【作者】 潘刚；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 肌肉生长与肌肉品质性状的改良一直是家畜育种工作的主要目标之一。作为动物体内最丰富的组织,其生长发育相关性状是肉用家畜最重要的性状。近年来,随着一批影响肌肉生长与肌肉品质主效基因(如氟烷敏感基因和酸肉基因)的分离与鉴定及分子标记技术的迅速发展,以分子标记辅助选择与渗入为特色的分子育种技术与常规育种技术相结合极大的加速了猪遗传改良的进程。而分子育种技术的有效开展依赖于大量影响猪肌肉生长及肌肉品质性状主效基因或分子标记的分离与鉴定。鉴于此,本研究充分利用猪QTLs定位信息、人-猪比较基因组学图谱及四月龄大白猪与梅山猪背最长肌基因差异表达结果,结合基因的生理生化功能及参与的信号通路,选取了四个影响猪肌肉生长发育和品质的功能候选基因进行了进一步研究。具体研究结果如下:1.猪melusin基因(1)全长cDNA序列的克隆与鉴定。结合EST序列拼接及cDNA末端快速扩增的方法,获得了猪melusin基因1253 bp的cDNA序列(GenBank序列号为DQ002920)。比较发现猪melusin与人、黑猩猩、猕猴、牛、大鼠、小鼠的同源性较高(＞86%)且与牛melusin的遗传进化关系最近。(2)品种特异性转录本的分离与鉴定。在大白猪背最长肌cDNA中发现了一个在大白猪中特异表达的转录本。该转录本由于外显子2转录了内含子1中的106 bp的序列而产生。经RACE等方法扩增,得到该转录本1359 bp的全长序列(FJ447492)。预测发现该转录本最大的读码框架为479 bp-1150 bp,并与野生型保持同一读码框架,编码223个氨基酸。同时对该转录本在不同猪种和不同时期的背最长肌的表达情况进行了鉴定。(3)全长基因组序列的分离与鉴定。根据不同物种melusin基因的同源性以及猪melusin基因cDNA序列,获取了猪melusin基因5872 bp的基因组序列,包含了全部11个外显子和10个内含子,且所有的内含子和外显子的拼接都符合GT/AG法则。(4)启动子区序列的获取及生物信息学分析。利用不同物种melusin基因上游序列的保守性,得到了797 bp的上游序列。该序列与经Gene2Promoter回收的人和鼠的该基因的启动子区的序列同源性达到了80%和73%。利用NNPP、MatInspetor、MotifFinder、CPGPLOT、SIGNALSCAN等软件对该基因核心启动子区、转录起始位点以及转录因子结合位点进行了预测和分析。(5)组织表达谱分析。利用半定量RT-PCR方法对猪melusin基因不同转录本在大白猪不同组织中的表达情况进行了分析。结果表明,猪melusin基因不同转录本在4月龄大白猪背最长肌、心脏和脾脏中高量表达,在肝脏和肾脏中有微弱表达,在肺脏和卵巢中只检测到了野生型转录本的表达,其中在卵巢中表达量高,在肺脏中微弱表达。在脂肪组织、小肠和胃中未检测到该基因的表达。(6)多态性检测。利用重叠引物分别在大白猪、梅山猪和长白猪中扩增melusin基因的基因组序列进行比对,结果共发现了25处潜在的SNP位点。经进一步验证后发现,在5’调控区的G419C、内含子1中A902G、内含子4中C3595T分别引起了PstI、RsaI、BstHHI酶切位点的改变。对这3个SNP位点分别在不同纯种猪中分型结果表明,在大白猪和长白猪中,只检测到419C-902G-3595C等位基因的存在。而在梅山猪中只存在419G-902A-3595T等位基因。由此确定,这3个SNP位点存在高度的连锁不平衡,即处于共分离装态。在“大白×梅山”资源家系F2代群体中的进一步分型验证了上述推测。(7)单倍型构建及遗传效应分析。对这3个SNP位点构建了单倍型并对不同基因型个体与性状间的相关性进行了分析。结果表明,不同基因型个体在骨率、瘦肥比率、肩部最后处背膘厚、6-7肋骨处背膘厚、胸腰椎间背膘厚、臀部背膘厚、平均背膘厚、板油重和花油重等性状存在极显著差异。在眼肌高、眼肌宽和皮率性状上存在显著差异。CGC/CGC基因型个体比GAT/GAT和CGC/GAT基因型个体相比具有较高的眼肌面积和眼肌宽,较低的背膘厚和眼肌高。与肉质性状的相关性分析表明,不同基因型个体在股二头肌色值、股二头肌大理石纹、背最长肌大理石纹、肌内脂肪和肌内水分等性状间存在显著或极显著相关。CGC个体具有较高的股二头肌色值和较高的肌内水分含量,而GAT个体具有较高的股二头肌大理石纹、背最长肌大理石纹和肌内脂肪含量,其基因效应主要以加性效应为主。2.猪APOM基因(1)cDNA序列的克隆与鉴定。结合EST序列拼接及物种同源性比较,获取了743 bp的猪APOM基因cDNA序列(DQ329240)。ClustalW比较发现猪APOM与人、黑猩猩、猕猴、牛、大鼠、小鼠等存在较高的同源性(＞85%),且与牛的APOM遗传进化关系最近。(2)全长基因组序列的分离与鉴定。根据BAC克隆(BX548169)序列信息,设计引物获取了3621 bp的猪APOM基因的全长基因组序列,其中包含1261bp的5’上游序列。该基因由6个外显子和5个内含子,且所有的内含子和外显子的拼接都符合GT/AG法则。(3)启动子区序列生物信息学分析。对获取的1261 bp的5’上游序列及完整的外显子1序列,利用NNPP、MatInspeto、MotifFinder、CPGPLOT、SIGNALSCAN等软件对该基因核心启动子区、转录起始位点以及转录因子结合位点进行了预测和分析。发现该区域存在典型的TATA box特征的核心启动子区,并将转录起始位点推测在1136bp处。(4)猪APOM基因的比较定位。根据人HAS 6p与SSC7p及着丝粒区存在的广泛的线性同源性,及对猪APOM基因附近的基因的定位信息的挖掘,发现猪APOM基因位于猪白细胞抗原基因(SLA)I型基因簇区,并将其定位在SSC7p1.1区域。(5)组织表达谱分析。利用半定量RT-PCR方法对猪APOM基因在大白猪不同组织中的表达情况进行了分析。结果发现猪APOM基因在肝脏和肾脏中高量表达,在脂肪组织和卵巢中检测到了微弱表达,在其他组织中未检测到该基因的表达。(6)多态型检测。比较大白猪、长白猪和梅山猪APOM基因的序列差异发现了16处潜在的SNP位点。其中内含子2中2289位的G/C突变引起了ECO130I酶切位点的改变。对该SNP位点在不同纯种猪中的分型结果表明,在国外品种大白猪和长白猪中,G等位基因占多数,而在国内品种梅山猪中C等位基因占多数。(7)G2289C遗传效应分析。对猪APOM基因G2289C位点在“大白×梅山”F2群体中进行了分型,并与胴体性状和肉质性状的相关进行了分析。结果表明,不同基因型个体在骨率、皮率、肩部最厚处背膘厚、胸腰椎间背膘厚、臀部背膘厚、平均背膘厚、板油重和眼肌宽等性状存在显著或极显著差异。GG基因型倾向于具有较高的骨率和皮率,较低的背膘厚,且基因的遗传效应以加性效应为主。与肉质性状的关联分析表明,不同基因型个体只在股二头肌pH性状上存在显著差异,在其它肉质性状中未检测的显著差异。