白羽王鸽产蛋期卵巢基质、卵泡、输卵管组织形态学和转录组学研究

发布时间：2018-04-22 02:17

本文选题：鸽子 + 卵巢基质　；参考：《南京农业大学》2016年博士论文

【摘要】：鸽子具有独特的繁殖特点,目前对鸽繁殖活动的认知还处于初级观察阶段,相关领域研究也非常少,仅限于少量繁殖相关的单基因功能研究,饲养管理及环境对其繁殖性状的影响。为了进一步认识鸽的繁殖过程,了解繁殖过程中繁殖相关组织的变化,本试验对鸽Q1期(排卵前1-2d )、Q2期(第二个卵刚被排出,即一个蛋已经产出,另一个蛋还在输卵管中)、Q3期(排卵后6 ～7d) 3个阶段的卵巢基质组织和输卵管膨大部组织,以及排卵前后的卵泡组织进行组织形态学观察,采用高通量转录组测序法对3阶段卵巢基质、输卵管膨大部组织以及排卵前后卵泡进行转录组学研究,以及卵清蛋白相关蛋白Y ( Ovalbumin-Related Y Protein, OVALY)基因克隆和序列分析。1.鸽排卵前后卵巢、卵泡、输卵管组织形态学观察本试验对鸽排卵前后3个时期卵巢组织进行形态学观察;对输卵管膨大部以及排卵后卵泡进行组织形态学观察,并进行凋亡率等检测。发现在鸽卵巢存在3个特殊阶段:只含等级卵泡和等级前卵泡、只含排卵后卵泡和等级前卵泡、仅有等级前卵泡的时期;排卵后卵泡的胶原质主要存在于膜外层中;组织退化可能与凋亡相关。鸽子输卵管膨大部管腔横切面积在3个时期中差异很大;膨大部粘膜上皮细胞中在排卵前后存在酸性黏液;排卵后输卵管膨大部组织中存在明显的凋亡现象。2.鸽排卵前后卵巢基质组织的转录组学研究本试验采用RNA-Seq测序技术分析鸽3个时期卵巢基质组织中基因表达情况。试验总共得到了 44,784,505个clean reads,与鸽子的基因组比对获得14,088个基因。在PI ( Q1期的卵巢基质组织)和P2 ( Q2期的卵巢基质组织)中共发现了 409个差异基因,其中有96个基因在P1中表达上调和313个基因在P2中表达上调。对P2中上调的基因进行GO功能富集分析:免疫应答(GO: 0006955 )、受体结合(GO:0005102)、免疫系统进程(GO: 0002376 )、生物粘附(GO: 0022610)和抗原加工与呈递(GO: 0019882 )等生物进程显著富集。KEGG通路富集显示,吞噬体(clv04145 )、溶酶体(clv04142)、细胞因子和细胞因子受体反应(clv04060)、细胞粘附分子(clv04514 )和Toll样受体信号通路(clv04620 )等通路显著富集。在P2中筛选到了53个与免疫相关,和22个与组织退化相关的候选基因,这些基因可能共同参与了排卵后卵泡组织的退化。3.排卵前后卵泡组织转录组研究本试验采用RNA-Seq测序技术分析鸽排卵前后卵泡组织中基因表达情况。平均每个样本获得49,487,038个raw reads,去掉接头序列、重复序列及低质量的序列后,最终获得47,270,639个高质量的clean reads,占原始数据的96%。与参考基因比对,共匹配到20,282个基因。排卵前后的卵泡组织中表达的基因中存在5,489个可变剪切事件,其中仅有0.26%的可变剪切发生了差异表达。共发现了 6,195个新转录本,492,060个SNP位点和33,945个InDel位点。差异表达基因分析显示,843个基因在P4 (Q1期卵泡组织)中表达上调,618个基因在P5 (Q2期卵泡组织)中表达上调。这些差异基因主要富集在胶原蛋白三聚体(G0:0005581 )、细胞外基质结构成分(G0:0005201 )、蛋白质聚合(G0:0051258 )、钙离子结合(G0:0005509)、蛋白结合桥连(G0:0030674)等功能中。KEGG富集分析显示:胞外基质受体相互作用(clv04512)、粘着斑(clv04510)、孕激素介导的卵母细胞成熟(clv04914)、心肌细胞的肾上腺素信号(clv04261 )、吞噬体(clv04145)、血管平滑肌收缩(clv04270)、缝隙连接(clv04540)、促性腺激素释放激素信号通路(clv04912)等通路显著富集。在P4中共筛选了 51个与胞外基质及其受体相关的候选基因,17个促进卵泡成熟、排卵紧密的相关基因;在P5中共筛选了 32个与组织退化相关的候选基因。4.鸽排卵前后输卵管膨大部组织转录组学研究本试验采用RNA-Seq测序技术分析鸽不同阶段输卵管膨大部组织中基因表达情况。本试验中平均每个样本获得56,354,181个rawreads,去掉接头序列、重复序列及低质量的序列后,最终获得54,764,938个高质量的clean reads,占原始数据的97.2%。与参考基因比对,共匹配到20,767个基因。在鸽子3个时间点输卵管膨大部组织表达的基因中存在大量的外显子跳跃,少量的外显子选择性跳跃和极少的内含子滞留,不存在第一个或最后一个外显子可变剪切事件。其中仅有3.8%的可变剪切发生了差异表达。共发现了 6,680个新转录本,819,137个SNP位点和53,702个InDe1位点。差异表达基因分析显示,在Cl (Q1期的输卵管膨大部组织)和C3 (Q3期的输卵管膨大部组织)间发现的差异表达基因最多,为3,966个,其中2,250个基因在C1中表达上调,1,716.个基因在C3中表达上调。C1中表达上调基因主要富集在细胞膜结构等功能。而C2(Q2期的输卵管组织)中表达上调差异基因主要富集在有机质反应、激素相关功能;C3中表达上调差异基因主要参与生物进程调节相关功能。KEGG通路富集分析显示,C1中表达上调的差异基因主要富集内质网蛋白加工(clv04141)、蛋白输出(clv03060)等通路中。C2中表达上调的差异基因主要富集到脂肪酸代谢(clv01212)、脂肪酸降解(clv00071 )、糖降解(clv00511 )等通路中。在C1中共发现了 62个与蛋白转运相关的候选基因;在C2中共发现了 40个与细胞凋亡相关基因,17个与蛋白质降解相关基因,以及18个与脂代谢相关的候选基因。这些基因可能与输卵管膨大部蛋白积累、组织退化紧密相关。在C1中与蛋清中主要蛋白相关的基因均表达上调,其中OV4LY基因表达量最高。5.OVA Y基因克隆与生物信息学分析试验获得白羽王鸽OVALY基因mRNA完整序列,该序列全长2068 bp,其中5'UTR序列180bp,3'UTR序列720bp和CDS序列1164bp ,共编码388个氨基酸序列,分子量约44.19kDa。与NCBI中岩鸽的OVALY基因XI亚型(GeneBank:XM-005509738.1 )相似性极高,只存在4个单碱基突变位点,分别位于参考序列376CT、730TC、835TC、11109TC 处。该序列与朱瀗(Nipponia nippon)、白尾鹰(Haliaeetus albicilla)和沙鸡(Pterocles gutturalis)的同源性较高,分别为 81%、80%、79%。该数据已提交到NCBI数据库,登录号为KX230792。OVALY氨基酸序列共有22个磷酸化位点和4个N-连接糖基化位点。经蛋白质保守结构域分析发现OVALY属于丝氨酸蛋白酶抑制剂超家族(Serpin superfamily),具有一个丝氨酸蛋白酶抑制剂家族的反应中心区域,未发现信号肽位点和跨膜结构。综上,本文对鸽三个时间点的卵巢基质、输卵管膨大部,以及排卵前后的卵泡组织进行了组织形态观察,对鸽繁殖系统有了进一步的认识。转录组学研究发现卵巢基质部分组织可能参与了卵泡退化;筛选到大量与卵泡生长、发育、成熟、退化相关候选基因,与输卵管蛋白合成、积累、组织退化等相关的候选基因。这些差异基因为繁殖系统中相关组织周期性变化研究提供一个可靠的参考基因库,为繁殖机理研究提供数据支持。
