【作者】 曹贵玲；

【导师】 储明星；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 本研究分为两部分:第一部分:缩短母羊的初情期,有助于减少后备母羊的生产投入。济宁青山羊是一个性早熟的山羊品种。本部分研究旨在利用抑制消减杂交技术(SSH)筛选济宁青山羊性早熟相关基因,为进一步深入开展山羊性早熟的分子机理研究及通过遗传选择缩短山羊初情期提供理论基础。应用抑制消减杂交技术成功构建了济宁青山羊与辽宁绒山羊幼年期、初情期、初情期同龄对照间下丘脑、垂体、卵巢-子宫、肾上腺的消减cDNA文库,并对差异表达基因进行了生物信息学分析。取得结果如下:1.应用SSH技术构建济宁青山羊与辽宁绒山羊幼年期、初情期、初情期同龄对照间下丘脑、垂体、卵巢-子宫、肾上腺共12个正向消减cDNA文库,以GAPDH为指标检测消减文库的消减效率在25以上,表明差异表达基因得到了有效的富集。经PCR和测序鉴定,插入片段大小在250-900 bp之间。2. 12组消减文库中共获得2046条差异表达EST,其中包括幼年期698条、初情期对照623条、初情期725条,在每个发育时期下丘脑和垂体中获得的差异表达EST多些,卵巢-子宫和肾上腺中较少一些。对各组差异表达EST按照已知基因、已知EST和未知EST进行了分类,并对差异表达EST进行了出现频次统计。3.将各组已知基因进行了在线功能分类。根据PANTHER数据库分析,差异表达基因主要涉及信号转导与细胞间通讯、物质转运、复制/转录/翻译相关、细胞结构与运动、代谢、细胞与机体防御。根据COG分类结果,获得的差异表达基因涉及细胞进程与信号、信息储存与加工、代谢3大类,含16小类。4.将已知基因提交KEGG Pathway数据库进行通路分析,发现差异表达基因参与最多的是Metabolic pathways,其次是Parkinson’s disease和Oxidative phosphorylation。5.将已知基因进行蛋白质互作关系分析,发现获得的差异表达基因形成了核糖体结构/蛋白质翻译和氧化磷酸化两个主要的作用网络。第二部分:KiSS-1和GPR54是调控动物初情期启动的重要基因。Lin28B基因内或附近的变异与女孩月经初潮年龄在基因组水平上相关。本部分研究旨在寻找新的山羊性早熟相关基因,了解其作用机制,期望能够用于缩短山羊初情期的分子育种。试验一:克隆了山羊KiSS-1基因4118 bp的序列,预测含408 bp的ORF,编码135 aa。该蛋白质含有一个17 aa的信号肽,是一个分泌型蛋白。其序列与牛、绵羊的同源性较高,分别为91.11%和95.24%,与人、小鼠、大鼠、猪的同源性较低,分别为60.53%、58.12%、59.66%、72.50%。在山羊KiSS-1内含子1中发现3个突变位点(G296C、G454T和T505A),外显子2中没有发现突变位点,内含子2中发现一个18 bp缺失(-)/插入(+)突变(1960-1977),外显子3中发现两个突变位点(G3433A[A86T]和C3688A)。这些突变位点在性早熟和性晚熟山羊品种之间的基因型分布没有规律性差异。296位点CC型济宁青山羊产羔数比GG、GC型分别多0.80只(P<0.01)和0.77只(P<0.01),GG与GC基因型个体间产羔数差异不显著。1960-1977位点-/-个体比+/+、+/-个体产羔数分别多0.77只(P<0.01和0.73只(P<0.01),+/+与+/-间差异不显著。其他4个位点基因型间产羔数差异不显著。本研究初步表明山羊KiSS-1基因296位点C等位基因和1960-1977位点的(-)等位基因可能与济宁青山羊的高产羔数有关。试验二:克隆了山羊GPR54基因4258 bp的序列,预测含1137 bp的ORF,编码378 aa,含有7个跨膜域。该蛋白序列与牛、绵羊的同源性最高,分别为94.57%和99.58%,和人、小鼠、大鼠、猪的同源性较高,分别为86.69%、84.88%、82.47%、88.99%。山羊GPR54基因外显子1-4上没有发现突变位点,外显子5上发现3个突变位点(G4014A[G328D]、G4136A[G368S]和C4152T[P373L],这3个突变位点可能与济宁青山羊的性早熟有一定关系。4152位点的TT和CT型济宁青山羊产羔数比CC型分别多1.02 (P<0.01)和0.84(P<0.01),TT与CT基因型个体间产羔数差异不显著。其他两个位点基因型间产羔数差异不显著。本研究初步表明山羊GPR54基因4152位点T等位基因可能与济宁青山羊的高产羔数有关。试验三:克隆获得山羊Lin28B基因mRNA序列916 bp,含有744 bp的CDS区,编码247 aa,预测山羊Lin28B蛋白含有一个冷休克结构域和一对锌指结构域。该蛋白序列与人、猕猴、大鼠、小鼠、牛的同源性分别为90. 80%、90.32%、76.19%、76.92%和94.09%。山羊Lin28B存在可变剪接,剪接位点发生在内含子3中,并且内含子3的3’-端含有一个微卫星序列。测序在外显子1-3上未发现突变位点,在外显子4上发现9个突变位点(G911A、C1026T、A2934T、C3053T、G3248A、C3414G、A3770T、C4478T和G4742A),全部位于3’-UTR。对其中8个位点在12个山羊品种中进行了检测,这8个位点的基因型分布在性早熟山羊品种和性晚熟山羊品种中没有明显规律。2934位点TT和AT基因型济宁青山羊产羔数比AA基因型分别多0.83只(P<0.01)和0.48只(P<0.05),TT与AT基因型个体间差异不显著,另外7个位点基因型个体间产羔数差异不显著。本研究初步表明山羊Lin28B基因2934位点T等位基因可能与济宁青山羊的高产羔数有关。更多还原

【Abstract】 This research contains two parts.PartⅠ: The cost of developing replacement nanny goats could be reduced by decreasing age at puberty because nanny goats can be brought into production at earlier age. Jining Grey goat is a sexual precocious breed. The objective of this study was to screen genes related with puberty to look into molecular mechanism of puberty and establish fundamental materials and techniques for further study and genetic selection.Subtracted cDNA libraries of hypothalamus, pituitary, ovary-uterus and adrenal gland of juvenile stage, puberty and the same age control of puberty between Jining Grey goats and Liaoning Cashmere goats were constructed using suppression subtractive hybridization (SSH). And the differential expression genes were analyzed by bioinformatics. The