【作者】 唐中林；

【导师】 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2006， 博士

【摘要】 中外猪种在生长、胴体和肉质性状等方面存在很大差异。从遗传的角度来讲，这些表型差异在很大程度由骨骼肌生长发育过程中基因的表达状况所决定。骨骼肌的生长发育过程十分复杂，涉及许多基因和转录因子的表达及精密的网络式调控机制控制。猪瘦肉产量和质量主要取决于骨髂肌肌纤维数量和横切面积，而肌纤维数量在出生前已经基本恒定。本研究利用长标签基因表达系列分析(Long Serial Analysis of Gene Expression，LongSAGE)技术分析中外两个不同猪品种——通城猪(Tongcheng pigs，简称T)和长白猪(Landrace，简称L)不同发育时期(妊娠33，65和90天)的胚胎骨骼肌转录谱，并结合电镜技术观察其肌细胞超微结构。同时，利用GLGI方法分析未知LongSAG标签，并对差异表达的四个猪新基因进行克隆、定位和时空表达研究，初步得到如下结果：1．构建并比较分析T33(50，450)、T65(53，927)、T90(53，761)、L33(53，104)、L65(54，483)和L90(51，188)6个LongSAGE转录谱文库，共包含317，115个标签(tag)，对应于98，437个单标签(unique tag)。其中，14，683个单标签至少在一个文库中检测到2个拷贝以上(含2个拷贝)，平均30.87％单标签不与参考数据库中的任何序列匹配。2．表达谱聚类分析表明，6个不同的骨骼肌表达谱分为3类。L65和L90最为相似，首先聚为一类，然后与T90形成一大类。L33和T33聚为一类，T65单独成一类。通城猪出生前骨骼肌发育过程中表达谱的变化相对平缓，而长白猪在发育33-65天时变化较65-90天时急剧。3．通城猪和长白猪骨骼肌发育过程中鉴定的差异表达单标签(unique tag)分别为1，400个和1，201个(P＜0.05)，其中832个特异地在通城猪中差异表达，683个特异地在长白猪中差异表达。这些品种内差异表达的标签都分别表现出八种不同的表达模式。4．骨骼肌发育过程中，差异表达基因所涉及的生物学过程在通城猪和长白猪中基本是相似的，主要涉及细胞生理过程(cellular physiological process)、代谢(metabolism)、定位(localization)、细胞通讯(cell communication)、刺激反应(response to stimulus)和发育(development)等。比较而言，通城猪中涉及的生物学过程比长白猪更多、更复杂。在通城猪中更多的差异表达基因与有机酸代谢(organic acid metabolism)(6.70％vs.0.79％，p=0.017)，胺代谢(amine metabolism)(4.47％vs.0.79％，p=0.08)和氨基酸及其衍生物的代谢(amino acid and derivative metabolism)(4.47％vs.0.79％，p=0.08)有关。在长白猪中更多的差异表达基因与酒精代谢(alcohol metabolism)(5.56％vs.0.56％，p=0.009)、核酸代谢(nucleic acidmetabolism) (18.99％vs.29.37％，p=0.039)和代谢正调控(positive regulation of metabolism) (2.38％vs.0.00％，p=0.069)有关。5．骨骼肌发育过程中差异表达基因涉及的通路主要有25个，两品种涉及基因最多的都是核糖体(ribosome)通路。比较而言，在长白猪中涉及较多基因的通路是钙离子信号通路(calcium signalling pathway)、调节肌动蛋白细胞骨架(regulation of actin cytoskeleton)和碳固定(carbon fixation)等通路。而在通城猪中，较多的基因涉及附着斑(Focal adhesion)、ECM受体互作(ECM receptor interaction)和细胞凋亡(apoptosis)等通路。6．通城猪和长白猪之间差异表达标签数最多的是在妊娠65天，有653个单标签。妊娠33天和90天差异表达(P＜0.05)的单标签数目分别为532个和459个。7．利用GLGI方法对67个未知的标签进行3’延伸，获得113条EST序列。BLAST分析表明，100条EST不与任何序列匹配，这些序列可能来源于未知基因或转录本。8．分离、克隆了TOB1、FSCN1、OLFML3和NPM基因的cDNA序列，其中OLFML3还获得了第二内含子序列，NPM基因获得了第四内含子序列。用猪辐射杂种板对这些基因进行染色体精细定位。TOB1、FSCN1、OLFML3和NPM基因分别定位在猪12、3、14和16号染色体上，并分别与SS04H11(LOD=5.52)、SSC10E12(LOD=8.03)、IGLV(LOD=5.26)、SW977(LOD=9.16)标记紧密连锁。体细胞杂种板的染色体区域定位结果表明NPM基因位于SSC16q21区段。9．用实时定量PCR分析TOB1、FSCN1和OLFML3等基因的时空表达谱。TOB1基因在通城猪骨骼肌发育过程中的妊娠65天时表达量最高，其次为成年期。该基因在成年五指山猪的各种组织中广泛表达，且在骨骼肌中表达丰富。在骨骼肌发育过程中，FSCN1基因在长白猪中妊娠65天时表达量最高，在通城猪中妊娠90天时表达量最高。在成年五指山猪的组织中以大脑中表达最丰富。OLFML3基因在骨骼肌发育过程中呈下调表达，在出生后的猪骨骼肌中几乎不表达。在成年五指山猪的胃、肝和脂肪等组织中表达丰富。10．透射电子显微镜观察肌细胞超微结构表明：长白猪肌纤维的肌节明显长于通城猪。妊娠33天时胚胎骨骼肌已具有一定的肌纤维结构特征，但长白猪特征更明显，具有明显的明暗相问的带纹。妊娠65天时肌纤维发育更完善，已能清楚分辨肌节结构，长白猪糖原颗粒相对丰富。妊娠90天长白猪肌原纤维排列较通城猪紧凑。更多还原

【Abstract】 Chinese indigenous pigs have great differences comparing to exotic pig breeds in meat production traits, such as growth, carcass and meat quality. Phenotype variations between breeds coming from different genetic backgrounds were directly determined by gene expression during skeletal muscle development. The muscle growth and development is complex processing involved in thousands of genes. Gene expression was subtly regulated in network mechanism. The production