【作者】 李楠；

【导师】 石德顺； 陆凤花；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 本研究主要探讨O-GlcNAc糖基化在水牛胚胎早期发育过程中的作用机理和水牛转基因克隆的相关影响因素,以便建立一套较为完善的水牛转基因克隆技术体系。具体的研究结果如下。1、建立了水牛早期胚胎O-GlcNAc糖基化蛋白和(beta-O-linked N-glycosylation, O-GlcNAcylation)相关基因的表达检测方法。免疫荧光染色抗体浓度优化的研究结果发现,当一抗浓度为1：200,二抗浓度为1：1000时,可清晰地观察到经抗体免疫后O-GlcNAc蛋白的分布,可用于O-GlcNAc蛋白的表达情况检测分析。为建立O-GlcNAc糖基化相关基因(GFPT、OGT和OGA)的mRNA表达荧光定量PCR检测分析方法,根据NCBI中GenBank所提供的人或牛GFPT、OGT、OGA基因的CDS序列设计水牛相关基因的PCR引物,克隆得到水牛的GFPT、OGT和OGA基因CDS序列,然后根据该序列设计合成实时荧光定量PCR引物。结果显示,PCR的CDS产物无杂带,条带均一,为特异性的目的片段扩增产物；QRT-PCR的熔解温度均一,熔解曲线表现为尖锐的单一峰,证实引物的有效性,说明该QRT-PCR体系可用于检测水牛早期胚胎GFPT、OGT和OGA基因的表达。2、以水牛孤雌激活(PA)、体外受精(IVF)和体细胞核移植(SCNT)早期胚胎为研究对象,研究分析了水牛早期胚胎发育过程中O-GlcNAc蛋白及相关基因(GFPT、OGT和OGA)的表达模式。免疫荧光染色结果显示,水牛PA、IVF和SCNT胚胎O-GlcNAc糖基化蛋白水平呈现相似地变化趋势,即从2-细胞期开始慢慢增加,到囊胚期达到最高；其中,IVF胚胎各阶段O-GlcNAc蛋白水平均显著高于SCNT和PA组(P<0.05),而SCNT胚胎各发育阶段O-GlcNAc蛋白水平显著高于PA组(P<0.05)。QRT-PCR的检测结果发现,水牛PA.IVF和SCNT胚胎的GFPT基因mRNA表达水平呈现先升后降的变化趋势,即从2-细胞到4-Cell期表达量升至最高,然后逐渐下降,到囊胚期达到最低;其中,GFPT的表达量在IVF胚胎各时期中表达量最高,均显著高于PA禾SCNT胚胎组(P<0.05),而SCNT胚胎组在8-细胞期后GFPT的表达量显著高于PA组(P<0.05)。OGT基因的mRNA表达量随着胚胎的发育逐渐增加,至囊胚期达到最高；其中,IVF胚胎各阶段OGT的mRNA表达量均显著高于PA胚胎(P<0.05),而在8-细胞期和桑椹胚期显著高于SCNT胚胎(P<0.05)。OGA基因的mRNA表达量从2-细胞期到4-细胞期呈下降趋势,而后又逐渐升高,到8-细胞期表达量达到最高,而后开始下降,到囊胚期降到最低,且IVF各个发育时期的胚胎OGA的mRNA表达量显著高于PA胚胎(P<0.05),但SCNT的8-细胞期和囊胚期胚胎OGA的mRNA表达量显著低于IVF胚胎(P<0.05)。3、研究探讨了糖基化底物—氨基葡糖(glucosamine, GlcN)对水牛早期胚胎发育、O-GlcNAc蛋白及相关基因表达水平的影响。当在PA、IVF和SCNT胚胎培养的0-72h添加不同浓度的GlcN(0、1、2和4mmol/L)时,2mmol/L组的囊胚率均显著高于对照组(23.75%vs13.38%；26.16%vs14.29%；25.52%vs12.70%,P<0.05),而各组之间分裂率差异不显著(P>0.05)。IF染色观察的结果显示,在各类水牛早期胚胎培养的0～72h添加2mmol/L GlcN, PA胚胎发育各阶段O-GlcNAc蛋白水平显著高于对照组(P<0.05)；IVF胚胎除囊胚期外,其它各发育阶段O-GlcNAc蛋白水平均显著高于对照组(P<0.05); SCNT胚胎除8-细胞和桑椹胚期外,其它各发育阶段均显著高于对照组(P<0.05)。QRT-PCR检测结果显示,在水牛胚胎培养的0-72h添加2mmol/L GlcN, PA胚胎各发育时期的GFPT基因mRNA表达量均显著高于对照组(P<0.05)；而IVF胚胎在4-细胞期和SCNT胚胎在4-细胞和8-细胞期的GFPT表达量均显著高于对照组(P<0.05),其它各发育阶段与对照组差异均不显著(P>0.05)。在胚胎OGT的表达方面,PA胚胎2mmol/L GlcN添加组在4-细胞期、IVF胚胎在囊胚期和SCNT胚胎2-细胞期和囊胚期的OGT表达量与对照组差异不显著(P>0.05),其余各阶段的OGT表达量均显著高于对照组(P<0.05)。在胚胎OGA的表达方面,IVF胚胎2mmol/L GlcN添加组在4-细胞期、8-细胞期、桑椹胚和囊胚期,SCNT胚胎在4-细胞期OGA表达量与对照组差异不显著(P>0.05),其它各阶段均显著低于对照组(P<0.05)。研究发现,在水牛胚胎发育0-72h间添加适宜浓度的O-GlcNAc蛋白糖基化底物(2mmol/L GlcN)有利于提高早期胚胎的发育能力；而且也可以促进早期胚胎O-GlcNAc蛋白的表达,上调OGT和GFPT基因,下调OGA基因的表达水平,从而促进胚胎的发育。4、主要探讨了影响供体细胞转染效率及转基因克隆胚胎发育的相关因素。电穿孔法、脂质体法、磷酸钙法及慢病毒法转染水牛胎儿成纤维细胞(BFF)筛选获得的转基因细胞作为核移植供体韵研究结果显示,电穿孔法构建的重构胚表达标记基因EGFP的阳性分裂率(48.00%)和阳性囊胚率(18.58%)极显著高于磷酸钙法(0%和0%)、慢病毒介导法(0%和0%)和脂质体包埋法(5.60%和3.72%)。采用电穿孔法二次转染BFF,其重构胚分裂率和囊胚率均略高于一次转染,但是差异不显著(71.23%vs68.37%；24.66%vs18.37%,P>0.05)。将内部核糖体接入位点(IRES)插入到表达载体与未插入IRES基因的表达载体比较发现,表达载体pEGFP-IRES-NEO的转基因克隆重构胚分裂率、阳性分裂率、阳性囊胚率均显著高于pEGFP-N1表达载体(71.43%vs57.77%；39.14%vs8.35%;17.64%vs5.20%,P<0.01)。将pEGFP-IRES-NEO表达载体进行单切或双切后分别转染供体细胞,单切载体重构胚的阳性分裂率和阳性囊胚率高于双酶切载体,但两组间差异不显著(37.39%vs32.41%；9.66%vs8.30%,P>0.05)。同时,在比较不同代数(6-10代)的供体细胞构建转基因克隆重构胚时发现,重构胚早期胚胎分裂和囊胚期EGFP表达率差异不显著(P>0.05)。不同性别的供体细胞转染EGFP基因用于构建核移植重构胚的结果显示,雌性细胞组阳性分裂率和阳性囊胚率显著高于雄性供体细胞组(67.63%vs42.76%；22.54%vs10.52%,P<0.05),分裂率和囊胚率也高于雄性供体细胞组,但差异不显著(71.10%vs68.42%；26.59%vs21.71%,P>0.05)。