花鲈HPG轴转录组与cyp19a基因的表达与功能分析
发布时间：2018-07-17 16:03
【摘要】：花鲈作为一种重要的水产经济鱼类,生长快速且能在较大温度范围和不同盐度下进行养殖,是一种极好的水产动物研究的模式生物。花鲈分布广,遍布在东亚沿海和河口。由于野生资源有限,花鲈在中国的养殖变得越来越重要。目前的研究主要集中在遗传标记、发育、基因图谱构建以及与生殖、环境压力、生长和免疫相关的功能基因分析。花鲈被淡化处理后,能够在淡水中进行养殖,然而,在完全脱离海水后,成鱼不能够实现完整的繁殖过程,这也是花鲈不能够在内陆大规模养殖的主要阻碍。本研究中,我们主要以花鲈为实验材料,基于HPG轴(Hypothalamus-pituitary-gonad axis),对花鲈的脑、性腺进行转录组测序,得到若干有明显表达差异的重要生殖基因,并对cyp19a结合其启动子DNA甲基化进行较为系统分析。主要研究结果如下:1.脑、性腺转录组数据分析取成年花鲈鱼脑、性腺,提取总RNA,采用Illumina HiSeq 2000高通量技术平台进行测序。共计产生78,256,909条clean reads,拼接得到274,909条contigs。根据序列同源性分析,注释得到31,683条unigenes,其中20,702条unigenes出现在8237个GO terms和3888个信号通路中。26,623条unigenes在所有的组织中均存在,然而,748,349和319条unigenes分别特异存在于脑、卵巢和精巢中。此外,在脑与卵巢、卵巢与精巢和脑与精巢之间比较,发现分别有671/367、496/315和1668/580条unigenes出现上调或下调。通过同源性分析,获得脑-性腺轴中表达的生殖相关基因,其中包括那些参与性别分化和性别维持的基因。这些数据为花鲈的研究提供了较为完整基因资源,为HPG轴基因的功能、生殖调节以及性别基因的表达奠定基础。2.下丘脑-垂体-性腺(HPG)轴相关的基因的鉴定及其表达分析通过转录组数据分析,对所得到的所有基因进行鉴定,其中,56个基因存在于Gn RH信号通路(KEGG No.map04912),31个基因存在于类固醇生成通路(KEGG No.map04912)中。例如Gn RH受体,LH,FSH,LH受体,LSH受体,雄激素受体(AR),雌激素受体(ER),GSDF,CYP19a等。其中6个基因在不同发育时期以及不同组织的表达实验表明,cyp19a主要在性腺以及脑中有表达,极少在其他组织中有表达;另一激素合成酶,3β-HSD广泛表达与所取的七个组织中;AR和ER在七个组织中也都有表达;与卵巢相比,GSDF更显著地表达与精巢;PIWI特异性地表达于精巢和卵巢。3.MSAP方法检测花鲈脑以及性腺组织基因组的DNA甲基化情况在花鲈鱼脑、精巢以及卵巢组织中,设计8对选择性扩增引物,在100-1000bp之间,分别共计产生596、580和796条扩增条带,其中每个个体8对组合引物平均获得149、145和199个条带,花鲈脑、精巢以及卵巢组织的基因组DNA甲基化指数分别为为34.23%、38.62%和47.74%。4.BSP法检测花鲈脑、性腺组织中cyp19a基因启动子的甲基化状态通过Genomic Walking方法,PCR扩增克隆得到花鲈cyp19a基因启动子序列,用亚硫酸盐分别处理脑、精巢以及卵巢基因组DNA,扩增分别得到处理后三组织的启动子序列,通过TA克隆、生工测序对三组织启动子序列Cp G位点进行甲基化分析。结果显示:cyp19a基因启动子在精巢以及脑组织中的甲基化程度明显高于卵巢,均约是其2倍。启动子甲基化水平和cyp19a基因在3组织中的表达呈负相关。5.cyp19a启动子体外转录功能分析通过构建PGL-Basic-cyp19a荧光素酶报告载体,我们对花鲈cyp19a启动子在体外活性进行分析。结果表明,去除-140至-46这段区域后,cyp19a启动子活性明显降低(降低约58.90%),所以较长的启动子片段表现出相对较高的转录活性。由此我们推断:包含若干转录因子结合位点SF-1,SOX,PPARE,CRE,and TATA box的区域对于调节cyp19a启动子活性起着非常关键性的作用,而Cp G甲基化能抑制启动子的转录活性。
[Abstract]:As an important aquatic fish fish, it grows fast and can be cultured at large temperature range and different salinity. It is an excellent model organism for aquatic animal research. The bass are widely distributed in the coastal and estuaries of East Asia. Because of the limited wild resources, the culture of bass in China is becoming more and more important. The study focuses on genetic markers, development, genetic mapping, and functional gene analysis related to reproduction, environmental stress, growth and immunity. After desalination of the perch, it can be cultured in fresh water. However, the adult can not achieve a complete reproduction process after complete isolation of the sea water, which is also the inability of the bass to be in the inland big rules. In this study, we mainly use the bass as experimental materials, based on the HPG axis (Hypothalamus-pituitary-gonad axis), to carry out a transcriptional sequence of the brain and gonads of the bass, to obtain some important reproductive genes that have obvious differences in expression, and to make a systematic analysis of the DNA methylation of the promoter of Cyp19a. The results are as follows: 1. brain, gonadal transcriptional group data analysis of adult flower bass brain, gonad, extraction of total RNA, Illumina HiSeq 2000 high throughput technology platform for sequencing. A total of 78256909 clean reads, splicing 274909 contigs. based on sequence homology analysis, annotated to get 31683 unigenes, 20702 unigenes In 8237 GO terms and 3888 signaling pathways,.26623 unigenes exists in all tissues. However, 748349 and 319 unigenes are specifically found in the brain, ovary and spermary. In addition, the comparison between the brain and ovary, the ovary and the spermary and the brain and the spermary is found to be 671/367496/315 and 1668/580 unigenes, respectively. Up or down. Through homology analysis, the reproduction related genes expressed in the brain and gonadal axis, including those involved in sex differentiation and gender maintenance, provide more complete genetic resources for the study of the bass, which lays the foundation for the function of the HPG axis gene, the genital joint and the expression of sex genes in the.2. hypothalamus. The identification of the genes