, Kocwin-Podsiadla M, Monin G Association of calpastatin (CAST/MspI) polymorphism with meat quality parameters of fatteners and its interaction with RYR1 genotypes. J Anim Breed Genet, 2005,122 (4): 251-258
91. Kulinskii V I, Kolesnichenko L S. Regulation of metabolic and energetic mitochondrial functions by hormones and signal transduction systems. Biomed Khim, 2006, 52 (5): 425-447
92. Le Roy P, Naveau J, Elsen J M, Sellier P. Evidence for a new major gene influencing meat quality in pigs. Genet Res, 1990,55: 33-40
93. Lefaucheursiov I. Myofiber typing and pig meat production. Slov Vet Res, 2001, 38 (1): 5-33
94. Lee S S, Chen Y, Moran C, Cepica S, Reiner G, Bartenschlager H, Moser G, Geldermann H. Linkage and QTL mapping for Sus scrofa chromosome 2. Journal of Animal Breeding Genetic, 2003,120 (Suppl. 1): 11-19
95. Lee Y J, Jeong S Y, Karbowski M, Smith C L, Youle R J. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opal in apoptosis. Mot Biol Cell, 2004,15 (11): 5001-5011
96. Leeuwenburgh C, Judge S. Cardiac Mitochondrial Bioenergetics, Oxidative Stress and Aging. Am J Physiol Cell Physiol, 2007, Mar 7, [Epub ahead of print]
97. Li J, Bai Y, Ianakova E, Grandis M, Uchwat F, Trostinskaia A, Krajewski K M, Garbern J, Kupsky W J, Shy M E. Major myelin protein gene (P0) mutation causes a novel form of axonal degeneration. J Comp Neurol, 2006, 498 (2): 252-265
98. Lion T. Control genes in reverse transcriptase-polymerase chain reaction assays. Leukemia, 1996,10: 1527-1528
99. Loeffen J, van den Heuvel L, Smeets R, Triepels R, Sengers R, Trijbels F, Smeitink J. cDNA sequence and chromosomal localization of the remaining three human nuclear encoded iron sulphur protein (IP) subunits of complex I: the human IP fraction is completed. Biochem Biophys Res Commun, 1998, 247 (3): 751- 758
100. Loeffen J, Smeets R, Smeitink J, Triepels R, Sengers R, Trijbels F, van den Heuvel L. The human NADH: ubiquinone oxidoreductase NDUFS5 (15 kDa) subunit: cDNA cloning, chromosomal localization, tissue distribution and the absence of mutations in isolated complex I-deficient patients. J Inherit Metab Dis, 1999, 22 (1): 19-28
101. Loeffen J, Elpeleg O, Smeitink J, Smeets R, Stockler-Ipsiroglu S, Mandel H, Sengers R, Trijbels F, van den Heuvel L. Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol, 2001, 49: 195-201
102. MacLennan D H. The genetic basis of malignant hyperthermia. Trends Pharmacol Sci, 1992, (13): 330-334
103. Malek M, Dekkers J C M, Lee H K, Baas T J, Rothschild MF. A molecular genome scan analysis to identify chromosomal regions influencing economic traits in the pig. I. Growth and body composition. Mamma lian Genome, 2001,12: 630- 636
104. Marone M, Mozzetti S, de Ritis D. Semi-quantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. J Biol Proc Online, 2001, 3 (1): 19-25
105. Martin M A, Blazquez A, Gutierrez-Solana L G, Fernandez-Moreira D, Briones P, Andreu A L, Garesse R, Campos Y, Arenas J. Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene. Arch Neurol, 2005, 62 (4): 659-661
106. McFarland R, Kirby D M, Fowler K J, Ohtake A, Ryan M T, Amor D J, Fletcher J M, Dixon J W, Collins F A, Turnbull D M, Taylor R W, Thorburn D R. De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial en-cephalopathy and complex I deficiency. Ann Neurol, 2004, 55: 58-64
107. Medhurst AD, Harrison D C, Read S J, Campbell C A, Robbins M J, Pangalos M N. The use of TaqMan RT-PCR assays for semiq- uantitative analysis of gene expression in CNS tissues and disease models. J Neuroscience Methods, 2000, 98 (1): 9-20