将得到的含有EGFP基因的6-8天囊胚进行胚胎移植,得到了表达EGFP的转基因克隆水牛5头,双胞胎中的一头不幸死亡。经过鉴定确认转基因克隆水牛表达EGFP基因。此外,用转干扰素(IFN)、生长素(GH)、促乳素(PRL)基因的供体细胞进行核移植,三种基因的转基因克隆胚胎分裂率、阳性分裂率、囊胚率和阳性囊胚率差异均不显著(66.44%vs62.50%、59.23%；54.11%vs57.35%、51.63%；17.81%vs19.12%、20.65%；16.44%vs13.60%、14.67%,P>0.05),且经胚胎移植给受体水牛后获得妊娠。上述结果表明：(1)电穿孔法较磷酸钙法、脂质体法和慢病毒转染法更适合制备转基因克隆水牛,使用该法能获得转基因克隆水牛；(2)水牛转基因克隆效率受供体细胞性别的影响,雌性胎儿成纤维细胞优于雄性,但不受供体细胞转染次数、代数以及载体的酶切方法的影响；(3)携带GH、IFN和PRL的转基因载体能够用于水牛转基因克隆胚胎生产,且胚胎移植后均可以在体内妊娠并进一步发育。5、探讨了Trichostatin A (TSA)处理供体细胞和转基因克隆重构胚对水牛转基因细胞表达EGFP及克隆重构胚发育的影响。流式细胞仪检测分离、纯化后的供体细胞结果显示,随着转EGFP供体细胞代数的增加(7-11代),表达EGFP细胞的数目有所降低,差异不显著(P>0.05)。将转EGFP基因的细胞分别用浓度为5nmol/L、10nmol/L、25nmol/L、50nmol/L TSA处理24h后显著提高细胞G0/G1期的比例,且从10nmol/L起表达EGFP细胞的数目开始显著地增加(P<0.05)。 TSA浓度为5nmol/L、10nmol/L时,乙酰化水平显著高于对照组(P<0.05),且供体细胞DNA甲基化水平显著低于对照组(P<0.05)。TSA处理24h供体细胞后进行核移植的结果发现,TSA浓度为10nmol/L时,重构胚的分裂率(85.71%vs60.40%)、阳性分裂率(84.82%vs55.45%)、囊胚率(49.11%vs26.73%)和阳性囊胚率(49.11%vs21.78%)显著高于对照组(P<0.05)。将转基因克隆重构胚后用5nmol/L、10nmol/Lv50nmol/L或100nmol/L的TSA处理6h后发现,50nmol/L TSA组的重构胚的分裂率(83.33%vs69.70%)、阳性分裂率(82.41%vs60.61%)、囊胚率(45.37%vs29.29%)和阳性囊胚率(43.52%vs21.21%)均显著高于对照组(P<0.05)。用50nmol/L TSA对重构胚分别处理3h、6h、9h或12h后发现,处理时间为9h的重构胚分裂率(86.17%vs74.31%)、阳性分裂率(82.20%vs70.64%)、囊胚率(49.15%vs33.94%)和阳性囊胚率(47.46%vs32.11%)均显著高于对照组(P<0.05),其余各组与对照组相比差异不显著。此外,水牛转基因克隆胚胎的乙酰化水平及相关基因的表达水平检测结果显示,50nmol/L TSA处理重构胚9h,能显著提高胚胎的乙酰化水平(P<0.05),显著提高HAT1基因的表达(P<0.05),显著降低HDAC1基因的表达(P<0.05)。以上结果表明：(1)转EGFP的水牛胎儿成纤维细胞随着细胞代数的增加(7-11代),表达EGFP细胞的数目呈逐渐降低的趋势；(2)采用10nmol/L的TSA处理供体细胞24h不但可以显著提高转EGFP基因水牛胎儿成纤维细胞处于G0/G1期的比例和表达EGFP细胞的数量,而且能提高细胞组蛋白H3K14组蛋白乙酰化水平,且降低细胞DNA甲基化水平,提高其核移植后的转基因克隆胚胎的体外发育率；(3)适当浓度(50nmol/L)的TSA处理水牛转基因克隆重构胚一定时间(9h),能提高水牛转基因克隆胚胎的乙酰化水平,且胚胎的HAT1表达水平显著上升,HDAC1的表达水平显著下降,从而促进水牛转基因克隆胚胎的发育能力。更多还原

【Abstract】 The objective of this research is to explore the glycosylation mechanism of buffalo preimplantation embryos and factors affecting the efficiency of buffalo transgenic cloning, so as to establish an effective technique system for producing transgenic cloning buffalos. Works are summarized in following.1. Methods to detect the expression of O-GlcNAc (beta-O-linked N-glycosylation, O-GlcNAcylation) gene in buffalo embryos was set up. In immunofluorescence (IF) analysis, the appropriate concentration of the first antibody was proved to be1:200and concentration of the second antibody was proved to be1:1000, in which the protein of O-GlcNAc can be observed clearly after IF staining. In order to set up a method to detect the mRNA expression of O-GlcNAc using real-time fluorescence quantitative PCR, the CDS sequences of buffalo glutamine:fructose6-phosphate amidotransferase (GFPT), O-GlcNAc transferase (OGT) and O-GlcNAc-selective N-acetyl-β-D-glucosaminidase (OGA) genes were cloned using the primers designed according to the CDS sequence of human and bovine GFPT, OGT and OGA genes in GenBank. Then, the primers were designed and synthesized for real-time fluorescence quantitative PCR according to the cDNA sequence