【摘要】：牦牛是青藏高原及毗鄰地區(qū)集役、肉、奶、皮、絨等為一體的特有畜種,是牧民生產和生活資料的主要來源,由于特殊自然環(huán)境的限制,牦牛生產性能及繁殖性能普遍較低。探索利用體外胚胎生產技術和體細胞核移植技術等現代生物技術應用于牦牛,提高優(yōu)秀牦牛個體的擴繁速度,降低繁育成本,保存優(yōu)良牦牛遺傳資源,為牦牛的選育改良提供技術支持。本研究優(yōu)化牦牛體外受精和體細胞核移植胚胎的培養(yǎng)體系;利用RNA-seq技術篩選牦牛體外受精囊胚和體細胞核移植囊胚差異表達基因,并探討組蛋白甲基轉移酶抑制劑通過促進核重編程提高牦�？寺⌒实姆椒胺c對牛附植前胚胎發(fā)育和凋亡的影響。主要研究內容和結果如下:1.優(yōu)化了牦牛體外受精、體細胞核移植體系(1)A級COCs體外成熟培養(yǎng)24 h,COCs成熟率、卵裂率和囊胚率顯著高于B級和C級COCs;用Percoll液梯度離心分離精子法和BO液上浮法分離牦牛精子,用含3 mg/m L BSA和2.5 mM Theophylline的BO受精液中精子密度在1-5×106mL-1范圍受精效果良好;受精卵用TCM 199+卵丘細胞或TCM199+輸卵管上皮細胞的共培養(yǎng)體系效果顯著高于G1/G2液和SOF液培養(yǎng)體系。(2)用于體細胞核移植的牦牛卵母細胞體外成熟培養(yǎng)20 h后去核,耳源成纖維細胞作為供體細胞傳代次數為6代以內,血清饑餓2 d進行核移植;重構胚電融合參數為1.4-1.6 kv/cm、兩次脈沖,脈沖時長20μs,脈沖間隔10μs;離子霉素聯合6-DMAP進行激活4 h后使用G1/G2胚胎培養(yǎng)液進行培養(yǎng)。2.利用RNA-seq技術挖掘牦牛IVF和SCNT囊胚的差異表達基因(1)IVF-Blastocyst文庫和SCNT-Blastocyst文庫進行單細胞測序,分別獲得52,365,530和52,365,908 raw reads,過濾后分別得到48,810,404(93.21%)和49,328,624(94.20%)clean reads;與牦�；蚪M的比對率分別為74.18%和74.54%,其中Unique match比對率分別為64.88%和64.66%。clean reads與牦牛已知基因的比對率分別為52.36%和51.00%,其中Unique match的比對率分別為47.17%和45.25%。(2)通過比對,共有14,352個基因,其中12,395個基因共表達,837和1,120個基因分別特異性表達于ivf-blastocyst和scnt-blastocyst中。采用fdr≤0.001和abs(log2(y/x))≥1的標準共篩選出576個差異表達基因,其中342個基因高表達于scnt-blastocyst,234個基因低表達于ivf-blastocyst。(3)go富集顯示,576個差異表達基因被富集于150個cellularcomponentgo項目、218個molecularfunctiongo項目和1042個biologicalprocessgo項目中;顯著性分析,有4個cellularcomponentgo項目和2個biologicalprocessgo項目顯著富集。經注釋后表明差異基因主要參與調節(jié)胞外區(qū)(go:0044421和go:0005576)、脂蛋白復合物(go:0032994和go:0034358)以及炎癥和創(chuàng)傷反應(go:0006954和go:0009611)。(4)kegg通路分析576個差異表達基因參與218條通路,主要包括代謝通路、癌癥中的轉錄失調、類固醇激素生物合成、神經營養(yǎng)因子信號通路、補體與凝血級聯和細胞粘附分子。經顯著分析發(fā)現,其中僅戊糖、葡萄糖醛酸轉換(ko00040)達到顯著水平(qvalue0.05)。(5)隨機選取20個差異表達基因進行熒光定量pcr驗證,結果顯示egr1、cpeb1、fgfr2、slc2a4基因在scnt組中的表達量顯著上調,hspb1、wnt2b、loc102279723、cdca7l、polr2a、crabp1、kifc3、cdc25a、ddit3、sdc1、loc102282421基因在ivf組中的表達量顯著上調,且與rna-seq數據基本一致。(6)ivf-blastocyst和scnt-blastocyst分別預測出1,435和1,499個新轉錄本,通過編碼潛能預測發(fā)現分別有23.62%和22.28%的轉錄本被預測出具有編碼潛能。3.組蛋白甲基化轉移酶抑制劑對牦牛scnt胚胎發(fā)育的影響(1)低劑量gsk126(10μm和20μm)處理牦牛成纖維細胞形態(tài)和細胞活力未發(fā)生明顯變化,高劑量(40μm和80μm)處理組細胞質中出現黑點,活力下降,g0/g1期比例增加、s期和g2/m期比例明顯降低,凋亡細胞數增加并影響到細胞增殖和代謝。(2)gsk126處理牦牛成纖維細胞后核移植,發(fā)現低劑量組核重構胚囊胚的發(fā)育率、te細胞數和icm細胞數顯著高于高劑量處理組;而囊胚中凋亡細胞數顯著低于高劑量組。依據細胞活力、囊胚發(fā)育率、囊胚發(fā)育質量綜合評價20μmgsk126處理牦牛成纖維細胞24h的作為供體細胞進行核移植效果最佳。(3)分析IVF囊胚、SCNT囊胚和20μM GSK126處理的SCNT囊胚中組蛋白甲基化水平,發(fā)現GSK126處理后能降低核移植囊胚中H3K27me2和H3K27me3組蛋白甲基化水平,而對H3K4me2組蛋白甲基化水平沒有顯著影響。(4)分析胚胎凋亡和發(fā)育相關基因,發(fā)現GSK126處理后囊胚中凋亡基因BCL-2mRNA表達水平下降,Caspase-9 mRNA表達水平上升,同時能上調胚胎發(fā)育基因NANOG、OCT4和GATA3基因mRNA的表達水平。4.NaF對附植前胚胎發(fā)育和凋亡的影響(1)NaF處理卵母細胞后,呈劑量依賴性顯著抑制卵母細胞的成熟率、卵裂率和囊胚發(fā)育率;高劑量的NaF處理后卵母細胞內ROS含量顯著增加并抑制卵母細胞內T-AOC、GSH-Px、CAT和GSH活性從而誘導卵母細胞氧化應激。(2)隨Na F劑量的增加,卵母細胞中Caspase-3和Caspase-9活性顯著增加;凋亡基因Bax、Caspase-3和Caspase-9的mRNA表達水平升高,抗凋亡基因Bcl-2mRNA表達水平降低;同時,卵母細胞內凋亡蛋白Bax表達水平增加,抗凋亡蛋白Bcl-2的表達水平則隨之下降。(3)NaF處理卵母細胞后體外受精,囊胚總細胞數和滋養(yǎng)層細胞數以及內細胞團數量隨NaF劑量的增加而顯著減少,囊胚細胞內凋亡細胞數隨之增加;胚胎發(fā)育相關基因Oct4、Nanog、Sox2和Cdx2的mRNA表達水平隨著NaF劑量的增加而顯著降低。
