【作者】 宋雪梅；

【导师】 杜立新；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 绵羊作为世界上重要的可再生资源，在农业生产中占有重要的地位。在鸡、牛和猪已经完成初步的基因组测序的情况下，绵羊基因组的研究现状呈现明显的滞后状态。基于比较基因组学的研究手段，在基因组图谱和测序的基础上，利用模式生物基因组研究中获得的信息推测畜禽品种中的基因数目、位置、功能、表达机理和物种进化，可以实现对畜禽品种重要经济性状候选基因的鉴定、克隆和特性的认识。本课题尝试在中国地方绵羊品种的功能基因组研究中引入比较基因组的研究方法，进行绵羊繁殖和疾病抗性相关基因的克隆，为中国地方绵羊的选育和品种资源保护提供理论依据，并用以指导绵羊品种的分子育种和品种资源的保护工作。本实验共克隆了绵羊YB-1、绵羊和山羊ISG15、TLR9、MSY2和CIB15个基因，并对各个基因进行了比较基因组学分析，结果如下：绵羊YB-1 mRNA全长1449bp（GenBank No．EF488163），ORF为867核苷酸，可编码288个氨基酸，该蛋白具有典型的CSD结构域，氨基酸序列与人、犬、小鼠、大鼠、黑猩猩、恒河猴等的一致性均在95％以上。绵羊YB-1为无TATA盒的启动子，5’-UTR分布有GATA-1、GATA-2、GATA-3和AML-1a转录因子的结合位点。多物种的YB-1 5’-UTR的转录因子结合位点分析表明，YB-1基因转录表达调控的复杂性与物种进化复杂程度可能存在一定的相关性。43个物种YB-1及其直向同源物蛋白的聚类与生物进化的方向基本一致，并且蛋白的3-D结构也同样保守，但各物种YB-1的非翻译区在基因调控及表达中发挥了不同作用，从而在进化方向上存在一定差异。建立了高效的绵羊YB-1原核表达体系，为进一步获得抗血清并应用到YB-1蛋白功能研究奠定了基础。小尾寒羊睾丸和卵巢中共克隆了75个非冗余的YB-1选择性剪接产物克隆，其中卵巢组织中26个，睾丸组织中49个，涵盖了5’端或3’端的选择性剪接、外显子跳跃及内含子保留等3种主要的剪接方式。其中34条编码的氨基酸残基超过20个，根据编码产物blastp分析的结果可将其分为6组。第一组共10个选择性剪接产物（卵巢中1个，睾丸中9个），编码蛋白均为YB-1长度不等的C端结构域。第二组共计17个（卵巢中4个，睾丸中13个）与犬中存在的另一种YB-1选择性剪接的蛋白序列一致性达到94％。第三组共3条序列，第四组共2条序列，均为睾丸中的选择性剪接产物，功能未知。第五组序列为1条，12个AA与多物种YB-1的C段结构域存在85％的一致性。第六组序列为卵巢中的1条选择性剪接方式，功能未知。绵羊YB-1基因在不同生殖系统中可能存在大量的非编码mRNA，睾丸非编码mRNA为42％（21/49），卵巢的非编码mRNA为84％（22/26）。7条150bp以下的YB-1扩增产物的RNA二级结构预测呈现一种类似的“茎泡”样结构，并随着序列碱基数目增加在主茎环结构外也增加了其他茎环结构，使整体的二级结构呈现“十字”型变化。小尾寒羊和济宁青山羊ISG15 DNA全长分别为1033bp和911bp，均由两个外显子构成。绵羊ISG15 mRNA序列与其他物种的相似性分别为，牛94％、黑猩猩77％、人77％、恒河猴77％、小鼠77％。蛋白的相似性分别为牛87％、黑猩猩66％、人66％、恒河猴67％、小鼠63％。多物种的ISG15蛋白均存在2个泛素同源结构域，并且其3-D结构也显示出较高的相似性。已克隆的小尾寒羊序列在904bp出现1个G的插入突变，13bp后又出现了1个G的缺失突变，由此造成小尾寒羊ORF框ORF从GenBank绵羊ISG15mRNA参照序列（NM₀01009735.1）的474bp延长至525bp，编码氨基酸残基的个数也从GenBank的参照序列NP₀01009735.1的157个氨基酸残基变为174个氨基酸残基，3’-UTR也相应地缩短为19bp。济宁青山羊的ISG15 ORF为402bp，编码133个氨基酸残基。ISG15的启动子区分析显示该基因为无TATA盒启动子，除犬DPE出现于mRNA的+41～+45bp外，多物种ISG15的DPE均以（GTGA）AGATG的形式出现于+23～+27。小尾寒羊和济宁青山羊的DNA预测出的蛋白由于DNA序列中存在的碱基插入或（和）缺失的现象，使其ISG15蛋白C末端结构并不存在Leu-Arg-Leu-Arg-Gly-Gly（LRLRGG）序列。绵羊基因组中TLR9以单拷贝形式出现，基因克隆产物长4192 bp，包括115 bp的5’-UTR、2个Exon和44bp的3’-UTR。小尾寒羊TLR9经基因预测后，与其他哺乳动物TLR9mRNA的相似性分别为，牛96％，猪87％，马86％，猫85％，犬85％，人83％，黑猩猩83％，恒河猴83％，大鼠78％，小鼠77％，和负鼠70％。TLR9蛋白与其他各物种的相似性分别为，牛95％，猪85％，马83％，猫84％，犬83％，人79％，黑猩猩79％，恒河猴79％，大鼠72％，小鼠73％，和负鼠60％。多物种TLR9蛋白功能域在数量、种类及相对位置上均有不同，因而造成恒河猴和小鼠在多物种TLR9蛋白功能域聚类分析与基因的系统进化树的聚类的位置上存在差异。绵羊基因组中MSY2以单拷贝形式出现，基因克隆产物长4927 bp，包括外显子2～8和493 bp的3’-UTR，在绵羊的精子和卵子发生过程中，MSY2可能在基因转录、RNA稳定性和RNA的翻译调控中发挥重要作用。CIB1基因组扩增产物为3Kb，包括7个外显子及50bp的5’-UTR和189bp的3’-UTR。CIB1睾丸cDNA扩增产物799 bp，ORF为576bp，编码191个氨基酸。多物种CIB1非常保守，绵羊与其他物种的CIB1 mRNA序列一致性分别为与牛为98％，犬95％，人、恒河猴、大鼠和小鼠均为93％。CIB1蛋白分子量为21.7kDa，等电点为4.36，具有与其它哺乳动物相同的EF-hand结构域，与其他物种的相似性分别为，牛97％，犬和恒河猴87％，人88％，大鼠和小鼠85％。更多还原