of three buffalo genes. The band of CDS was uniform and defined as the specific PCR product of target gene segment. The liquating temperature of QRT-PCR was uniform and liquating curve displayed a single peak, indicating that the primer is efficient and can be employed for detecting the expression of GFPT, OGT and OGA genes in the early buffalo embryos.2. The expression patterns of O-GlcNAc and expression of GFPT, OGT and OGA in the development of buffalo embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) was investigated using IF and QRT-PCR. The O-GlcNAc protein displayed a similar change pattern in the development of PA, IVF and SCNT embryos from the2-cell stage to the blastocyst stage, in which the O-GlcNAc begun to increase from the2-cell stage and arrived at a peak at the blastocyst stage. The level of O-GlcNAc in IVF embryos was significantly higher than PA and SCNT embryos (P<0.05), and level of O-GlcNAc in SCNT embryos was also significantly higher than PA embryos (P<0.05). QRT-PCR revealed that the level of GFPT mRNA in the three types of embryos increased from the2-cell stage to the4-cell stage, and then decreased till to the blastocyst stage. Among the three types of embryos, the GFPT expression level in IVF embryos was significantly higher than PA embryos and SCNT embryos (P<0.05), and SCNT embryos from the8-cell stage to the blastocyst stage was significantly higher than PA embryos (P<0.05). The expression of OGT increased gradually from the2-cell to the blastocyst stage, and arrived at the highest level in the blastocyst stage in the three type embryos. The OGT expression level in IVF embryos was significantly higher than PA embryos (P<0.05) and SCNT embryos from the8-cell stage to the morula stage (P<0.05). The OGA mRNA levels decreased from the2-cell to the4-cell stage, increased from the4-cell stage to the8-celln stage, and then decreased again. The OGA expression level in IVF embryos was significantly higher than PA embryos (P<0.05) and SCNT embryos at the8-cell stage and blastocyst stage (P<0.05).3. Effects of glucosamine (GlcN) on the in vitro development of buffalo PA, IVF and NT embryos, and expression of O-GlcNAc and its related genes were investigated. When buffalo embryos derived from PA, IVF and SCNT were cultured in the medium supplemented with different concentrations of GlcN (0mmol/L,1mmol/L,2mmol/L, and4mmol/L) during0to72h, addition of2mmol/L GlcN resulted in significantly higher blastocyst yield in comparison with other groups (PA:23.75%vs13.38%; IVF:26.16%vs14.29%; SCNT:25.52%vs12.70%, P<0.05). Therer was not significant difference in cleavage rate among the groups (P>0.05). Meanwhile, the O-GlcNAc level of embryos was higher than control group (P<0.05) with the exceptions of IVF blastocysts, eight-cell and morula SCNT embryos. In addition, the expression level of GFPT was also significantly higher than control (P<0.05) with the exceptions of IVF embryos at8-cell, morula and