related to the axis of the brain pituitary gonadal (HPG) axis and its expression analysis are identified by the analysis of the transcriptional data. Among them, 56 genes exist in the Gn RH signaling pathway (KEGG No.map04912), and the 31 genes exist in the steroid generation pathway (KEGG No.map04912), such as Gn RH receptor, LH, FSH, LH receptor. Receptor, androgen receptor (AR), estrogen receptor (ER), GSDF, CYP19a, etc., of which 6 genes expressed in different developmental stages and different tissues showed that Cyp19a was mainly expressed in the gonads and brain, rarely expressed in other tissues; the other hormone synthase, 3 beta -HSD were widely expressed in seven tissues, and AR and ER in seven. GSDF is more expressed in the tissues; compared with the ovary, GSDF is more expressed with the spermary. The PIWI specificity of the DNA methylation of the perch and the gonadal genome in the spermary and ovary is more specific than the ovary. The 8 pairs of selective amplification primers are designed in the brain, the spermary and the ovarian tissue of the perch and the ovary and the 100-1000bp, respectively. 596580 and 796 bands were produced, of which 149145 and 199 bands were obtained by 8 pairs of primers for each individual. The genomic DNA methylation index of the perch, spermary and ovarian tissue was 34.23%, 38.62% and 47.74%.4.BSP, respectively. The methylation status of Cyp19a promoter in the gonadal tissues passed through Genomic W ALKING method, PCR amplification and cloning of the Cyp19a gene promoter sequence of bass bass, the brain, the spermary and the ovarian genome DNA were treated with sulfite respectively. The promoter sequence of the three tissues after the treatment was amplified respectively. The TA cloning was used to analyze the Cp G loci of the promoter sequence of the three tissue. The results showed that the Cyp19a gene was started. The methylation level in the spermary and the brain tissue is obviously higher than that of the ovary. It is about 2 times higher than that of the ovary. The level of promoter methylation and the expression of Cyp19a gene in 3 tissues are negatively correlated with.5.cyp19a promoter in vitro transcriptional function analysis by constructing PGL-Basic-cyp19a luciferase reporter carrier, and we live in vitro on the Cyp19a promoter of bass bass. The results showed that after the removal of -140 to -46, the activity of Cyp19a promoter was significantly reduced (about 58.90%), so the longer promoter fragment showed relatively high transcriptional activity. Therefore, we infer that the region containing a number of transcription factor binding sites, SF-1, SOX, PPARE, CRE, and TATA box, is for Cyp19a startup. Subactivity plays a key role, and Cp G methylation can inhibit the transcriptional activity of the promoter.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2130185
[Abstract]:As an important aquatic fish fish, it grows fast and can be cultured at large temperature range and different salinity. It is an excellent model organism for aquatic animal research. The bass are widely distributed in the coastal and estuaries of East Asia. Because of the limited wild resources, the culture of bass in China is becoming more and more important. The study focuses on genetic markers, development, genetic mapping, and functional gene analysis related to reproduction, environmental stress, growth and immunity. After desalination of the perch, it can be cultured in fresh water. However, the adult can not achieve a complete reproduction process after complete isolation of the sea water, which is also the inability of the bass to be in the inland big rules. In this study, we mainly use the bass as experimental materials, based on the HPG axis (Hypothalamus-pituitary-gonad axis), to carry out a transcriptional sequence of the brain and gonads of the bass, to obtain some important reproductive genes that have obvious differences in expression, and to make a systematic analysis of the DNA methylation of the promoter of Cyp19a. The results are as follows: 1. brain, gonadal transcriptional