108. Nguyen M H, Dudycha S J, Jafri M S. The Effects of Ca~(2+) on Cardiac Mitochon- drial Energy Production is Modulated by Na~+ and H~+ Dynamics. Am J Physiol Cell Physiol, 2007, Mar 7, [Epub ahead of print]
109. OVilo C, Oliver A, Noguera J L, Clop A, Barragan C, Varona L, Rodriguez C, Toro M, Sanchez A, Perez-Enciso M, Silio L. Test for positional candidate genes for body composition on pig chromosome 6. Genet Sel Evol, 2002,34 (4): 465- 479
110. Prieur I, Lunardi J, Dupuis A. Evidence for a quinone binding site close to the interface between NUOD and NUOB subunits of Complex I. Biochimica et Biophysica Acta (BBA) Bioenergetics, 2001,1504 (2-3): 173-178
111. Procaccio V, de Sury R, Martinez P, Depetris D, Rabilloud T, Soularue P, Lunardi J, Issartel J. Mapping to 1q23 of the human gene (NDUFS2) encoding the 49 KDa subunit of the mitochondrial respiratory Complex I and immunodetection of the mature protein in mitochondria. Mamtn Genome, 1998, 9 (6): 482-484
112. Procaccio V, Lescuyer P, Bourges I, Beugnot R, Duborjal H, Depetris D, Mousson B, Montfort M F, Smeets H, De Coo R, Issarte J P. Human NDUFS3 gene coding for the 30-kDa subunit of mitochondrial Complex I: genomic organization and expression. Mammalian Genome, 2000,11 (9): 808-810
113. Quensel C, Kramer J, Cardoso M C, Leonhardt H. Smoothelin contains a novel actin cytoskeleton localization sequence with similarity to troponin T. J Cell Biochem, 2002, 85 (2): 403-409
114. Rensen S S, Thijssen V L, De Vries C J, Doevendans P A, Detera Wadleigh S D, Van Eys G J. Expression of the smoothelin gene is mediated by alternative promoters. Cardiovasc Res, 2002, 55 (4): 850-863
115. Rensen S S, Niessen P M, Long X, Doevendans P A, Miano J M, van Eys G J. Contribution of serum response factor and myocardin to transcriptional regulation of smoothelins. Cardiovasc Res, 2006, 70 (1): 136-145
116. Rice C I, Whitehead R. Phis Chem, 1965, 69: 4017
117. Roher G A, Keele J. Identification of quantitative trait loci affecting carcass composition in swine II Muscling and wholesale product trait yield traits. J Anim Sci, 1998, 76: 2255-2262
118. Runswick M J, Fearnley I M, Skehel J M, Walker J E. Presence of an acyl carrier protein in NADH: ubiquinone oxidoreductase from bovine heart mitochondria. FEBS Lett, 1991, 286 (1-2): 121-124
119. Ruoslahti E, Pierschbacher M D. Arg-Gly-Asp: A versatile cell recognition signal. Cell, 1986,44 (4): 517-518
120. Schneider R, Massow M, Lisowsky T, Weiss H. Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactiveation of the gene encoding the mitochondrial acyl carrier protein. Curr Genet. 1995, 29: 10 -17
121. Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, Stockler-Ipsiroglu S, van den Heuvel L. Mutant NDUFVl subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet, 1999, 21: 260- 261
122. Schuler F, Casida J E. Functional coupling of PSST and ND1 subunits in NADH: ubiquinone oxidoreductase established by photoaffinity labeling. Biochim Biophys Acta, 2001,1506 (1): 79-87
123. Shoubridge E A. Nuclear genetic defects of oxidative phosphorylation. Hum Mol Genet, 2001,10: 2277-2284
124. Skehel J M, Fearnley I M, Walker J E. NADH: ubiquinone oxidoreductase from bovine heart mitochondria: sequence of a novel 17.2-kDa subunit. FEBS Lett, 1998, 438: 301-305
125. Smeitink J, van den Heuvel L. Human Mitochondrial compound I in Health and Disease. Am J Hum Genet, 1999, 64:1505-1510
126. Tharp D L, Wamhoff B R, Turk J R, Bowles D K. Upregulation of intermediate conductance Ca~(2+)-activated K~+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle. Am J Physiol Heart Circ Physiol, 2006, 291 (5): H2493 -2503