blastocyst stage, SCNT embryos at morula and blastocyst stage. As to the OGT expression, addition of2mmol/L GlcN to culture medium from0h to72h resulted in higher expression of OGT in the three type embryos with the exceptions of PA embryos at4-cell stage, IVF embryos at blastocyst stage and SCNT embryos at2-cell and blastocyst stage. The OGA expression of embryos cultured in the medium supplemented with2mmol/L GlcN during0-72h was lower than control groups (P<0.05) with the exceptions of IVF embryos. These results indicate that supplementation of2mmol/L GlcN into medium during culture of0-72h can improve the development of buffalo embryos, increases the expression of O-GlcNAc, OGT and GFPT, decreased the expression of OGA.4. Factors affecting the transfection effciciency of somatic cells and in vitro development of buffalo transgenic cloning embryos were investigated. Plasmide containing marker gene EGFP was transfected into the buffalo fetal fibroblast cells (BFF) by CaHPO4, liposome, electro-transfection and lentivirus respectively, and the transgenic-cells were used as donor cells for nuclear transfer. The percentage of EGFP-positive cleaved oocytes (48.00%) and EGFP-positive blastocysts (18.58%) was higher in SCNT embryos derived from donor cells by electro-transfection than lentivirus infection (0%&0%), CaHPO4transfection (0%&0%) and liposome transfection (5.60%&3.72%)(P<0.01). There was not different in cleavage rate and blastocyst yield between BFF transfected one and two times with electro-transfection (71.23%vs68.37%;24.66%vs18.37%, P>0.05). The cleavage rate, EGFP-positive cleavage rate and EGFP-positive blastocyst rate of SCNT embryos reconstructed with BFF containing pEGFP-IRES-NEO were significantly higher than pEGFP-N1group (71.43%vs57.77%;39.14%vs8.35%;17.64%vs5.20%, P<0.01). The cleavage and blastocyst rate of transgenic cloning embryos from pEGFP-IRES-NEO vector digested with double enzymes was not different from the vector digested with BamH I single enzyme (37.39%vs32.41%;9.66%vs8.30%, P>0.05). There was not significantly different in the embryonic development among embryos reconstructed with fetal fibroblasts derived from different generation (6-10generation)(P>0.05). The EGFP-positive cleavage rate and blastocyst rate of embryos derived from female BFF was significantly higher than male BFF (67.63%vs42.76%;22.54%vs10.52%; P<0.05). Five transgenic cloning swamp buffalos that express GFP gene were obtained following embryo transfer and one of them died at delivery. When buffalo fibroblast cells that had been transfected with IFN, GH and PRL were used as donor cells for production of transgenic cloning embryos, there was not significantly different in the cleavage rate, EGFP-positive cleaved eggs, blastocyst yield and EGFP-positive blastocysts among the three transgenic donor cells (66.44%vs62.50%,59.23%;54.11%vs57.35%,51.63%;17.81%vs19.12%,20.65%;16.44%vs13.60%,14.67%, P>0.05), and pregnancies were