[Abstract]:Dove has unique reproductive characteristics. At present, the cognition of pigeon reproduction is still in the primary observation stage, and there are few studies in related fields. It is limited to a small number of single gene functional studies related to reproduction, and the influence of breeding management and environment on its reproductive traits. In order to further recognize the breeding process of pigeons, understand the reproduction phase during the breeding process. In this experiment, the test was performed on pigeon Q1 (pre ovulation 1-2D), Q2 phase (second eggs had been discharged, another egg was produced, another egg was in the fallopian tube), Q3 (6 to 7d after ovulation) in 3 stages of ovarian tissue and oviduct enlargement tissue, and the histomorphology of the follicle tissue before and after ovulation. A transcriptional study of 3 stage ovarian stroma, oviduct expanded tissue and pre and post ovulation follicles, and the cloning and sequence analysis of the ovalbumin related protein Y (Ovalbumin-Related Y Protein, OVALY) gene of the ovaries, follicles and oviduct histomorphology before and after ovulation in pigeon pigeons, the ovulation of pigeons was observed. Morphological observation of ovarian tissue in the 3 periods, histomorphological observation of oviduct enlargement and oviposit follicles were observed, and apoptosis rate was detected. It was found that there were 3 special stages in pigeon ovaries, including only grade follicles and pre grade follicles, only after ovulation and preovulatory follicles and only the stage of pre grade follicles. After ovulation, the collagen mainly exists in the outer layer of the membrane; the degeneration of the tissue may be associated with apoptosis. The area of the transverse section of the enlarged lumen of the dove oviduct is very different in 3 periods; there is an acidic mucus in the epithelial cells of the enlarged mucous epithelium before and after ovulation, and there is a obvious apoptotic phenomenon of.2. pigeon ovulation in the tissue of the oviduct after ovulation. The transcriptional study of ovarian stromal tissue in this experiment was conducted by RNA-Seq sequencing technology to analyze the gene expression in the ovarian stroma tissues of 3 pigeons. A total of 44784505 clean reads were obtained, and 14088 genes were obtained by comparison with the dove genome. In PI (Q1 period egg nest stroma) and P2 (Q2 phase ovarian matrix) 409 differentially expressed genes were found in the group, of which 96 genes were up-regulated in P1 and 313 genes were up-regulated in P2. GO function enrichment analysis for the up regulated genes in P2: immune response (GO: 0006955), receptor binding (GO:0005102), immune system process (GO: 0002376), bioadhesion (GO: 0022610) and antigen processing and presentation A significant enrichment of.KEGG pathway in biological processes, such as (GO: 0019882), showed that phagosome (clv04145), lysosome (clv04142), cytokine and cytokine receptor reaction (clv04060), cell adhesion molecules (clv04514) and Toll like receptor signaling pathway (clv04620) were significantly enriched. 