摘要
绵羊的多产性状是养羊业获得更多经济效益的重要途径,为研究蒙古羊及国内外绵羊多产性状的分子遗传机理,以蒙古羊为主要研究对象,采用分子标记技术和抑制消减杂交技术(SSH),从候选基因和差异基因筛选入手,研究蒙古羊和其他绵羊品种产羔性状的遗传多态性,探讨蒙古羊产双羔的形成因素及其分子遗传机理:
1、绵羊繁殖性状候选基因的研究
应用PCR-SSCP和RFLP技术,以蒙古羊、中国美利奴(科尔沁型)、内蒙古细毛羊、呼伦贝尔羊、萨福克、道塞特和德国肉用美利奴7个绵羊品种共375个样本为实验材料,通过分析BMP15、BMPRIB和GDF9基因共5个位点的遗传多态性,结果表明:(1)国内4个绵羊品种和德国肉用美利奴中均检测到BMP15外显子1的突变,呈现三种基因型,在第28-30位的碱基缺失,导致编码区第10号亮氨酸缺失,形成B1突变体,其它两个品种没有突变;在BMP15基因的B2和B4位点没有检测到突变;(2)中国美利奴羊(科尔沁型)BMPRIB基因编码序列第746位碱基处发生了与Booroola Merino绵羊相同的突变(A746G),而其它五个绵羊品种没有发生突变;(3)对GDF9基因的G8位点进行了SNP检测,结果在7个绵羊品种中均没有发现突变位点。
2、蒙古羊卵巢差异表达基因的筛选和特征分析
(1)用抑制性消减杂交技术(SSH)成功构建了经产单、双羔蒙古羊的正反向差减cDNA文库,在两个文库中分别随机挑取了384个克隆。利用PCR技术进行鉴定,分别获得了768个阳性克隆,插入片段长度主要分布在150～1000 bp之间。
(2)正、反向消减cDNA作探针对两消减cDNA文库中的阳性克隆进行了筛选。在以经产双羔蒙古羊为Tester、经产单羔蒙古羊为Drive的消减cDNA文库中,利用斑点杂交筛选、测序分析和序列比对,我们揭示了47个差异EST,10个在绵羊中为己知基因,4个未知基因,其中27个与其它物种的基因有较高的同源性(80%以上),6个未找到明显的同源性。
3、影响绵羊排卵全长基因BMP15的克隆与序列分析
成功克隆了影响绵羊排卵全长基因BMP15,序列全长6642bp,包括两个外显子和一个内含子,外显子长1182bp,编码393个氨基酸,经序列比对分析,发现蒙古羊和其它物种的BMP15基因的序列同源性很高,而与禽类和鱼类动物之间,同源性较低,20%以上,同时预测了蒙古羊BMP15氨基酸序列的高级结构。
The trait of prolificacy of sheep is an important approach to increase more economic benefit, in order to research the molecule genetic mechanism of fertility character of Mongolian sheep and other sheep breeds at home and aboard, we take Mongolian sheep as our main research object, adopting techniques such as molecule genetic markers as well as SSH. Starting with candidate genes and selecting different genes. We Studied moleculer genetic Polymorphism of Mongolian sheep and other sheep breeds, and discussed the factors and molecule genetic mechanism of multiparous biparous sheep:
1 .Study of candidate genes on sheep prolificacy
By the methods of PCR-SSCP and RFLP, we studied polymorphism on 5 sites of BMPRIB, BMP 15 gene and GDF 9 genes from 7 sheep breeds, 375 individuals, such as Mongolian sheep, Hulunbeier, Chinese Merino, Suffolk, Dorset, German Merino and Inner Mongolian fine wool sheep .The results showed that (1) the genotype of BMP15 gene exon 1 was delected in four domestic breeds and German Merino, three genotypes were displayed the deletion of 3 base pairs in 28-30, causes the deletion of tenth amino acid, the other two breeds have not any variation. (2). the genotype of BMPRIB gene has the same variations as Booroola Merino (A746G), and the other five breeds have not any variation. (3). The analysis of PCR-SSCP shows that G8 sites of GDF9 Gene didn’t existed any variations in seven sheep breeds .
2、Identication and Characterization of the differentially expressed Genes between Mongolian sheep ovary
(1) we constructed forward and reverse subtracted cDNA libraries between ovaries from multiparous single and biparous Mongolian sheep by suppression subtractive hybridization(SSH) technique successfully, thus, 384 clones were randomly selected from each cDNA subtracted library, and identified by PCR, obtaining 768 masculine clones,The Length of the sequences was between 150bp～1000bp.
(2) Using forward subtractive cDNA and reversal subtractive cDNA as Probe,in the subtracted cDNA library, multiparous biparous is tester and multiparous single Mongolian sheep is driver, 47 clones were selected by blot dot technology , through sequencing and blast, they represent 10 ESTs of sheep, known in sheep, 4 of them are unknown but 27 of them have high similarity with human and other species genes, and 6 have no obvious similarity with known genes.
3、Cloning and sequence analysis Mongolian sheep full length BMP15 gene
Full length BMP15 gene was cloned in Mongolian sheep, including 2 exons and 1 intron, full-length DNA 6642 bp, ORF 1182bp, coding 393 aa, by sequence analysis we found it has a high similarity with human and other species genes, but it has a less similarity with fish and birds, about 20%. At the same time, we also forecaste the advanced structure of BMP15
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