【作者】 刘振山；

【导师】 谢庄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 牦牛是青藏高原及其毗邻地区不可或缺的全能家畜,对高寒气候等恶劣环境有极强的适应性,但其生产性能较低。为了提高牦牛的生产性能,我国从上世纪50年代开始采用良种黄牛与牦牛进行杂交,杂种一代犏牛表现出很强的杂种优势,但其雄性不育使得杂种优势的利用受到了极大的限制,成为牦牛杂交改良的“拦路虎”。为了探讨犏牛雄性不育问题,本研究以成年健康雄性犏牛、黄牛和牦牛为研究对象,运用同源扩增、PCR克隆测序方法获得牦牛印记基因或精子发生相关基因差异甲基化区（DMR）序列,利用Real-time PCR技术检测了这些基因在犏牛及其亲本睾丸组织中的表达差异,运用亚硫酸氢钠测序法检测了相关基因DMR甲基化状态,为研究犏牛雄性不育与DNA甲基化的关系提供理论基础。1.牦牛H19/IGF2（?）记控制区（ICR）的分子克隆及序列分析本实验根据黄牛H19基因DNA序列NW₀01494548设计引物扩增出牦牛H19基因长约6.5kb的序列（GenBank登录号：EU502716）,该序列包含完整的印记控制区（ICR）、启动子和部分第一外显子。用生物信息学方法,将该序列与黄牛、绵羊猪、人类和小鼠的相应序列比对分析,发现牦牛与他们的相似度分别为97%、83.13%、48%、47%、42%；该扩增序列中共有6个CpG岛,长度分别为1182、1186、707、277、281和1311bp；有6个锌指蛋白CTCF结合位点,均位于CpG岛内,分别定位于转录起始部位上游5.5kb、5.1kb、4.1kb、3.7kb、2.7kb和1.3kb；H19基因5′端存在核心启动子区,TATA box、GC box及Sp1、Sp2、AP2、NF-κB、STAT5、P53、 WT1、GFI1、MTBP、CPBP、ZF9、E2F知Myc-Max等识别位点。2.犏牛及其亲本睾丸组织中H19基因mRNA表达水平、DNA甲基化状态差异分析本实验运用Real-time PCR技术对犏牛及其亲本睾丸组织中H19基因表达水平进行了分析,结果发现,犏牛的表达水平极显著高于黄牛和牦牛（P<0.01）,而黄牛和牦牛两者之间表达差异不显著（P>0.05）。采用亚硫酸氢钠测序法检测了包含CTCF结合位点3（CTCF-binding site III）的H19ICR的甲基化状态,结果发现,犏牛H19ICR的甲基化水平（48.82%）极显著低于黄牛（70%）（P<0.01）,显著低于牦牛（59.10%）（P<0.05）,但黄牛的甲基化水平显著高于牦牛（P<0.05）；犏牛CTCF结合位点3的甲基化水平（48.33%）极显著低于黄牛（75%）和牦牛（68%）（P<0.01）。说明H19ICR及ICR中的CTCF结合位点对H19基因表达调节具有重要作用。3.犏牛及其亲本睾丸组织中IGF2基因mRNA表达水平、DNA甲基化状态差异分析本实验根据黄牛IGF2基因组序列DQ298740设计引物扩增出了牦牛IGF2基因第十外显子部分序列,GenBank登陆号为FJ152104；通过Real-time PCR检测了犏牛及其亲本睾丸组织中IGF2基因的表达水平,运用亚硫酸氢钠测序法检测了睾丸组织中IGF2基因第十外显子DMR的甲基化状态。结果发现,犏牛睾丸组织中IGF2基因mRNA表达水平最低,与亲本的表达水平差异极显著（P<0.01）；黄牛、牦牛和犏牛睾丸中IGF2DMR的甲基化水平分别为90%、90.7%和92.0%,三组之间均未见显著差异（P>0.05）。实验结果说明IGF2基因mRNA表达水平与牛精子发生有关,可能在牛的精子发生过程中发挥重要作用,并可能与犏牛雄性不育有关；IGF2基因第十外显子DMR区的甲基化与IGF2基因在犏牛及其双亲睾丸中的表达差异无关,可能是其他的机制参与调节IGF2基因的表达。4.犏牛及其亲本睾丸组织中印记基因SNRPN的表达及DMR甲基化分析本实验根据黄牛SNRPN基因序列NW935049设计引物扩增出了牦牛SNRPN基因5′端序列,长为1137bp,与黄牛参照序列相似度达98.2%；经生物信息学分析,发现含有YY1和SP1等甲基化敏感位点。通过Real-time PCR检测了犏牛及其亲本睾丸组织中SNRPN基因的表达水平,运用亚硫酸氢钠测序法检测了睾丸组织中SNRPN基因5′端DMR的甲基化状态。结果发现,黄牛、牦牛和犏牛睾丸组织中SNRPN基因mRNA表达差异不显著（P>0.05）,但黄牛和牦牛SNRPN基因的表达高于犏牛；犏牛SNRPN DMR的甲基化水平（38.53%）极显著高于黄牛（21.08%）和牦牛（20.81%）的（P<0.01）。说明犏牛SNRPN DMR区的高甲基化本身还不足以引起SNRPN基因表达水平的显著变化,还有其它机制参与SNRPN基因的表达调节。5. DAZL基因5′端CpG岛DNA甲基化水平及其与犏牛雄性不育的关系研究前期研究发现LDAZL基因与犏牛雄性不育相关,是犏牛雄性不育的候选基因。为了进一步研究调控DAZL基因表达的机制,本实验根据黄牛DAZL基因序列（NC₀07299, complement:141791034..141818671）设计引物扩增了牦牛DAZL基因的5′端序列,并对牦牛、黄牛和犏牛DAZL基因的甲基化水平进行了分析,结果发现：牦牛DAZL基因的5′端存在CpG岛,CpG岛长1744bp（EU686694：650-2393）,包含启动子区、第一外显子和第一内含子；犏牛DAZL5′端DNA甲基化水平（85.6%）极显著高于黄牛（71.4%）和牦牛（69.8%）（P<0.01）。可见犏牛LDAZL基因的高甲基化与其nRNA表达缺乏、雄性不育的表型是一致的,说明DAZL基因的甲基化对DAZL基因mRNA的表达调控起重要作用,可能对犏牛生精细胞减数分裂、雄性不育有重要影响。更多还原

【Abstract】 The yak, known as the "almighty livestock", is indispensable in the region of the Qinghai-Tibet Plateau and nearby areas, and is well adapted to high altitude environments. However, it is primitive and grows slowly, resulting in low production performance. To improve its productivity, hybridization between cattle and yaks has been carried out since the1950s. Cattle yaks, the F1hybrid between cattle and yaks, exhibit significant hybrid vigor. However, the males are sterile, which greatly restricts the