main results obtained were listed as follows:1. The 12 forward subtracted cDNA libraries of hypothalamus, pituitary, ovary-uterus and adrenal gland of juvenile stage, puberty and same age control of puberty between Jining Grey goats and Liaoning Cashmere goats were constructed using suppression subtractive hybridization (SSH). The subtraction efficiency was estimated by GAPDH gene, and the results showed that the GAPDH was subtracted efficiently at 25 folds at least, which demonstrated that differential expression genes were also enriched efficiently. The size of inserted fragments was 250-900 bp identified by PCR and sequencing.2. There were 2046 differentially expressed ESTs in 12 subtracted cDNA libraries, which contained 689 ESTs at juvenile stage, 725 ESTs at puberty and 623 ESTs at the same age control of puberty. The number of differential expressed ESTs from hypothalamus and pituitary was a little more than ovary-uterus and adrenal gland at every stage. The differentially expressed ESTs in each subtracted cDNA libraries were classified to high homology genes, high homology ESTs and novel ESTs according to sequence homology in the GenBank nr and EST database.3. The high homology genes were carried out functional classification at the internet. The high homology genes were related to signal transduction and cell-cell communication, material transport, replication/transcription/translation correlated, cell structures and motion, metabolism, defension of cell and organism according to PANTHER site. And based on COG classification, the high homology genes were related to cellular processes and signaling, information storage and processing and metabolism, three main types, 16 subdivisions.4. Pathway analysis in KEGG pathway database of the high homology genes revealed the most three pathways with most genes involving: metabolic pathways, Parkinson’s disease and oxidative phosphorylation.5. Protein interaction analysis of the high homology genes revealed the most dominant network: structure of ribosome/protein translation and oxidative phosphorylation. PartⅡ:The kisspeptin/GPR54 pathway is crucial in the process of puberty onset. Variations in or near Lin28B reached a genome-wide association with age at menarche. The aim of the current research was to find new genes associated with sexual precocity of goat and understand the mechanism of action, and provide some useful information for the molecular breeding to shorten the timing of puberty of goat.In the first experiment, a DNA fragment with 4118 bp of goat KiSS-1 was obtained, which contains an open reading frame (ORF) of 408 bp and encodes 135 amino acids, having 91.11% and 95.24% identity with the protein sequence of bovine and sheep, respectively, and 60.53%, 58.12%, 59.66%, 72.50% with human, mouse, rat and pig, respectively. The protein was predicted containing a signal peptide of 17 amino acids. There were two mutations (G3433A [A86T] and C3688A) in exon 3, three mutations (G296C, G454T and T505A) in intron 1 and a 18 bp deletion (-)/insertion (+) (1960-1977) in intron 2 and no mutation in exon 2. The genotype distribution of the six mutations didn’t show obvious difference between sexual precocious and sexual late-maturing goat breeds and no