【作者】 唐文花；

【导师】 李奎； 彭克美；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【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。更多还原

【Abstract】 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.更多还原