【作者】 何小龙；

【导师】 荣威恒；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 绵羊的繁殖性状一般分为受胎率、多胎率及羔羊存活率三类性状,繁殖性能的高低直接关系到养羊业生产成本和生产效率,具有重大的经济意义。蒙古羊是我国最古老的地方绵羊品种之一,乌珠穆沁系蒙古羊是在当地生态环境条件下,经长期选育形成的一个优良类群,具有许多优良特性,主要以抗病力强、产肉率高和繁殖性能高而闻名。为了揭示蒙古羊高繁殖性能,使蒙古羊这一宝贵资源能得到有效利用,本研究以乌珠穆沁系蒙古羊为实验材料,首先以BMPR-IB和FSHβ基因作为蒙古羊高繁殖力候选基因,对其进行了克隆及序列分析,并采用实时荧光定量PCR(RQ-PCR)的相对定量方法对BMPR-IB和FSHβ基因在单羔蒙古羊非发情期、发情期及产双羔蒙古羊怀孕后期的卵巢、子宫、输卵管、下丘脑和垂体组织进行了差异表达研究;其次采用抑制性消减杂交技术(SSH)对经产单、双羔蒙古羊的卵巢组织差异表达基因进行了筛选并采用电子克隆加RT-PCR方法对部分差异表达基因进行了克隆和验证。通过以上研究,为提高我国肉用绵羊繁殖力及加快肉羊新品种选育提供了重要的理论依据,并获得以下主要研究结果:(1)采用RT-PCR方法分别从蒙古羊卵巢组织中扩增出BMPR-IB、FSHβ基因的cDNA序列。蒙古羊BMPR-IB基因全长1567bp,包括完整的1509bp开放读码框(ORF),共编码502个氨基酸。通过对单、多胎蒙古羊BMPR-IB基因测序结果分析发现,在单、多胎蒙古羊BMPR-IB基因编码区第746位和第1113位碱基相一致,分别为“A”和“C”,并没有发生同多胎Booroola羊相同的A→G突变和C→A突变。蒙古羊FSHβ基因全长1021bp,包括完整的390bp ORF,共编码129个氨基酸。(2)采用相对定量的双标准曲线法建立了蒙古羊β-Actin、BMPR-IB和FSHβ基因的RQ-PCR反应体系。以非发情期单羔蒙古羊的卵巢组织定量结果为对照,计算得到FSHβ基因在单羔蒙古羊发情期下丘脑组织中表达量最高,在发情期的输卵管组织中表达量最低;BMPR-IB基因在单羔蒙古羊发情期卵巢组织中表达量最高,在发情期的子宫组织中表达量最低。在卵巢组织中,FSHβ基因在发情期卵巢组织中的表达量是非发情期的1.31倍,双羔羊是单羔羊的0.70倍;BMPR-IB基因在发情期卵巢组织中的表达量是非发情期的2.65倍,双羔羊是单羔羊的2.16倍。证明BMPR-IB基因是蒙古羊多胎主要候选基因,FSHβ基因虽然在绵羊繁殖过程中具有重要的作用,但并非蒙古羊多胎主效基因,可能与多胎性状的主基因或QTL相连锁。(3)采用SSH技术成功构建了单、双羔蒙古羊卵巢组织的正、反向消减cDNA文库,并从两个消减cDNA质粒文库中筛选得到768个有效阳性克隆,插入片段长度主要分布于150~1000bp之间。结合斑点杂交技术获得到差异克隆373个,通过测序和同源性检索获得已知基因序列185条,10条已知的差异表达EST和4条未知的EST序列。(4)对差异表达基因功能分析表明,本实验所获得的基因主要由以下几大类组成:机体和细胞防御、免疫类14个,代谢类53个,物质运输类20个,核酸修饰类12个,细胞发育类18个,信号传导类12个,细胞结构类9个,未分类47个,其中代谢类基因占据了最大部分占28.65%。将这些差异表达基因和已报道的与繁殖性状相关的基因比较发现,经过初步筛选得到12个差异表达基因与已报道的繁殖性状相关基因相一致,这些基因分别为ITGB1、BMP6、MHC-I、ACVRL1、IGFBP5、SPON1、FABP5、ADAMTS1、FAM46A、THY1、ARP3和Id3基因。(5)分别以SSH获得的差异表达ADAMTS1和Id3基因序列片段为种子序列在绵羊的EST数据库中进行blastn同源性检索,得到相应的UniGene编号分别为Oar.7453和Oar.5068,经过序列拼接分别得到2418bp和1099bp的蒙古羊ADAMTS1、Id3基因电子克隆cDNA序列。采用RT-PCR方法实验克隆得到2420bp的蒙古羊ADAMTS1基因序列和1045bp的蒙古羊Id3基因序列,经同源性比对发现,实验所得序列与电子延伸序列的同源性分别为99.4%和99.3%,并将序列提交至GenBank获得注册号分别为:GU437212和GU437213。更多还原

【Abstract】 The reproduction traits of ovine are generally divided into conception rate, prolificacy and lambs survival, it is directly related to production cost and efficiency of sheep industry and it has a great economic significance. The Mongolian Sheep is one of the ancient indigenous sheep breeds in our country. Ujumuqin sheep is a good group of Mongolian Sheep through a long breeding under the local ecological environment and it has many fine characteristics, it is especially famous for its high disease resistance, dressing percentage and reproductive performance. In order to reveal its high reproductive performance and to make an effective utilization of the Mongolian Sheep precious resource, this research used Ujumuqin sheep as the experiment material. First, as candidate genes for prolificacy, the BMPR-IB and FSHβgene were cloned, sequence analyzed and differentially expressed in ovary, uterus, oviduct, hypothalamus, pituitary tissues of normal and estrus physiological stage of single Mongolian Sheep, late pregnancy physiological stage of biparous Mongolian Sheep by relative quantitative Real-time Quantitative PCR (RQ-PCR). Secondly, differentially expressed genes between ovaries from single and biparous Mongolian sheep were selected by suppression subtractive hybridization (SSH) technique and some differentially expressed genes were further cloned and verified by silicon cloning and RT-PCR. Given all the above research, important theoretical basis were provided for improving fecundity and breeding new varieties of meat sheep in our country, and the following mainly results were obtained:(1) The BMPR-IB and FSHβgene cDNA sequence were amplified