consistency within the sexual late-maturing breeds. For the 296 locus, the Jining Grey goats with genotype CC had 0.80 (P<0.01) or 0.77 (P<0.01) kids more than those with genotype GG or GC, respectively. No significant difference (P>0.05) was found in litter size between GG and GC. For the 1960-1977 locus, the Jining Grey goat with genotype -/- had 0.77 (P<0.01) or 0.73 (P<0.01) kids more than those with +/+ or +/-, respectively. No significant difference (P>0.05) was found in litter size between +/+ and +/- genotypes. For the other four loci, no significant difference (P>0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele C of the 296 locus and allele - of the 1960-1977 locus in KiSS-1 and high litter size in Jining Grey goats.In the second experiment, a DNA fragment of 4258 bp of goat GPR54 was obtained, which contains an ORF of 1137 bp and encodes 378 amino acids, having 94.57% and 99.58% identity with the protein sequence of bovine and sheep, respectively, and 86.69%, 84.88%, 82.47%, 88.99% with human, mouse, rat and pig, respectively. The protein was predicted to have seven transmembrane regions. There were no base pair variation in exons 1-4 and three base changes (G4014A [G328D], G4136A [G368S] and C4152T [P373L]) in exon 5 by sequencing and the three mutations may have some correlation with sexual precocity in goats. For the 4152 locus, the Jining Grey goat with genotype TT and CT had 1.02 (P<0.01) and 0.84 (P<0.01) kids more than those with genotype CC, respectively. No significant difference (P>0.05) was found in litter size between TT and CT genotypes in Jining Grey goat. For the other two loci, no significant difference (P>0.05) was found in litter size between different genotypes. The present study preliminarily indicated an association between allele T of the 4152 locus in GPR54 and high litter size in Jining Grey goats.In the third experiment, an mRNA sequence with 916 bp of goat Lin28B was obtained, containing a CDS of 744 bp, encoding 247 amino acids, which was predicted having a cold shock domain and a pair of zinc finger domain. The 247-amino acid has 90. 80%, 90.32%, 76.19%, 76.92% and 94.09% identify with the protein sequence of human, macaque, rat, mouse and bovine, respectively. Two alternative transcripts were detected expressing in goat tissues. And the alternative splicing site lies in intron 3, which contain a microsatellite sequence in its 3’- terminal. There was no mutation in exons 1-3 and nine mutations found in exon 4 (G911A, C1026T, A2934T, C3053T, G3248A, C3414G, A3770T, C4478T and G4742A), which were all in 3’-UTR, by sequencing. The genotype distribution of the eight sites detected didn’t show obvious difference between sexual precocious and sexual late-maturing goat breeds and no consistency within the sexual late-maturing breeds. For the 2934 locus, Jining Grey goats with genotype TT and AT had 0.83(P<0.01) and 0.48(P<0.05) kids more than the goats with genotype AA, and no significant difference (P>0.05) was found in litter size between TT and AT genotypes in Jining Grey goat. For the other seven loci, no significant difference (P>0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele T of the 2934 locus in Lin28B and high litter size in Jining Grey goats.更多还原