本文選題：鴿子 + 卵巢基質(zhì)��； 參考：《南京農(nóng)業(yè)大學》2016年博士論文

【摘要】：鴿子具有獨特的繁殖特點,目前對鴿繁殖活動的認知還處于初級觀察階段,相關(guān)領(lǐng)域研究也非常少,僅限于少量繁殖相關(guān)的單基因功能研究,飼養(yǎng)管理及環(huán)境對其繁殖性狀的影響。為了進一步認識鴿的繁殖過程,了解繁殖過程中繁殖相關(guān)組織的變化,本試驗對鴿Q1期(排卵前1-2d )、Q2期(第二個卵剛被排出,即一個蛋已經(jīng)產(chǎn)出,另一個蛋還在輸卵管中)、Q3期(排卵后6 ～7d) 3個階段的卵巢基質(zhì)組織和輸卵管膨大部組織,以及排卵前后的卵泡組織進行組織形態(tài)學觀察,采用高通量轉(zhuǎn)錄組測序法對3階段卵巢基質(zhì)、輸卵管膨大部組織以及排卵前后卵泡進行轉(zhuǎn)錄組學研究,以及卵清蛋白相關(guān)蛋白Y ( Ovalbumin-Related Y Protein, OVALY)基因克隆和序列分析。1.鴿排卵前后卵巢、卵泡、輸卵管組織形態(tài)學觀察本試驗對鴿排卵前后3個時期卵巢組織進行形態(tài)學觀察;對輸卵管膨大部以及排卵后卵泡進行組織形態(tài)學觀察,并進行凋亡率等檢測。發(fā)現(xiàn)在鴿卵巢存在3個特殊階段:只含等級卵泡和等級前卵泡、只含排卵后卵泡和等級前卵泡、僅有等級前卵泡的時期;排卵后卵泡的膠原質(zhì)主要存在于膜外層中;組織退化可能與凋亡相關(guān)。鴿子輸卵管膨大部管腔橫切面積在3個時期中差異很大;膨大部粘膜上皮細胞中在排卵前后存在酸性黏液;排卵后輸卵管膨大部組織中存在明顯的凋亡現(xiàn)象。2.鴿排卵前后卵巢基質(zhì)組織的轉(zhuǎn)錄組學研究本試驗采用RNA-Seq測序技術(shù)分析鴿3個時期卵巢基質(zhì)組織中基因表達情況。試驗總共得到了 44,784,505個clean reads,與鴿子的基因組比對獲得14,088個基因。在PI ( Q1期的卵巢基質(zhì)組織)和P2 ( Q2期的卵巢基質(zhì)組織)中共發(fā)現(xiàn)了 409個差異基因,其中有96個基因在P1中表達上調(diào)和313個基因在P2中表達上調(diào)。對P2中上調(diào)的基因進行GO功能富集分析:免疫應(yīng)答(GO: 0006955 )、受體結(jié)合(GO:0005102)、免疫系統(tǒng)進程(GO: 0002376 )、生物粘附(GO: 0022610)和抗原加工與呈遞(GO: 0019882 )等生物進程顯著富集。KEGG通路富集顯示,吞噬體(clv04145 )、溶酶體(clv04142)、細胞因子和細胞因子受體反應(yīng)(clv04060)、細胞粘附分子(clv04514 )和Toll樣受體信號通路(clv04620 )等通路顯著富集。在P2中篩選到了53個與免疫相關(guān),和22個與組織退化相關(guān)的候選基因,這些基因可能共同參與了排卵后卵泡組織的退化。3.排卵前后卵泡組織轉(zhuǎn)錄組研究本試驗采用RNA-Seq測序技術(shù)分析鴿排卵前后卵泡組織中基因表達情況。平均每個樣本獲得49,487,038個raw reads,去掉接頭序列、重復(fù)序列及低質(zhì)量的序列后,最終獲得47,270,639個高質(zhì)量的clean reads,占原始數(shù)據(jù)的96%。與參考基因比對,共匹配到20,282個基因。排卵前后的卵泡組織中表達的基因中存在5,489個可變剪切事件,其中僅有0.26%的可變剪切發(fā)生了差異表達。共發(fā)現(xiàn)了 6,195個新轉(zhuǎn)錄本,492,060個SNP位點和33,945個InDel位點。差異表達基因分析顯示,843個基因在P4 (Q1期卵泡組織)中表達上調(diào),618個基因在P5 (Q2期卵泡組織)中表達上調(diào)。這些差異基因主要富集在膠原蛋白三聚體(G0:0005581 )、細胞外基質(zhì)結(jié)構(gòu)成分(G0:0005201 )、蛋白質(zhì)聚合(G0:0051258 )、鈣離子結(jié)合(G0:0005509)、蛋白結(jié)合橋連(G0:0030674)等功能中。KEGG富集分析顯示:胞外基質(zhì)受體相互作用(clv04512)、粘著斑(clv04510)、孕激素介導的卵母細胞成熟(clv04914)、心肌細胞的腎上腺素信號(clv04261 )、吞噬體(clv04145)、血管平滑肌收縮(clv04270)、縫隙連接(clv04540)、促性腺激素釋放激素信號通路(clv04912)等通路顯著富集。