3.猪DMPK基因(1)背最长肌中差异表达验证。该基因是从利用基因芯片技术筛选四月龄大白猪和梅山猪背最长肌差异表达基因时筛选出来的。其在大白猪中的表达量要显著高于在梅山猪中的表达量。设计引物分别用半定量RT-PCR和实时定量RT-PCR技术鉴定了猪DMPK基因在背最长肌中的差异表达。(2)cDNA序列的分离与鉴定。根据物种同源性及EST拼接序列,设计引物扩增得到了2673 bp的猪DMPK基因的cDNA序列,编码624个氨基酸(3)全长基因组序列的分离与鉴定。根据不同物种该基因的同源性及获取的猪DMPK基因cDNA序列与人DMPK基因组序列的Spidey比对结果,设计引物获取了11742 bp的猪DMPK基因全基因组序列,其中包含1745 bp的上游调控区序列。经比对发现,该基因有15个外显子组成,且其外显子与内含子的拼接位点的序列均符合GT/AG法则。(4)启动子区序列生物信息学分析。对获取的1745 bp的5’上游序列及完整的外显子1序列,利用NNPP、MatInspeto、MotifFinder、CPGPLOT、SIGNALSCAN等软件对该基因核心启动子区、转录起始位点以及转录因子结合位点进行了预测和分析。(5)组织表达谱分析。利用半定量RT-PCR方法对猪DMPK基因在大白猪不同组织中的表达情况进行了分析。结果发现猪DMPK基因在背最长肌中表达量最高,在心脏、脾脏、脂肪组织中中量表达,在胃和卵巢中微弱表达。4.猪G6PT基因(1)背最长肌中差异表达验证。该基因是从利用基因芯片技术筛选四月龄大白猪和梅山猪背最长肌差异表达基因时筛选出来的。其在大白猪中的表达量要显著高于在梅山猪中的表达量。设计引物分别用半定量RT-PCR和实时定量RT-PCR技术鉴定了猪G6PT基因在背最长肌中的差异表达。(2)cDNA序列的分离与鉴定。结合EST序列拼接和RACE技术获取1925bp的猪G6PT基因的cDNA序列。比较发现猪G6PT与人、狗、大鼠和小鼠存在高的同源性(＞93%),且遗传进化关系与人和狗最近。(3)全长基因组序列的分离与鉴定。根据不同物种该基因的比对结果及获取的1925 bp的cDNA序列,获取了5077 bp的全基因组序列。经比对发现,该基因由9个外显子和8个内含子组成,且其外显子与内含子拼接位点的序列均符合GT/AG法则(4)组织表达谱分析。利用半定量RT-PCR方法对猪G6PT基因在大白猪不同组织中的表达情况进行了分析。结果发现猪G6PT基因背最长肌中高量表达,在心脏中量表达,在肝脏和肾脏中有微弱表达,在脾脏、肺脏、脂肪组织、胃、小肠、卵巢中不表达。更多还原

【Abstract】 Improvement of muscle growth and meat quality has always been one of the main goals for livestock genetics and breeding. As the most abundant tissue in animals, its related growth and development traits are the most important ones to the livestock. In rencent years, with the rapid development of molecular biology and the isolation, identification of several major functional genes related to muscle growth and meat qualities such as RYR1 and RN, the molecular breeding techniques such as marker-assisted selection （MAS） and marker-assisted introgression （MAI） combining with traditional breeding methods have greatly promoted the process of genetic improvement in pigs. The bases for effective MAS rely on the identification of more major genes or markers which are closely connected with the significant economic traits. So in this research, combining the QTLs mapping results, the comparative genome map of pig and human, the differential expression profile between Large White and Meishan pigs of four month old in skeletal muscle and the physiological and biochemical function of candidate genes, four genes were chosen as candidate genes for muscle growth and meat quality traits and studied on. The main results are as follows:1. melusin （Integrin beta 1 binding protein 2）1) Isolation and characterization of full length cDNA sequence. A 1253 bp cDNA were acquired through contig-assembling from EST data and the RACE method, and submitted to GenBank database with DQ002920. Predicted porcine melusin shares high homology （>86%） with that of human, chimpanzee, monkey, cattle, rat and mouse proteins and has the closest genetic relationship with cow.2) Breed specific transcript variant identification. One transcript variant with exon 2 extends from 50 bp to 156 bp and specially expressed in longissimus doris muscle of Large White pigs was isolated. Through RACE method, the 1359 bp in length cDNA sequence which has the maximum ORF extending from 479 bp to 1150 bp and encoding a protein of 223 amino acids was acquired （FJ447492）. The expression pattern in different pig breeds and ages in longissimus doris muscle was also studied by semi quantitative RT-PCR method. 