【Abstract】 As an important renewable resource in the world, sheep holds the important station in agricultural production. In case of The Human, Chicken, Cow and Pig Genome Initial Sequencing already having been completed, the research on sheep genome presents obvious laggard status. Based on tools of comparative genomics, the information from model organisms can be used to confer quantities, positions, function, and expression model and species evolution of genes in domestic animal and poultry variety. So important economical character candidate genes can be appraised, cloned and gotten characteristic. We attempt to induct the tools of comparative genomics on function genome study of Chinese local sheep variety to clone sheep reproduction and the disease resistance correlation genes, in order to provide the theory basis for local sheep variety breeding or resources conservation, and instruct the sheep variety molecular breeding and resources protection work-Sheep YB-1, sheep and goat ISG15, TLR9, MSY2 and CIB1 were cloned, and analyzed by methods of comparative genomics. The results as followed:Small Tail Han Sheep YB-1 mRNA was 1449bp in length (GenBank No. EF488163). ORF consists of 867 nucleotides, which codes 288 amino acids. Sheep YB-1 is composed of typical CSD motif. The comparison of amino acid sequence revealed the sequence identities over 95%. Sheep YB-1 is TATA-less promoter. GATA-1, GATA-2, GATA-3 and AML-la distribute on 5’-UTR. Species potential transcription factors binding sites Analysis of YB-1 5’-UTR indicated that, the complexity of regulation on YB-1 expression and direction of species evolution had some kind of relativity. Phylogenetic relationship of 43 species based on YB-1 and its ortholog uses amino acid sequences was parallel to species biological evolution direction. And species protein 3-D structures were also conservative. Because YB-1 untranslated regions of species played the different role in gene regulation and expression, untranslated regions showed different evolution direction.YB-1 recombinant prokaryotic expression was constructed to obtain the antiserum or for the further YB-1 protein function research.We got 75 non-redundant alternative splicing (AS) production, which 26 were from ovary, and 49 from testicle. The molecular mechanisms of YB-1 AS included exon skip, alternative 5’ or 3’ splicing and intron retention. 34 non-redundant AS sequences could code in excess of 20 amino acids. According to blastp results, the protein production can be grouped in six. The first group of AS(1 from ovary, and 9 from testicle) consisting of 10 sequences, could code various length of YB-1 C motif. The second group (4 from ovary, and 13 from testicle) consisting of 17 sequences, could code the similar sequence of another YB-1 AS in Canis familiaris. The third group (consisting of 3 sequences) and the fourth groups (consisting of 4 sequences) were both from testicle, and the function of protein production was unclear. 