【作者】 朱广琴；

【导师】 曹斌云；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 产羔性状是山羊生产中重要的经济性状之一,提高产羔性能直接关系到奶山羊养殖业的生产成本和生产效率的高低。然而,产羔性状是多基因和环境因素互作的结果,目前,对其调控机理还知之甚少。卵巢是动物繁殖中最重要的器官,其内部卵泡在每个发情周期中经历着细胞生长、分化和凋亡。这种规律性的生理变化直接影响或决定着山羊的排卵数、受精率和产羔数的多少。为了深入探索奶山羊卵巢基因的表达模式,本研究以发情期多羔和单羔关中奶山羊卵巢组织为试验材料,利用抑制消减杂交技术构建正反向cDNA消减文库,对其基因的表达谱进行了研究,取得如下结果:1.利用SSH技术构建了多羔和单羔关中奶山羊发情期卵巢组织的正反向cDNA消减文库:M-U(以多羔羊卵巢组织为Tester,单羔羊卵巢组织为Driver)和U-M(以单羔羊卵巢组织为Tester,多羔羊卵巢组织为Driver)cDNA消减文库;用看家基因GAPDH(甘油三磷酸脱氢酶)检测两个cDNA文库的消减效率分别为211和28,表明两个文库中差异表达的基因也富集了211和28倍。2.在利用SSH技术成功构建多羔和单羔关中奶山羊卵巢组织cDNA消减文库的基础上,采用斑点杂交技术对两个文库进行了筛选和鉴定。经测序,在M-U cDNA消减文库中得到77个完全不同的ESTs,其中4个ESTs是山羊的已知序列;53个ESTs是山羊中的未知序列,但与绵羊、牛、猪等其它家畜具有较高的相似性;其余20个ESTs在NCBI数据库中尚未找到其相似性序列。在U-M cDNA消减文库中得到50个ESTs,其中没有山羊的已知序列,30个ESTs是山羊中未知序列,但与绵羊、牛、猪等其它家畜具有较高的相似性;其余20个ESTs在NCBI数据库中尚未找到其相似性序列。得到的77个正库序列共参与了139个不同的生物过程,而反库中的50个序列只参与了22个不同的生物过程;经分析,正库中序列参与的生物过程主要有如下过程被富集:细胞过程、代谢过程、生物调节、应激反应、多细胞协调过程、发育过程、信号转导、细胞定位、细胞原件合成、细胞原件组织、繁殖、细胞增殖、生长、死亡、生物黏附、转运和定位共17个类别;而反库中序列参与的生物过程仅有4个被富集,分别是细胞过程、代谢过程、生物调节和应激反应。3.利用实时定量PCR技术对关中奶山羊卵巢组织抑制消减cDNA文库中筛选出来的12个基因进行检测与分析,结果表明12个基因在多羔奶山羊卵巢组织中的表达丰度较单羔奶山羊卵巢组织分别提高:16.03(DCN)、11.87(OAZ1)、5.28(EPHX1)、5.68(FSHR)、5.36(TIMP1)、9.67(RUNX1)、3.90(ADAMTS1)、7.97(BMP6)、2.91(RPL5)、6.01(VIM)、2.23(ZNF207)和4.91(SF3B5)倍。对基因的相对表达量和产羔数进行相关性分析,发现只有DCN、OAZ1、EPHX1和FSHR与产羔数呈显著正相关(R2=0.9783,0.9747,0.9669和0.902);并对这4个基因进行了克隆,首次从关中奶山羊中获得了相应的CDS全长并提交到GenBank:FSHR(ORF 2055bp,Accession No:HQ326239)、OAZ1(ORF 577bp,Accession No:HQ326240)、EPHX1(ORF 1356bp,Accession No:HQ326238)和DCN(ORF 1083bp,Accession No:HQ326237)。4.本研究利用了多个生物信息学的分析软件,对已经筛选到的差异表达的4个基因DCN、OAZ1、EPHX1和FSHR进行山羊、绵羊、牛、人、鼠和猪物种间的同源性分析、氨基酸结构分析、潜在功能位点预测、蛋白质的二级、三级结构等进行了分析比较,结果发现山羊、绵羊、牛、人、鼠和猪这4个基因的ORF区和氨基酸序列高度同源,所编码蛋白的结构和潜在功能位点基本相同。山羊、绵羊、牛、人、鼠和猪的DCN和FSHR具有信号肽;EPHX1和OAZ1没有信号肽,没有卷曲,因此推断这两个基因没有复杂的空间结构。这4个基因都具有N-糖基化位点。只有OAZ1没有跨膜螺旋。更多还原

【Abstract】 Reproductive rate is an important economic trait in goat reproduction. To improve the fecundity, also known as litter size, is of special meaning in the selection of animals with high reproductivity. But the litter size is a result of interaction between genetic and environmental factors, how precisely the litter size is controlled remains a critical and important question in reproductive biology. Ovary is the main functional organ in reproduction. During each estrous cycle, ovaries experience the changes of proliferation, invasion, differentiation and cell apoptosis, and these normal physiological changes of ovaries more or less directly affect and/or determine the ovulation, the rate of fertilization and the litter size of goats. But Studies of gene expressions using ovaries during the estrous cycle of goats are limited. Thus we collected ovary samples of multiparous and uniparous Guanzhong dairy goats during the estrous cycle to construct forward and reverse subtracted cDNA libraries of ovaries, and identified differentially expressed genes enriched in multiparous and uniparous Guanzhong dairy goats. The main results were as follows:1. Forward and reverse subtracted cDNA libraries of ovaries at estrous stage were constructed using suppression subtractive hybridization technology, called M-U (ovary of multiparous Guanzhong dairy goat as Tester, and ovary of uniparous Guanzhong dairy goats as Driver) and U-M (ovary of uniparous Guanzhong dairy goat as Tester, and ovary of multiparous Guanzhong dairy goat as Driver) subtracted cDNA library respectively. The subtraction efficiency was estimated by a housekeeping gene GAPDH, and the result showed that GAPDH was subtracted efficiently by 211 and 28 folds for M-U and U-M subtracted cDNA library respectively, which indicated that differentially expressed genes were also enriched by the same corresponding