utilization of this hybrid vigor, and is a "stumbling block" in the varietal improvement of the yak. To investigate the mechanism of male sterility of cattle yaks, in present study, the differentially methylated region （DMR） sequences of imprinting genes and spermato genesis-related gene in yak were obtained using homogenetic amplification, the expression of genes in cattle yaks and their parents was investigated by Real-time PCR, and the methylation patterns were examined using bisulfite sequencing. This would provide evidence for the epigenetic mechanism of sterility of cattle yaks.1. Molecular Cloning and Sequence Analysis of H19/IGF2Imprinting Control Region （ICR） in Yaks （Bos grunniens）Primers were designed from the sequence of the cattle H19DNA （NW₀01494548）, sequencing and assembly led to the establishment of the complete nucleotide sequence （6.5kb） of the H19ICR, promoter, and partial exon1. The sequence was submitted to GenBank under accession number EU502716. Sequence analysis revealed that the homology between yak and cattle, sheep, pig, human and mouse, was97%,83.13%,48%,47%,42%, respectively; six CpG islands were identified, and their lengths were1182,1186,707,277,281, and1311bp, respectively;6CTCF-binding sites were identified in HI9ICR, and they were all present in the CpG islands and located at approximately5.5kb （site1）,5.1kb （site2）,4.1kb （site3）,3.7kb （site4）,2.7kb （site5）, and1.3kb （site6） upstream of the transcription initiation site, respectively; computer assisted analysis revealed that the 5’-flanking region consisted of a putative core promoter region,2GC boxes, and several binding sites for Sp1, Sp2, AP2, NF-κB, STAT5, P53, WT1, GFI1, MTBP, CPBP, ZF9, E2F, and Myc-Max.2. Analysis of H19mRNA expression and its methylation status in testes between cattle yaks and their parentsBy Real-time PCR the expression of testes H19mRNA in cattle yaks and their parents was investigated. The result showed that the H19expression in cattle yaks was the lowest; and there was statistically significant difference between cattle yaks and their parents （P<0.01）, but no significant difference between cattle and yak （P>0.05）.The methylation patterns of H19ICR which contained CTCF-binding site III was examined in testis using bisulfite sequencing. The result showed that the methylation level of H19ICR in cattle yaks （48.82%） was significantly lower than that in cattle （70%）（P<0.01） and yaks （59.10%）（P<0.05）, and there was significant difference between cattle and yaks （P<0.05）; the methylation level of CTCF-binding site III in cattle yaks （48.33%） was significantly lower than that in cattle （75%） and yaks （68%）（P<0.01）. This indicated that the methylation status of H19ICR and CTCF-binding sites played an important role in transcriptional regulation.3. Analysis of IGF2mRNA expression and its methylation status in testes between cattle yaks and their parentsBased on the cattle IGF2gene （DQ298740）, primers were designed, and the partial sequence of IGF2exon10in