3) Isolation and characterization of full-length genomic DNA sequence. Based on the homology between species and the acquired porcine melusin cDNA sequence, a 5872 full-length genomic DNA sequence of melusin gene was obtained. Comparison analysis with homogenes revealed that porcine melusin gene consists of 11 exons and 10 introns and the location of splice donor/acceptor sites conform to the GT/AG rule.4) Isolation and bioinformatics analysis of promoter. A 797 bp 5’ flanking region of porcine melusin was acquired by conservation comparison between different species. The acquired 797-bp sequence shares 80% and 73% homology with that of human and mouse retrieved by Gene2Promoter. NNPP, MatInspector, CPGPLOT, SIGNALSCAN softwares were used for prediction of promoter, initial position of transcription, distribution of CpG island and transfactos binding to promoter.5) Tissue expression profile anlysis. Tissue expression profile analysis in Large White pigs showed that 2 transcriptional variants were predominantly expressed in skeletal muscle, cardiac muscle, and spleen, weakly in liver and kidney. And only the wild type transcript is detected in lung and ovary, with it highly expressed in lung and weakly expressed in ovary.6) Polymorphism detection. Through sequence comparison of porcine melusin genomic sequences among Meishan pigs, Large White pigs and Landrance pigs, 25 putative SNP sites were identified. Three SNPs denoted G419C, A902G, T3595C can be detected by PstI, RsaI, BstHHI-PCR-RFLP assay respectively. The frequencies of the allele distribution were analyzed in different pure pig breeds. Only haplotype 419C-902G-3595C was detected in Large White and Landrance pigs, whereas the haplotype 419G-902A-3595T was only presented in Meishan pigs, showing this region is in extremely linkage disquilibrium status. SNP detection in the Large White×Meishan F₂ population further confirmed our conclusion.7) Association analysis for different haplotypes. Association analysis of different haplotype with carcass traits showed significant association of the genotypes with BP, RLF, SFT, RFT, TFT, BFT, ABF, LFW, CFW （p<0.01）, LEH, LEW, SP （p<0.05）. Pigs with genotype CGC/CGC trends to have higher LEA and LEW, and lower backfat thickness and LEH. Association analysis of different genotypes with meat quality traits revealed that significant associations of the genotypes with MCV2, MM2, MM1, IMF and WM were observed. Pigs with genotype of CGC have higer MCV2 and WM, whereas pigs with GAT have higher MM2, MM1 and IMF with the mainly genetic effect to be additive.2. APOM （Apolipoprotein M）1) Isolation and characterization of full-length cDNA