53 immune related and 22 proteins were screened in P2. Candidate genes associated with tissue degradation, these genes may participate in the degenerative.3. ovulation after ovulation after ovulation. The RNA-Seq sequencing technique was used to analyze the gene expression in the follicle tissue of pigeons before and after ovulation. The average of 49487038 raw reads in each sample was obtained and the joint sequence was removed. After repeated sequences and low quality sequences, 47270639 high quality clean reads were obtained, which accounted for the 96%. of the original data and the comparison of the reference genes to 20282 genes. There were 5489 variable shear events in the gene expressed in the follicular tissue before and after ovulation, of which only 0.26% of the variable splices were differentially expressed. 6195 new transcripts, 492060 SNP loci and 33945 InDel loci were present. Differential expression gene analysis showed that 843 genes were up-regulated in P4 (Q1 follicle tissue), and 618 genes were up-regulated in P5 (Q2 follicle tissue). These differentially expressed genes were mainly enriched in the colloid protein trimer (G0:0005581) and the extracellular matrix structure. Components (G0:0005201), protein polymerization (G0:0051258), calcium ion binding (G0:0005509), protein binding bridge (G0:0030674) and other functions of.KEGG enrichment analysis showed that extracellular matrix receptor interaction (clv04512), adhesion plaque (clv04510), progesterone mediated oocyte maturation (clv04914), adrenaline signal (clv04261) of cardiac myocytes (clv04261), and swallowing. Phagocytic (clv04145), vascular smooth muscle contraction (clv04270), gap junction (clv04540), gonadotropin releasing hormone signaling pathway (clv04912) and other pathways were significantly enriched. In P4, 51 candidate genes related to extracellular matrix and its receptor were screened, and 17 related genes promoting the maturation of ovule and the close ovulating genes were selected, and 32 genes were screened in P5. Tissue degradation related candidate genes.4. pigeon oviduct bulking tissue transcriptional group before and after ovulation, RNA-Seq sequencing was used to analyze the gene expression in the oviductal tissue of different stages of pigeons. In this experiment, 56354181 rawreads were obtained for each sample to remove the joint sequence, repeat sequence and low quality. After the sequence, 54764938 high quality clean reads were finally obtained, which accounted for the 97.2%. of the original data and the comparison of the reference genes to 20767 genes. In the 3 time points of dove, there were a large number of exons leaping in the genes expressed in the oviduct enlargement tissue, and a small amount of exon selective jumping and minimal intron retention, no existence. The first or last exon variable shear events. Only 3.8% of the variable splices were differentially expressed. 