[Abstract]:Yak is a special breed of livestock in Qinghai-Tibet Plateau and its adjacent areas, which integrates meat, milk, skin and cashmere. It is the main source of herdsmen's production and livelihood. Due to the limitation of special natural environment, the production performance and reproductive performance of yak are generally low. In this study, we optimized the culture system of in vitro fertilization and somatic cell nuclear transfer embryos of yak, screened the embryos of in vitro fertilization blastocysts and somatic cell nuclear transfer blastocysts of yak by RNA-seq technology. The main contents and results were as follows: 1. Optimizing in vitro fertilization of yaks, somatic cell nuclear transfer system (1) A-class COCs maturation culture in vitro for 24 hours, C OCs maturation rate, cleavage rate and blastocyst rate were significantly higher than those of grade B and grade C COCs; yak sperm were separated by Percoll fluid gradient centrifugation and BO fluid floatation, and the fertilization effect was good when the sperm density of BO fertilized with 3 mg/mL BSA and 2.5 mM Theophylline ranged from 1 to 5 x 106 mL-1; fertilized eggs were fertilized with TCM 199 + cumulus cells or TCM 199 + fallopian tubes. The effect of co-culture system of epithelial cells was significantly higher than that of G1/G2 and SOF. (2) Yak oocytes used for somatic cell nuclear transfer matured in vitro and cultured for 20 hours, then were denucleated. Otological fibroblasts were subcultured within 6 generations as donor cells, and serum starved for 2 days. Secondary pulses lasted 20 microseconds with a pulse interval of 10 microseconds; ionomycin combined with 6-DMAP was activated for 4 hours and then cultured in G1/G2 embryo culture medium. 2. Differentially expressed genes (1) IVF-Blastocyst library and SCNT-Blastocyst library were digged by RNA-seq technique to obtain 52,365,530 and 52,365,9,respectively. 08 raw reads, 48,810,404 (93.21%) and 49,328,624 (94.20%) clean reads were obtained after filtration, and the ratio of clean reads to yak genome was 74.18% and 74.54%, respectively. The ratio of Unique match was 64.88% and 64.66% respectively. The ratio of clean reads to known yak genes was 52.36% and 51.00%, respectively. 47.17% and 45.25%. (2) A total of 14,352 genes were identified, of which 12,395 genes were co-expressed, 837 and 1,120 genes were specifically expressed in ivf-blastocyst and scnt-blastocyst respectively. A total of 576 differentially expressed genes were screened by FDR < 0.001 and ABS (log2 (y/x) > 1, of which 342 genes were highly expressed in scnt-blastocyst and 2,120 genes were highly expressed in scnt-blastocyst. 34 genes were low expressed in ivf-blastocyst. (3) go enrichment showed that 576 differentially expressed genes were enriched in 150 cellular component go projects, 218 molecular function go projects and 1 042 biologic process go projects; significant analysis showed that 4 cellular component go projects and 2 biologic process go projects were significantly enriched. These results indicated that the differentially expressed genes were involved in regulating extracellular domain (go:0044421 and go:0005576), lipoprotein complexes (go:0032994 and go:0034358) and inflammatory and traumatic reactions (go:0006954 and go:0009611). (4) KEGG pathway analysis showed that 576 differentially expressed genes were involved in 218 pathways, including metabolic pathways, transcriptional disorders in cancer, steroid hormone production. Biosynthesis, neurotrophic factor signaling pathway, complement cascade, coagulation cascade and cell adhesion molecule. significant analysis showed that