from Mongolian Sheep ovary tissues by RT-PCR respectively. The full-length of Mongolian Sheep BMP-IB gene is 1567bp, including complete 1509bp open reading frame (ORF) and encoding 502 amino acids. The bases were“A”and“C”in the 746 and 1113 sites of coding region, not occurred the same mutations (A→G) and (C→A) of BMPR-IB gene in Booroola Sheep by the sequence analysis of single and biparous Mongolian Sheep BMPR-IB gene. The full-length of Mongolian Sheep FSHβgene is 1021bp, including complete 390bp ORF and encoding 129 amino acids.(2) Theβ-Actin, BMPR-IB and FSHβgene RQ-PCR reaction systems were established by relative quantitative double standard curve method. Calculated with ovary tissue quantitative result of normal physiological stage of single Mongolian Sheep, the FSHβ gene expression was the highest in hypothalamus and the lowest in oviduct of estrus physiological stage of single Mongolian Sheep, the BMPR-IB gene expression was the highest in ovary and lowest in uterus of estrus physiological stage of single Mongolian Sheep. For the ovary tissue, the expression of FSHβgene in estrus physiological stage was 1.31 times than that in normal physiological stage and biparous sheep was 0.70 times than that in single sheep; the expression of BMPR-IB gene in estrus physiological stage was 2.65 times than that in normal physiological stage and biparous sheep was 2.16 times than that in single sheep. The results proved that the BMPR-IB gene was the mainly prolificacy candidate gene of Mongolian Sheep. Although the FSHβgene have an important role in reproduction, but it’s not the prolificacy candidate gene of Mongolian Sheep and may be linkage with the prolificacy major genes or QTL.(3) Forward and reverse subtracted cDNA libraries between ovaries from single and biparous Mongolian sheep were constructed by SSH technology, and 768 masculine clones from the two subtracted cDNA libraries were obtained. The Length of inserted fragments were between 150~1000bp. Combined with dot blot technology 373 differentially clones were obtained and 185 genes sequence, 10 ESTs, 4 unknown ESTs were obtained from the differentially clones by sequencing and homology blast.(4) According to the analysis of the differentially expressed genes function, genes obtained from the experiment were mainly grouped into the following groups: cell/body defense or immune, 14 genes, metabolism, 53 genes, transporters, 20 genes, modified nucleic acid, 12 genes, cell development, 18 genes, signaling transduction, 12 genes, cell structure, 9 genes and the unknown or unclassed, 47 genes. The metabolism genes was the biggest clusters and the percentage was 28.65%. There were 12 differentially expressed genes consistent with the reproduction traits related to the genes that had been reported by comparing and screening, and the gene were ITGB1、BMP6、MHC-Ⅰ、ACVRL1、IGFBP5、SPON1、FABP5、ADAMTS1、FAM46A、THY1、ARP3 and Id3.(5) The UniGene number Oar.7453 and Oar.5068 were obtained by homology blastn in the ovine EST database using the differentially expressed ADAMTS1 and Id3 gene fragment obtained in SSH as seed sequence, the length of Mongolian Sheep ADAMTS1 and Id3 gene silicon cloning cDNA sequence were 2418bp and 1099bp respectively by sequence assembly. The length of Mongolian Sheep ADAMTS1 and Id3 gene were 2420bp and 1045bp respectively by RT-PCR of experimental cloning, the homology were 99.4% and 99.3% respectively with the silicon cloning sequence. The sequence has been registered in the GenBank and the accession were GU437212 and GU437213.更多还原