本文選題：羅非魚 + 低氧性狀　； 參考：《中山大學(xué)》2017年碩士論文

【摘要】：羅非魚(Oreochromis niloticus)原產(chǎn)于非洲,形似鯽魚,俗稱非洲鯽魚,是世界水產(chǎn)業(yè)重點培養(yǎng)的淡水魚類,被譽為未來動物性蛋白質(zhì)的主要來源之一。羅非魚有很強的繁殖能力和環(huán)境適應(yīng)能力。低氧對水生生物乃至整個水生生態(tài)具有嚴(yán)重的危害,是水產(chǎn)養(yǎng)殖面臨的最為嚴(yán)重的挑戰(zhàn)之一。然而關(guān)于魚類低氧適應(yīng)的分子機制仍不是很清楚。羅非魚對低溶氧環(huán)境具有較強的適應(yīng)性,是研究低溶氧問題的理想材料。本論文以羅非魚為研究材料從三個角度對低氧性狀的遺傳網(wǎng)絡(luò)進(jìn)行了解析。我們首先對保守的低氧相關(guān)候選基因,HIF1α及其抑制劑HIF1αn,進(jìn)行了克隆、結(jié)構(gòu)和功能分析;其次,對羅非魚低氧相關(guān)轉(zhuǎn)錄組差異表達(dá)基因和差異表達(dá)外顯子進(jìn)行了鑒定和功能分析;最后,對一個尼羅羅非魚家系中控制低氧性狀的基因組QTL進(jìn)行了鑒定與分析。本研究獲得的相關(guān)結(jié)果如下:1.低氧相關(guān)候選基因的克隆、結(jié)構(gòu)和功能分析。對HIF1α基因及其抑制基因HIF1αn的全CDS序列進(jìn)行了克隆測序,并分析了其基因和蛋白序列的保守結(jié)構(gòu)特征。利用SigniSite 2.1Server分別在HIF1α和HIF1αn蛋白序列上找到21個和2個與水生和陸生表型顯著相關(guān)的氨基酸位點。利用PAML軟件對HIF1α和HIF1αn序列進(jìn)行分析發(fā)現(xiàn)兩個基因都不存在強的正選擇效應(yīng),但HIF1α序列上部分位點可能受到選擇壓力。時空表達(dá)結(jié)果顯示,低氧條件下HIF1α基因在不同組織不同低氧處理時間點具有復(fù)雜的調(diào)節(jié)模式,而HIF1αn基因在低氧下的表達(dá)總體上調(diào)。2.轉(zhuǎn)錄組差異表達(dá)的基因和外顯子鑒定以及功能分析。對羅非魚低氧處理12h的心臟組織和鰓組織進(jìn)行了轉(zhuǎn)錄組測序。利用cuffdiff軟件鑒定了低氧處理組和正常組差異表達(dá)的基因和外顯子。在心臟組織中鑒定了284個差異表達(dá)基因和103個具有差異表達(dá)外顯子的選擇性剪接基因;在鰓組織中鑒定了239個差異表達(dá)基因和34個具有差異表達(dá)外顯子的選擇性剪接基因。熒光定量PCR實驗成功驗證了幾乎所有選取的基因及外顯子的差異表達(dá)。GO注釋和Pathway通路分析揭示這些差異表達(dá)基因和選擇性剪接基因在與能量保存、蛋白合成和免疫反應(yīng)等相關(guān)的過程中顯著富集。同時,還發(fā)現(xiàn)這些差異表達(dá)基因和選擇性剪接基因存在不同的富集分類。研究說明兩者在低氧反應(yīng)中具有不同的調(diào)節(jié)作用。3.羅非魚低氧耐受性狀基因組QTL分析。利用QTLseq和ddRADseq兩種高通量測序方法對羅非魚低氧性狀的遺傳調(diào)控網(wǎng)絡(luò)進(jìn)行了分析。兩種方法均在LG3和LG14連鎖群上各鑒定了一個與低氧性狀顯著關(guān)聯(lián)的QTL。為了驗證鑒定的QTLs的準(zhǔn)確性,在兩個QTL區(qū)間內(nèi)開發(fā)了4個微衛(wèi)星標(biāo)記。研究證實這兩個QTL區(qū)間標(biāo)記基因型/耐低氧表型間存在顯著相關(guān)。進(jìn)一步分析在QTL區(qū)間內(nèi)發(fā)現(xiàn)兩個低氧候選基因,即LG3 QTL區(qū)間的GPR132基因以及LG14QTL區(qū)間的ABCG4基因。對這兩個基因進(jìn)行結(jié)構(gòu)和功能分析顯示兩個基因的部分外顯子上的SNPs與低氧性狀存在顯著相關(guān)性。熒光定量PCR實驗顯示這兩個基因的表達(dá)量與羅非魚低氧耐受性相關(guān)。結(jié)果表明這兩個基因很有可能與羅非魚低氧耐受性狀相關(guān)。通過采用多種方法對羅非魚低氧遺傳網(wǎng)絡(luò)進(jìn)行綜合分析,我們鑒定了羅非魚基因組低氧性狀相關(guān)的重要QTL區(qū)間及候選功能基因,獲得了性狀緊密連鎖的分子標(biāo)記。該研究為進(jìn)一步通過應(yīng)用分子標(biāo)記(或基因)輔助育種技術(shù)開展羅非魚耐低氧新品種選育奠定了基礎(chǔ)。
[Abstract]:Oreochromis niloticus, native to Africa, resembles crucian carp and is commonly known as the African crucian carp, is a major freshwater fish cultivated in the world's water industry. It is known as one of the main sources of the future animal protein. The tilapia has strong breeding ability and environmental adaptability. Hypoxia has serious danger to aquatic organisms and even the whole aquatic ecology. Damage is one of the most serious challenges in aquaculture. However, the molecular mechanism of hypoxia adaptation is still not very clear. Tilapia has strong adaptability to low dissolved oxygen environment. It is an ideal material