only pentose and glucuronic acid conversion (ko00040) reached significant levels (qvalue 0.05). (5) 20 differentially expressed genes were randomly selected for fluorescence quantitative PCR verification, and the results showed that egr1, cpeb1, fgfr2, slc2a4 genes in SCNT The expression levels of hspb1, wnt2b, loc102279723, cdca7l, polr2a, crabp1, kifc3, cdc25a, ddit3, sdc1, loc102282421 genes in IVF group were significantly up-regulated, and were consistent with the RNA-seq data. (6) ivf-blastocyst and scnt-blastocyst predicted 1,435 and 1,499 new transcripts respectively. 23.62% and 22.28% of transcripts were predicted to have coding potential respectively. 3. Effects of histone methylation transferase inhibitors on embryonic development of yak SCNT (1) The morphology and cell viability of yak fibroblasts treated with low doses of gsk126 (10 and 20 microns) did not change significantly, and black spots appeared in cytoplasm of yak fibroblasts treated with high doses (40 and 80 microns). (2) After nuclear transplantation of yak fibroblasts treated with gsk126, it was found that the development rate of nuclear reconstituted blastocysts in low dose group, the number of te cells and the number of ICM cells in blastocysts were significantly higher than those in high dose group. According to the cell viability, blastocyst development rate and blastocyst development quality, the best effect of nuclear transplantation was obtained when the yak fibroblasts were treated with 20 micron gsk126 for 24 hours. (3) Histone methylation levels in IVF blastocysts, SCNT blastocysts and SCNT blastocysts treated with 20 micron GSK126 were analyzed. H3K27me2 and H3K27me3 histone methylation levels in nuclear transfer blastocysts were decreased, but H3K4me2 histone methylation levels were not significantly affected. (4) Analysis of embryo apoptosis and development-related genes, it was found that GSK126 treatment of blastocysts apoptotic gene BCL-2 mRNA expression level decreased, Caspase-9 mRNA expression level increased, and at the same time, it could up-regulate embryo development. Effects of NaF on the development and apoptosis of preimplantation embryos (1) After treated with NaF, the maturation rate, cleavage rate and blastocyst development rate of oocytes were significantly inhibited in a dose-dependent manner; ROS content in oocytes significantly increased and T-AO was inhibited after treated with high dose of NaF. The activities of C, GSH-Px, CAT and GSH induced oxidative stress in oocytes. (2) The activities of Caspase-3 and Caspase-9 in oocytes increased significantly with the increase of Na F dosage, the mRNA expression levels of apoptotic genes Bax, Caspase-3 and Caspase-9 increased, and the expression levels of anti-apoptotic genes Bcl-2 mRNA decreased. With the increase of NaF dosage, the total number of blastocysts and trophoblast cells and the number of inner cell mass decreased significantly, and the number of apoptotic cells in blastocysts increased. The mRNA expression of embryonic development-related genes Oct4, Nanog, Sox2 and CDx2 increased. The level of NaF decreased significantly with the increase of dose.
【學位授予單位】：甘肅農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823.85
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本文編號：2228810