established following transfer of these embryos into recipients. In conclusions of these results,(1) electro-transfection is more suitable in preparing transgenic cloning buffalos than lentivirus, liposome and CaHPO4, and transgenic cloning buffalos can were obtained using this method;(2) buffalo transgenic cloning efficiency is related to the donor cell sex rather than transfection times, enzyme digestion method of vectors and passages of donor cells, female donor cells are superior to male donor cells;(3) buffalo transgenic cloning embryos can be produced using the plasmide containing IFN, GH or PRL genes, and can develop further after transfer into recipients.5Effects of TSA on the development of buffalo transgenic cloning embryos and EGFP expression were investigated. Results of flow cytometry analysis indicated that percentage of cells expressed EGFP trended to decrease with the increase of cell passages (7-11passages). Treatment of cells with5nmol/L,10nmol/L,25nmol/L or50nmol/L TSA resulted in higher proportion of G0/G1cells and EGFP-positive cells (P<0.05). In addition, treatment of cells with5nmol/L or10nmol/L decreased their DNA methylation and increased their histone acetylation (P<0.05). When donor cells were treated with10nmol/L TSA for24h before nuclear transfer, the cleavage rate (85.71%vs60.40%), EGFP-positive cleavage eggs (84.82%vs55.45%), blastocyst yield (49.11%vs26.73%) and EGFP-positive blastocysts (49.11%vs21.78%) were increased in comparison with the control group (P<0.05). When the transgenic cloning embryos were treated with5nmol/L,25nmol/L,50nmol/L and100nmol/L TSA for6h respectively, treatment with50nmol/L TSA resulted in higher cleavage rate (83.33%vs69.70%), EGFP-positive cleaved eggs (82.41%vs60.61%), blastocyst yield (45.37%vs29.29%) and EGFP-positive blastocysts (43.52%vs21.21%) in comparison with control group (P<0.05). When reconstructed embryos were treated with50nmol/L TSA for3h,6h,9h and12h, treatment for9h resulted in higher cleavage rate (86.17%vs74.31%), EGFP-positive cleaved eggs (82.20%vs70.64%), blastocyst yield (49.15%vs33.94%) and EGFP-positive blastocysts (47.46%vs32.11%) in comparison with control group (P<0.05). In addition, treatment of transgenic cloning embryos with50nmol/L TSA for9h increased their acetylation level of histone and HAT1expression, decreased the expression of HDAC1(P<0.05). In conclusions,(1) proportion of BFF expressed EGFP trends to decrease as their passage number increases;(2) treatment of donor cells with10nmol/L TSA can increase their proportion in G0/G1phases, expressed EGFP and H3K14acetylation of histone, reduce DNA methylation level, and then improve the development of transgenic cloning embryos reconstructed with these cells;(3) treatment of transgenic cloning embryos with50nmol/L TSA for9can increase their histone acetylation level, expression level of HAT1, reduce the expression of FIDAC1,and then improve their subsequent development.更多还原