group data analysis of adult flower bass brain, gonad, extraction of total RNA, Illumina HiSeq 2000 high throughput technology platform for sequencing. A total of 78256909 clean reads, splicing 274909 contigs. based on sequence homology analysis, annotated to get 31683 unigenes, 20702 unigenes In 8237 GO terms and 3888 signaling pathways,.26623 unigenes exists in all tissues. However, 748349 and 319 unigenes are specifically found in the brain, ovary and spermary. In addition, the comparison between the brain and ovary, the ovary and the spermary and the brain and the spermary is found to be 671/367496/315 and 1668/580 unigenes, respectively. Up or down. Through homology analysis, the reproduction related genes expressed in the brain and gonadal axis, including those involved in sex differentiation and gender maintenance, provide more complete genetic resources for the study of the bass, which lays the foundation for the function of the HPG axis gene, the genital joint and the expression of sex genes in the.2. hypothalamus. The identification of the genes related to the axis of the brain pituitary gonadal (HPG) axis and its expression analysis are identified by the analysis of the transcriptional data. Among them, 56 genes exist in the Gn RH signaling pathway (KEGG No.map04912), and the 31 genes exist in the steroid generation pathway (KEGG No.map04912), such as Gn RH receptor, LH, FSH, LH receptor. Receptor, androgen receptor (AR), estrogen receptor (ER), GSDF, CYP19a, etc., of which 6 genes expressed in different developmental stages and different tissues showed that Cyp19a was mainly expressed in the gonads and brain, rarely expressed in other tissues; the other hormone synthase, 3 beta -HSD were widely expressed in seven tissues, and AR and ER in seven. GSDF is more expressed in the tissues; compared with the ovary, GSDF is more expressed with the spermary. The PIWI specificity of the DNA methylation of the perch and the gonadal genome in the spermary and ovary is more specific than the ovary. The 8 pairs of selective amplification primers are designed in the brain, the spermary and the ovarian tissue of the perch and the ovary and the 100-1000bp, respectively. 596580 and 796 bands were produced, of which 149145 and 199 bands were obtained by 8 pairs of primers for each individual. The genomic DNA methylation index of the perch, spermary and ovarian tissue was 34.23%, 38.62% and 47.74%.4.BSP, respectively. The methylation status of Cyp19a promoter in the gonadal tissues passed through Genomic W ALKING method, PCR amplification and cloning of the Cyp19a gene promoter sequence of bass bass, the brain, the spermary and the ovarian genome DNA were treated with sulfite respectively. The promoter sequence of the three tissues after the treatment was amplified respectively. The TA cloning was used to analyze the Cp G loci of the promoter sequence of the three tissue. The results showed that the Cyp19a gene was started. The methylation level in the spermary and the brain tissue is obviously higher than that of the ovary. It is about 2 times higher than that of the ovary. The level of promoter methylation and the expression of Cyp19a gene in 3 tissues are negatively correlated with.5.cyp19a promoter in vitro transcriptional function analysis by constructing PGL-Basic-cyp19a luciferase reporter carrier, and we live in vitro on the Cyp19a promoter of bass bass. The results showed that after the removal of -140 to -46, the activity of Cyp19a promoter was significantly reduced (about 58.90%), so the longer promoter fragment showed relatively high transcriptional activity. Therefore, we infer that the region containing a number of transcription factor binding sites, SF-1, SOX, PPARE, CRE, and TATA box, is for Cyp19a startup. Subactivity plays a key role, and Cp G methylation can inhibit the transcriptional activity of the promoter.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
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2 宫时玉;蒋曹德;邓昌彦;;DNA甲基化及其生物学功能[J];华中农业大学学报;2005年06期
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