sequence. A 743 bp cDNA of porcine APOM gene was acquired by EST contig assembling and submitted to GenBank database with DQ329240. Predicted porcine APOM shares high homology （>85%） with that of human, chimpanzee, monkey, cattle, rat, mouse proteins, and has the closest relationship with cow.2) Isolation and characterization of full-length genomic DNA sequence. Based on the BAC colone （BX548169）, a 3621 bp genomic DNA sequence of porcine APOM gene including a 1261-bp 5’ flanking region was obtained. Comparison analysis with homogenes revealed that porcine APOM gene consists of 6 exons and 5 introns and the location of splice donor/acceptor sites in all introns conform to the GT/AG rule.3) Isolation and bioinformatic analysis of promoter. A sequence encompassing the 1261-bp 5’ flanking region and the first exon was subjected to several online sofewares including NNPP, MatInspector, CPGPLOT, SIGNALSCAN to predict the core promoter region, initial position of transcription, distribution of CpG island and transfactors binding sites. One core promoter region with classical TATA box was predicted and the TSS was assigned to 1236 bp.4) Comparative mapping of porcine APOM gene. Based on the extensive synteny between HSA 6p and the p arm and the centromeric region of SSC7, the previously mapped genes surrounding APOM and the recently finished SSC7 reference sequence, the porcine APOM was predicted to lie in SLA-I region in SSC7p1.1.5) Tissue expression profile of porcine APOM mRNA. Semi quantitative RT-PCR results have revealed that porcine APOM is predominantly expressed in liver and kidney, and weakly expressed in fat and ovary with no detected expression in other tissues.6) Polymorphism identification. Through sequence comparasion among Large White pigs, Landrance pigs and Meishan pigs, 16 putative SNP sites were predicted and one mutation site denoted G2289C in intron 2 can be detected by ECO130I-PCR-RFLP assay. Allele G was found to be dominant in Large White and Landrance pigs; while in comparison, the other allele C was found to be dominant in Meishan pigs.7) Association analysis for G2289C mutation. Association analysis of different genotypes with carcass traits showed significant association of the genotypes with BP, SP, SFT, TFT, BFT, ABF, LFW, LEW. Pigs with genotype GG trend to have higher SP, BP, lower backfat thickness and the genetic effect of this loucs is mainly of additive. Association analysis of different genotypes with meat quality traits revealed that only pH （BF） was found to be significantly associated with different genotypes.3.DMPK1) Identification of differentially expressed porcine DMPK gene in skeletal muscle. On Affymetrix Porcine Chip, expression of EST （CO949753） was significantly higher in Large White pigs than in Meishan pigs. Through contig-assembling from EST data, the differentially expressed EST was defined as porcine DMPK gene. Following semi-quantitative and real-time RT-PCR analysis confirmed this