yaks was obtained （GenBank No. FJ152104）. the IGF2mRNA expression of testes in cattle yaks and their parents was investigated by Real-time PCR and the methylation patterns of IGF2DMR in exon10was examined in testes using bisulfite sequencing. The results showed that IGF2expression in cattle yaks’testes was lower than their parents （significant, P<0.01）. The IGF2DMR was highly methylated （cattle, yak and cattle yak was90%,90.7%and92.0%respectively） in testes, with the highest level in cattle yaks （not significant, P>0.05）. Our study showed that IGF2played an important role in bovine spermatogenesis and might be involved in cattle yak male sterility. The methylation level of the IGF2DMR was irrelevant to the lower expression of IGF2in cattle yaks; other factors perhaps play roles in its expression.4. Analysis of SNRPN mRNA expression and its methylation status in testes between cattle yaks and their parentsPrimers were designed from the sequence of the cattle SNRPN DNA （NW₉35049）, the5’-flanking sequence （1137bp） in yaks was obtained, the homology between yaks and cattle was98.2%, and computer assisted analysis suggested that the region consisted of the putative methylation-sensitive binding sites, including YY1and SP1sites. SNRPN mRNA expression in cattle yaks and their parents and the methylation patterns of the SNRPN DMR were examined in testes. The results showed that there was no significant difference between cattle yaks and their parents in SNRPN expression, but the expression level in cattle yaks was lower than that in their parents; the methylation level of SNRPN gene in cattle yaks （38.53%） was very significantly greater than that in cattle （21.08%） and yaks （20.81%）（P<0.01）. This indicated that the methylation status of SNRPN5’-flanking in cattle yaks couldn’t lead to apparent different expression alone, other factors maybe play an important role in the SNRPN expression,.5. Relationship between Methylation Status of DAZL5’CpG Island and Male Sterility of Cattle YakIn our prophase research, we have found that DAZL gene was a candidate gene for male infertility in cattle yaks. To study the mechanism of DAZL gene expression regulation in cattle yaks, the yaks DAZL5’region sequence was firstly cloned and DAZL gene methylation patterns in cattle, yaks and cattle yaks were examined using bisulfite sequencing. The results showed that within the5’region of DAZL there was a CpG island which contained the promoter region, exon1and intron1; the methylation level of DAZL gene in cattle yaks （85.6%） was very significantly greater than that in cattle （69.8%） and yaks （71.4%）（P<0.01）. These results demonstrated that the hypermethylation of DAZL gene was in accordance with the phenotype of DAZL deficient expression and male infertility in cattle yaks, and indicated that the methylation status of DAZL gene played an important role in DAZL transcriptional regulation and maybe have a severe effect on meiotic process of spermatogenesis and male sterility in cattle yaks.更多还原