and meat quality of pork were largely detemined by the total number and cross sectionarea of skeletal muscle fiber. However, total number of skeletal muscle fibers was fixed at prenatal stage. In this study, transcriptome of skeletal muscle from pig embryo of Tongcheng (T) and Landrace (L) pigs at different stages (at 33,65 and 90days of gestation) were analyzed using long serial analysis of gene expression technique (LongSAGE), and cell ultrastructure of skeletal muscle was also observed through electronic transmission microscope. The novel LongSAGE tags were further extended using GLGI (generation of longer cDNA fragments from SAGE tags for gene identification) technique. Four novel swine genes that were represented by LongSAGE unique tags differentially expressed in libraries were cloned, mapped, and the temporal and spatial expression of these genes was detected. The results of this study were presented as follows:1. Transcrptional profiling of six Long SAGE libraries from T33 (50,450 tags), T65 (53,927 tags), T90 (53,761 tags), L33 (53,104 tags), L65 (54,483 tags), and L90 (51,188 tags) were generated and compared. A total of 317,115 tags, corresponding to 98,437 unique tags were obtained. A total of 14,683 unique tags with more than one copy at least in one library were detected. On average, 30.87% of unique tags could not be assigned to UniGene entries in NCBI database.2. The clustering results of the LongSAGE libraries showed that T65 was distinctive from other transcriptional profiles. L65 and L90 were merged firstly into a single cluster, and then T33 and L33 formed another group. Finally, T90, L65 and L90 formed a big group. The expression pattern of Tongcheng pigs changed more smoothly than those of Landrace. And the expression pattern changed more sharply at early stages (33-65) than later stages (65-90) during skeletal muscle development in Landrace. 3. The comparative analysis of the transcriptomes at the three different gestation stages identified 1,400 and 1,201 differentially expressed unique tags in Tongcheng and Landrace pigs, respectively. Of these differentially expressed unique tags, 832 (p＜0.05) were exclusively differentially expressed in Tongcheng pigs and 683 (p＜0.05) were in Landrace, respectively. The expression patterns of differentially expressed unique tags could be grouped into eight expression modes.4. The main biological process in which these differently expressed genes participate in myogenesis of Tongcheng pigs generally similar to Landrace. These processes mainly included cellular physiological process, metabolism, localization, cell communication, response to stimulus and development. More biological processes were found to be represented in Tongchenhg pigs. Furthermore, more differentially expressed genes be concerned with organic acid metabolism (6.70%vs.0.79%, p=0.017), amine metabolism (4.47%vs.0.79%, p=0.08), as well as amino acid and derivative metabolism (4.47%vs.0.79%, p=0.08) in Tongcheng pigs than Landrace. In contrary, more genes were involved in alcohol metabolism (5.56%vs.0.56%, p=0.009), nucleic acid metabolism (18.99%vs.29.37%, p=0.039) and positive regulation of metabolism (2.38%vs.0.00%, p=0.069) in Landrace than Tongcheng pigs.5. Signal pathway analysis indicated that there were 25 primary pathways involved in genes differentially expressed, and ribosome pathway was the leading pathway during skeletal muscle development in both Tongcheng pigs and Landrace. A number of genes associated with calcium signalling pathway, regulation of actin cytoskeleton and carbon fixation pathway were found in Landrace, while a number of genes associated with focal adhesion, ECM receptor interaction and apoptosis pathway were in Tongcheng pigs.6. A total of 653, 532, and 459 unqiue tags were differentially expressed at 65, 33, and 90 days of gestation between Landrace and Tongcheng pigs, respectively. Most unique tags were differentially expressed at 65 day of gestation between Tongcheng pigs and Landrace.7. A total of 113 ESTs were obtained from 67 novel LongSAGE tags using GLGI method. BLAST analysis showed that 100 ESTs did not assigne to any sequence, and which indicated that these ESTs may potentially deriv from novel genes or transcripts.8. The cDNA sequence of TOB1、FSCN1、OLFML3 and NPM genes were isolated and cloned together with the second intron of OLFML3 and fourth intron of NPM. TOB1, FSCN1, OLFML3 and NPMgene were assigned to SSC12, SSC3, SSC14 and SSC16, and linked to SS04H11 (LOD=5.52), SSC10E12 (LED=8.03), IGLV (LED=5.26) and SW977 (LOD=9.16) by IMpRH panel, respectively. Furthermore, NPM gene was mapped to SSC16q21 using SCHP panel.9. The temporal and spatial expression of TOB1、FSCN1 and OLFML3 genes were examined using quantitative real time PCR (qPCR) method. Expression level of gene TOB1 is highest at 65 day of gestation in Tongcheng pigs. Secondly, it was highly expressed in adult pig. Spatial expression profile of TOB1 showed that it was expressed in many tissues from Wuzhishan pigs, while it had abundant expression in skeletal muscle. Expression of FSCN1 peaked in prenatal skeletal muscle at 65 days of Landrace and 90 days of Tongcheng pigs, respectively. And FSCNI was most highly expressed in brain from Wuzhishan pigs. OLFML3 gene was down-regulated during skeletal muscle development, and hardly was examined in postnatal skeletal muscle. This gene was abundantly expressed in stomach, liver and fat tissues, and rarely expressed in skeletal muscle from adult Wuzhishan pigs.10. The ultrastructure of embroynic skeletal muscle via electronic transmission microscope suggested that Landrace had longer sarcomere and more obvious structure of myofiber than Tongcheng pigs. However, compared with Toncheng pigs, more typical characteristic were observed in Landrace, such as sarcomere with A and I region, and Z-lines. Muscle cell had more mature structure at 65 days than 33 days of gestation, and more abundant hepatin granules were distributed in Landrace than Tongcheng pigs. The assignment of myofibrils in Landrace was more compact than in Tongcheng pigs at 90 days of gestation.更多还原