folds.2. Screening and identification of the constructed M-U and U-M libraries. Dot blot hybridization was used for the screening of the differentially expressed clones. From the sequenced clones we identified 77 completely different genes in M-U subtracted cDNA library, of which 4 were known in goats, 53 were unknown in goats but have high homology with Ovis aries, Bos taurus, Sus scrofa or other species, and the rest 20 were unknown genes. Whereas we identified 50 completely different sequences from the sequenced clones in the U-M library, of which no one of them was known in goats, 30 were unknown in goats but have high homology with Ovis aries, Bos taurus, Sus scrofa or other species, and 20 were completely unknown genes. The 77 goat ESTs in M-U subtracted cDNA library involved in 139 biological processes based on the GO annotation for biological process; whereas, there were only 22 biological processes involved for the sequences identified in the U-M library. Enrichment analysis of the biological processes involved by the 77 sequences was carried out, and 17 different processes were overrepresented, which included biological regulation, biological adhesion, death, growth, locomotion, metabolic process, cell proliferation, signaling, localization, cellular component biogenesis, cellular component organization, immune system process, reproduction, multicellular organismal process, developmental process, response to stimulus and cellular process. The same analysis was done to the sequences from the U-M subtracted cDNA library, but only 4 biological processes were enriched, which comprised response to stimulus, cellular process, biological regulation and metabolic process.3. Real-time reverse transcriptase-polymerase chain reaction was used to analyse the changes of mRNA expression levels of 12 genes selected from M-U subtracted cDNA library in ovaries of multiparous and uniparous Guanzhong dairy goats. The results showed that they increased 16.03 (DCN), 11.87 (OAZ1), 5.28 (EPHX1), 5.68 (FSHR), 5.36 (TIMP1), 9.67 (RUNX1), 3.90 (ADAMTS1), 7.97 (BMP6), 2.91 (RPL5), 6.01 (VIM), 2.23 (ZNF207) and 4.91 (SF3B5) folds in ovaries of multiparous than that of uniparous Guanzhong dairy goats. Aggression analysis was performed to investigate the correlation between gene expression levels and litter size in multiparous goats. Of the 12 genes, only DCN, OAZ1, EPHX1 and FSHR were significantly positively correlated with litter size, with R2= 0.9783, 0.9747, 0.9669 and 0.902 respectively. We then cloned the complete coding region of DCN, OAZ1, EPHX1 and FSHR from goats, namely, Capra hircus Decorin (ORF 1083bp, Accession No:HQ326237), Capra hircus Epoxide Hydrolase 1 (ORF 1356bp, Accession No:HQ326238), Capra hircus Follicle Stimulating Hormone Receptor (ORF 2055bp, Accessoin No:HQ326239) and Capra hircus Ornithine Decarboxylase Antizyme 1 (ORF 577bp, Accession No:HQ326240), which laied a foundation for further functional research.4. Sequence homology, amino acid structure, the potential functional site prediction, protein secondary and tertiary structure of DCN, OAZ1, EPHX1 and FSHR were compared among goat, sheep, bovine, human, mouse and pig using bioinformatics softwares. We found the ORFs and amino acid constitutions of each of the 4 genes in different species had high homology; the protein structures and potential functional domains of each gene among species were similar. The signal peptides were predicted for DCN and FSHR of goat, sheep, bovine, human, mouse and pig. No signal peptides and curls of EPHX1 and OAZ1 were found, so there were no complex spatial structures. These four genes have N-glycosylation sites. Only OAZ1 does not have transmembrane helix.更多还原