result.2) Isolation and characterization of the cDNA sequence. Based on the EST contig information and sequence comparison between different species, a 2673 bp fragment containing a 1874 bp open reading frame was isolated. The encoded protein is 624 amimo acids in length with a calculated molecular mass of 69.2 kDa and an isoelectric point of 4.67. Conserved between species, porcine DMPK is predicted to have a Serine/Threonine proterin kinases catalytic domain and a DMPK coiled coil domain like. It shares 78%, 74% homology with that of human and mouse protein respectively.3) Isolation and characterization of genomic DNA sequence. Based on the homology between species and the acquired porcine DMPK cDNA sequence, a total of 11.2 kb genomic sequence including 1745 bp 5’ flanking region was isolated which spans all the 15 exons. The locations of splice donor and acceptor sites in all introns conform to the GT/AG rule.4) Isolation and bioinformatics analysis of the 5’ regulatory sequence. The isolated 1745 bp 5’ regulatory sequence plus comeplet exonl was subjected to several online bioinformatic analysis software （NNPP, MotifFinder, TFSEARCH, CPGPLOT and SignalScan） to predict potential promoter, initial positon of transhicription, distribution of CpG isand.5) Tissue expression profile analysis of porcine DMPK gene. Semi quantitative RT-PCR experiments showed that DMPK transcripts are expressed predominantly in skeletal muscle, mildly in heart, spleen and fat and weakly in stomach, ovary. 4. G6PT1) Identification of differentially expressed porcine G6PT gene in skeletal muscle. On Affymetrix Porcine Chip, expression of EST （BX672651） was significantly higher in Large White pigs than in Meishan pigs. Through contig-assembling from EST data, the differentially expressed EST was defined as porcine G6PT gene. Following real-time RT-PCR analysis confirmed this result.2) Isolation and characterization of the cDNA sequence. A 1925 bp cDNA of porcine G6PT gene with complete 3’ UTR was acquired by EST retrieval and 3’-RACE method. It contains a 1356 bp open reading frame encoding a protein of 451 amino acids with a calculated molecular mass of 48.7 kDa and an isoelectric point of 8.73. A conserved sugar transporter domain exists in this gene. The putative protein, which shares 95%, 93%, 93% homology with that of human, mouse and rat proteins respectively, has a 22 amino acids insertion mutation between 328 and 329 AA.3) Isolation and characterization of genomic DNA sequence. A total of 5077 bp full-length genomic sequence was isolated. Comparison analysis with homogenes revealed that porcine G6PT gene consists of 9 exons and 8 introns. The locations of splice donor and acceptor sites in all introns conform to the GT/AG rule. 4) Tissue expression profile analysis of porcine G6PT gene. Expression analysis applying semi quantitative RT-PCR assays showed that G6PT transcripts are expressed predominantly in skeletal muscle, mildly in heart, liver and kidney.更多还原