127. Thomsen H, Lee H K, Rothschild M F, Malek M, Dekkers J C M. Characterization of quantitative trait loci for growth and meat quality in a cross between commercial breeds of swine. J Anim Sci, 2004, 82: 2213-2228
128. Tondera D, Czauderna F, Paulick K, Schwarzer R, Kaufmann J, Santel A. The mitochondrial protein MTP18 contributes to mitochondrial fission in mammalian cells. J Cell Sci, 2005, 118 (Pt 14): 3049-3059
129. Triepels R H, van den Heuvel L P, Loeffen J L, Buskens C A, Smeets R J, Rubio Gozalbo M E, Budde S M, Mariman E C, Wijburg F A, Barth P G, Trijbels J M, Smeitink J A. Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I. Ann Neurol, 1999a, 45 (6): 787- 790
130. Triepels R, Smeitink J, Loeffen J, Smeets R, Buskens C, Trijbels F, van den Heuvel L. The human nuclear-encoded acyl carrier subunit (NDUFAB1) of the mitochon-drial complex I in human pathology. J Inherit Metab Dis, 1999b, 22 (2): 163-173
131. Ugalde C, Triepels R H, Coenen M J, van den Heuvel L P, Smeets R, Uusimaa J, Briones P,Campistol J, Majamaa K, Smeitink J A, Nijtmans LG. Impaired complex I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene. Ann Neurol, 2003, 54: 665-669
132. Ugalde C, Janssen R J, van den Heuvel L P, Smeitink J A, Nijtmans L G Differences in assembly or stability of compound I and other mitochondrial OXPHOS complexes in inherited compound I deficiency. Hum Mol Genet, 2004,13 (6): 659- 667
133. van der Loop F T, Schaart G, Timmer E D, Ramaekers F C, van Eys G J. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol, 1996, 134 (2): 401-411
134. van Wijk H J, Dibbits B, Baron E E, Brings A D, Harlizius B,Groenen M A, Knol E F, Bovenhuis H. Detection of quantitative trait loci for carcass composition and pork quality traits in a commercial finishing cross. J Anim Sci, 2006, 84 (4): 789-799
135. Wagenknecht D, Bartenschlager H, Van Poucke M, Geldermann H, Peelman L J, Majzlik I, Stratil A. Linkage and radiation hybrid mapping of the porcine MPZ gene to chromosome 4q. Animal Genetics, 2005,36 (2): 181-182
136. Walker J E. The NADH: ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys, 1992, 25: 253-324
137. Weidner U, Geier S, Ptock A, Friedrich T, Leif H, Weiss H. The gene locus of the proton translocating NADH: ubiquinone oxidoreductase in Escherichia coli: organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex 1.7 Mol Biol, 1993, 233: 109-122
138. Wu Y Q, Wu J S, Zhao X F, Guo X L, Xu N Y. Correlation between porcine CAST gene polymorphism with muscle fiber histological traits and carcass characteristics. Yi Chuan, 2007, 29 (1): 65-69
139. Yanai I, Benjamin H, Shmoish M, Chalifa-Caspi V, Shklar M, Ophir R, Bar-Even A, Horn-Saban S, Safran M, Domany E, Lancet D, Shmueli O. Genome wide midrange transcription profiles reveal expression level relationships in human tissue specification. Bioinformatics, 2005, 21 (5): 650-659
140. Yano A, Kubota M, Iguchik K, Usui S, Hirano K. Buformin Suppresses the Expression of Glyceraldehyde 3- Phosphate Dehydrogenase. Bio Pharm Bull, 2006, 29 (5): 1006-1009
141. Yano T, Ohnishi T. The origin of cluster N2 of the energy-transducing NADH-quinone oxidoreductase: comparisons of phylogenetically related enzymes. J Bioenerg Biomembr, 2001, 33 (3): 213-222
142. Zhang L, Joshi A K, Smith S. Cloning, expression, characterization, and interacttion of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein. J Biol Chem, 2003, 278 (41): 40067- 40074
143. Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride H M. The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J Cell Sci, 2007, Mar 6, [Epub ahead of print]