6680 new transcripts, 819137 SNP loci and 53702 InDe1 loci were found. Differential expression gene analysis showed that in Cl (Q1 oviduct enlargement tissue) and C3 (Q3 oviduct enlargement tissue) The most differentially expressed genes are 3966, of which 2250 genes are up-regulated in C1, 1716. genes are expressed in C3, and the up-regulated genes are mainly enriched in the cell membrane structure, while C2 (Q2 stage oviduct tissue) is mainly enriched in organic matter, hormone related functions, and C3. The accumulation of differential genes involved in biological process regulation related function.KEGG pathway enrichment analysis showed that the differential genes up regulated in C1 mainly enriched endoplasmic reticulum protein processing (clv04141), and the differential genes up regulated in.C2 were mainly enriched in fatty acid metabolism (clv01212) and fatty acid degradation (clv000). 71), in the pathway of sugar degradation (clv00511), 62 candidate genes associated with protein transport were found in C1; 40 genes associated with apoptosis were found in C2, 17 genes associated with protein degradation and 18 candidate genes associated with lipid metabolism. These genes may be accumulated and retreated with the oviduct enlargement protein. The genes related to the main proteins in the egg white were up-regulated in C1. The OV4LY gene expression was the highest.5.OVA Y gene clone and bioinformatics analysis test to obtain the complete mRNA sequence of the OVALY gene of the white feather king pigeon. The sequence was 2068 BP, in which the 5'UTR sequence was 180bp, 3'UTR sequence 720bp and CDS sequences were 3. The 88 amino acid sequences, the molecular weight of about 44.19kDa. and the OVALY gene XI subtype (GeneBank:XM-005509738.1) of the NCBI pigeon are very similar. Only 4 single base mutation sites exist, which are located in the reference sequence 376CT, 730TC, 835TC, and 11109TC respectively. This sequence is with Zhu Xian (Nipponia nippon), white tailed eagle and sandy chicken. The homology of utturalis is high, which is 81%, 80%, and 79%., which has been submitted to the NCBI database. The login number of the KX230792.OVALY amino acid sequence has 22 phosphorylation sites and 4 N- junction glycosylation sites. By protein conservative domain analysis, it is found that OVALY belongs to the serine egg white enzyme inhibitor superfamily (Serpin superfamily). In the central region of the serine protease inhibitor family, the signal peptide site and the transmembrane structure were not found. To sum up, the tissue morphology of the ovarian matrix, the oviduct expansion and the follicular tissue before and after ovulation at three time points of pigeons was observed, and the reproductive system of pigeons was further recognized. Ovarian stromal tissues may be involved in follicular degeneration, screening candidate genes associated with follicle growth, development, maturation, degradation, and oviduct protein synthesis, accumulation, and tissue degradation. These differentially genes provide a reliable reference gene for the study of periodic changes in the related tissues in the reproductive system. Provide data support for the study of reproductive mechanism.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S836

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