【摘要】：花鱸作為一種重要的水產(chǎn)經(jīng)濟(jì)魚類,生長快速且能在較大溫度范圍和不同鹽度下進(jìn)行養(yǎng)殖,是一種極好的水產(chǎn)動物研究的模式生物�；|分布廣,遍布在東亞沿海和河口。由于野生資源有限,花鱸在中國的養(yǎng)殖變得越來越重要。目前的研究主要集中在遺傳標(biāo)記、發(fā)育、基因圖譜構(gòu)建以及與生殖、環(huán)境壓力、生長和免疫相關(guān)的功能基因分析�；|被淡化處理后,能夠在淡水中進(jìn)行養(yǎng)殖,然而,在完全脫離海水后,成魚不能夠?qū)崿F(xiàn)完整的繁殖過程,這也是花鱸不能夠在內(nèi)陸大規(guī)模養(yǎng)殖的主要阻礙。本研究中,我們主要以花鱸為實驗材料,基于HPG軸(Hypothalamus-pituitary-gonad axis),對花鱸的腦、性腺進(jìn)行轉(zhuǎn)錄組測序,得到若干有明顯表達(dá)差異的重要生殖基因,并對cyp19a結(jié)合其啟動子DNA甲基化進(jìn)行較為系統(tǒng)分析。主要研究結(jié)果如下:1.腦、性腺轉(zhuǎn)錄組數(shù)據(jù)分析取成年花鱸魚腦、性腺,提取總RNA,采用Illumina HiSeq 2000高通量技術(shù)平臺進(jìn)行測序。共計產(chǎn)生78,256,909條clean reads,拼接得到274,909條contigs。根據(jù)序列同源性分析,注釋得到31,683條unigenes,其中20,702條unigenes出現(xiàn)在8237個GO terms和3888個信號通路中。26,623條unigenes在所有的組織中均存在,然而,748,349和319條unigenes分別特異存在于腦、卵巢和精巢中。此外,在腦與卵巢、卵巢與精巢和腦與精巢之間比較,發(fā)現(xiàn)分別有671/367、496/315和1668/580條unigenes出現(xiàn)上調(diào)或下調(diào)。通過同源性分析,獲得腦-性腺軸中表達(dá)的生殖相關(guān)基因,其中包括那些參與性別分化和性別維持的基因。這些數(shù)據(jù)為花鱸的研究提供了較為完整基因資源,為HPG軸基因的功能、生殖調(diào)節(jié)以及性別基因的表達(dá)奠定基礎(chǔ)。2.下丘腦-垂體-性腺(HPG)軸相關(guān)的基因的鑒定及其表達(dá)分析通過轉(zhuǎn)錄組數(shù)據(jù)分析,對所得到的所有基因進(jìn)行鑒定,其中,56個基因存在于Gn RH信號通路(KEGG No.map04912),31個基因存在于類固醇生成通路(KEGG No.map04912)中。例如Gn RH受體,LH,FSH,LH受體,LSH受體,雄激素受體(AR),雌激素受體(ER),GSDF,CYP19a等。其中6個基因在不同發(fā)育時期以及不同組織的表達(dá)實驗表明,cyp19a主要在性腺以及腦中有表達(dá),極少在其他組織中有表達(dá);另一激素合成酶,3β-HSD廣泛表達(dá)與所取的七個組織中;AR和ER在七個組織中也都有表達(dá);與卵巢相比,GSDF更顯著地表達(dá)與精巢;PIWI特異性地表達(dá)于精巢和卵巢。3.MSAP方法檢測花鱸腦以及性腺組織基因組的DNA甲基化情況在花鱸魚腦、精巢以及卵巢組織中,設(shè)計8對選擇性擴(kuò)增引物,在100-1000bp之間,分別共計產(chǎn)生596、580和796條擴(kuò)增條帶,其中每個個體8對組合引物平均獲得149、145和199個條帶,花鱸腦、精巢以及卵巢組織的基因組DNA甲基化指數(shù)分別為為34.23%、38.62%和47.74%。4.BSP法檢測花鱸腦、性腺組織中cyp19a基因啟動子的甲基化狀態(tài)通過Genomic Walking方法,PCR擴(kuò)增克隆得到花鱸cyp19a基因啟動子序列,用亞硫酸鹽分別處理腦、精巢以及卵巢基因組DNA,擴(kuò)增分別得到處理后三組織的啟動子序列,通過TA克隆、生工測序?qū)θM織啟動子序列Cp G位點進(jìn)行甲基化分析。結(jié)果顯示:cyp19a基因啟動子在精巢以及腦組織中的甲基化程度明顯高于卵巢,均約是其2倍。啟動子甲基化水平和cyp19a基因在3組織中的表達(dá)呈負(fù)相關(guān)。5.cyp19a啟動子體外轉(zhuǎn)錄功能分析通過構(gòu)建PGL-Basic-cyp19a熒光素酶報告載體,我們對花鱸cyp19a啟動子在體外活性進(jìn)行分析。結(jié)果表明,去除-140至-46這段區(qū)域后,cyp19a啟動子活性明顯降低(降低約58.90%),所以較長的啟動子片段表現(xiàn)出相對較高的轉(zhuǎn)錄活性。由此我們推斷:包含若干轉(zhuǎn)錄因子結(jié)合位點SF-1,SOX,PPARE,CRE,and TATA box的區(qū)域?qū)τ谡{(diào)節(jié)cyp19a啟動子活性起著非常關(guān)鍵性的作用,而Cp G甲基化能抑制啟動子的轉(zhuǎn)錄活性。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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本文編號：2130185