在P4中共篩選了 51個與胞外基質(zhì)及其受體相關(guān)的候選基因,17個促進卵泡成熟、排卵緊密的相關(guān)基因;在P5中共篩選了 32個與組織退化相關(guān)的候選基因。4.鴿排卵前后輸卵管膨大部組織轉(zhuǎn)錄組學研究本試驗采用RNA-Seq測序技術(shù)分析鴿不同階段輸卵管膨大部組織中基因表達情況。本試驗中平均每個樣本獲得56,354,181個rawreads,去掉接頭序列、重復(fù)序列及低質(zhì)量的序列后,最終獲得54,764,938個高質(zhì)量的clean reads,占原始數(shù)據(jù)的97.2%。與參考基因比對,共匹配到20,767個基因。在鴿子3個時間點輸卵管膨大部組織表達的基因中存在大量的外顯子跳躍,少量的外顯子選擇性跳躍和極少的內(nèi)含子滯留,不存在第一個或最后一個外顯子可變剪切事件。其中僅有3.8%的可變剪切發(fā)生了差異表達。共發(fā)現(xiàn)了 6,680個新轉(zhuǎn)錄本,819,137個SNP位點和53,702個InDe1位點。差異表達基因分析顯示,在Cl (Q1期的輸卵管膨大部組織)和C3 (Q3期的輸卵管膨大部組織)間發(fā)現(xiàn)的差異表達基因最多,為3,966個,其中2,250個基因在C1中表達上調(diào),1,716.個基因在C3中表達上調(diào)。C1中表達上調(diào)基因主要富集在細胞膜結(jié)構(gòu)等功能。而C2(Q2期的輸卵管組織)中表達上調(diào)差異基因主要富集在有機質(zhì)反應(yīng)、激素相關(guān)功能;C3中表達上調(diào)差異基因主要參與生物進程調(diào)節(jié)相關(guān)功能。KEGG通路富集分析顯示,C1中表達上調(diào)的差異基因主要富集內(nèi)質(zhì)網(wǎng)蛋白加工(clv04141)、蛋白輸出(clv03060)等通路中。C2中表達上調(diào)的差異基因主要富集到脂肪酸代謝(clv01212)、脂肪酸降解(clv00071 )、糖降解(clv00511 )等通路中。在C1中共發(fā)現(xiàn)了 62個與蛋白轉(zhuǎn)運相關(guān)的候選基因;在C2中共發(fā)現(xiàn)了 40個與細胞凋亡相關(guān)基因,17個與蛋白質(zhì)降解相關(guān)基因,以及18個與脂代謝相關(guān)的候選基因。這些基因可能與輸卵管膨大部蛋白積累、組織退化緊密相關(guān)。在C1中與蛋清中主要蛋白相關(guān)的基因均表達上調(diào),其中OV4LY基因表達量最高。5.OVA Y基因克隆與生物信息學分析試驗獲得白羽王鴿OVALY基因mRNA完整序列,該序列全長2068 bp,其中5'UTR序列180bp,3'UTR序列720bp和CDS序列1164bp ,共編碼388個氨基酸序列,分子量約44.19kDa。與NCBI中巖鴿的OVALY基因XI亞型(GeneBank:XM-005509738.1 )相似性極高,只存在4個單堿基突變位點,分別位于參考序列376CT、730TC、835TC、11109TC 處。該序列與朱瀗(Nipponia nippon)、白尾鷹(Haliaeetus albicilla)和沙雞(Pterocles gutturalis)的同源性較高,分別為 81%、80%、79%。該數(shù)據(jù)已提交到NCBI數(shù)據(jù)庫,登錄號為KX230792。OVALY氨基酸序列共有22個磷酸化位點和4個N-連接糖基化位點。經(jīng)蛋白質(zhì)保守結(jié)構(gòu)域分析發(fā)現(xiàn)OVALY屬于絲氨酸蛋白酶抑制劑超家族(Serpin superfamily),具有一個絲氨酸蛋白酶抑制劑家族的反應(yīng)中心區(qū)域,未發(fā)現(xiàn)信號肽位點和跨膜結(jié)構(gòu)。綜上,本文對鴿三個時間點的卵巢基質(zhì)、輸卵管膨大部,以及排卵前后的卵泡組織進行了組織形態(tài)觀察,對鴿繁殖系統(tǒng)有了進一步的認識。轉(zhuǎn)錄組學研究發(fā)現(xiàn)卵巢基質(zhì)部分組織可能參與了卵泡退化;篩選到大量與卵泡生長、發(fā)育、成熟、退化相關(guān)候選基因,與輸卵管蛋白合成、積累、組織退化等相關(guān)的候選基因。這些差異基因為繁殖系統(tǒng)中相關(guān)組織周期性變化研究提供一個可靠的參考基因庫,為繁殖機理研究提供數(shù)據(jù)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.

【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S836

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