12 AA on the fifth group(l from testicle) shared 85% similar to YB-1 C motif. The sixth group was from ovary, and the function of protein production was unclear. There were a lot of ncRNA in sheep ovary (21/49) and testicle (22/26). Predicted 7 RNA secondary structure of YB-1 AS productiom length less than 150bp present the structures just like "stem bubbles". With base numbers of AS production increase, the structures appeared like "the cross".ISG15 full length of Small Tail Han Sheep and Jining black goat is 1033bp and 911bp, respectively, constitutes by two exons. The sheep ISG15 mRNA sequence had 94% identity with the cow ISG15 mRNA, and chimp 77%, human 77%, rhesus 77%, house mouse 77%. The sheep ISG15 protein sequence had 87% identity with the cow, and chimp 66%, human 66%, rhesus 67%, house mouse 63%. ISG15 contains two domains with structural homology close to ubiquitin, and species protein 3-D structures were also conservative. There existed a "G" insertion mutation after 904bp, and a "G" deletion mutation appeared 13bp behind. ORF of Small Tail Han Sheep ISG15 enlarged into 525bp, which coded 174aa. The 3’-UTR shortened into 19bp. ORF of Jining black goat ISG15 was 402bp, which coded 174aa. The 3’-UTR shortened into 19bp. ISG15 is TATA-less promoter. Expect that DPE of dog ISG15 appeared at +41~+45bp, the others appeared at +23～+27 showing as (GTGA) AGATG. Because of the mutations, ISG15 C-terminal of Small Tail Han Sheep and Jining black goat did not present LRLRGG motif.TLR9 shows a single copy in sheep genome. Gene cloning product was 4192bp, consisted of 5’-UTR(115 bp), 2 exons and 3’-UTR(44bp). After gene predicted, The sheep TLR9 mRNA sequence had 96% identity with the cow TLR9 mRNA, and pig 87%, horse 86%, cat 85%, dog 85%, chimp 83%,, rhesus 83%, human 83%, rat 78%, house mouse 77%, and short-tailed opossum 70%. The sheep TLR9 protein sequence had 95% identity with the cow TLR9protein, and pig 85%, horse 83%, cat 84%, dog 83%, chimp 79%,, rhesus 79%, human 79%, rat 72%, house mouse 73%, and short-tailed opossum 60%. The difference on the quantity, type and the relative position of TLR9 protein motif caused rhesus and house mouse location diversity between TLR9 Phylogenetic relationship and cluster analysis on TLR9 conserved domain.MSY2 shows a single copy in sheep genome. Gene clone product was 4927bp, consisted of exon 2 to exon 8, and 3’-UTR (493bp). During the Spermatogenesis and Ovogenesis, MSY2 possibly plays the vital role on gene transcription, RNA stability and RNA translation regulation.CIB1 genome cloning production was 3Kb in length, consisted of 5’-UTR (50bp), 7 exons and 3’-UTR (189bp), while the testicle cDNA cloning product was 799bp, which coded 191aa. The sheep CIB1 mRNA sequence had 98% identity with the cow CIB1 mRNA; and dog 95%; human, rhesus, house mouse and rat 93%. CIB1 of species is composed of typical EF-hand motif. The protein sequence had 97% identity with the cow CIB1 protein; dog and rhesus 95%; human 88%, house mouse and rat 85%.更多还原