for the study of low dissolved oxygen. This paper uses tilapia as a research material for the genetic network of hypoxic characters from three angles. Analysis. We first clone, structure and functional analysis of the conservative hypoxic related candidate genes, HIF1 alpha and its inhibitor HIF1 alpha n. Secondly, the differential expression gene and the differential expression exons of the low oxygen related transcriptional group of tilapia were identified and functional analysis were carried out, and then the low oxygen trait was controlled in a Nile tilapia family. The genomic QTL was identified and analyzed. The results obtained in this study were as follows: 1. cloning, structure and function analysis of low oxygen related candidate genes. The whole CDS sequence of the HIF1 alpha gene and its suppressor HIF1 alpha N was cloned and sequenced, and the conservative structural characteristics of the gene and protein sequence were analyzed. The SigniSite 2.1Server score was used. 21 and 2 amino acid sites associated with aquatic and terrestrial phenotypes were not found on the sequence of HIF1 alpha and HIF1 alpha N protein. The analysis of HIF1 alpha and HIF1 alpha n sequences by PAML software found that there was no strong positive selection effect in two genes, but some of the loci on HIF1 a sequence may be subjected to selective pressure. Under oxygen conditions, the HIF1 alpha gene has a complex regulation mode in different tissues and different hypoxia treatment time points, while the expression of HIF1 alpha N gene under hypoxia generally up-regulated the differentially expressed genes and exons of the.2. transcriptional group and the functional analysis. The transcriptional sequence of the cardiac tissue and gill tissue of 12h in the hypoxic treatment of tilapia was sequenced. C The uffdiff software identified the differentially expressed genes and exons in the hypoxia treatment group and the normal group. In the cardiac tissue, 284 differentially expressed genes and 103 selective splicing genes with differentially expressed exons were identified, and 239 differentially expressed genes and 34 selective splicing groups with differentially expressed exons were identified in the gill tissue. The difference expression of almost all the selected genes and exons was verified by the fluorescence quantitative PCR experiment. The.GO annotation and Pathway pathway analysis revealed that these differentially expressed genes and selective splicing genes were significantly enriched in the processes related to energy preservation, protein synthesis and immune response. There are different enrichment and classification of selective splicing genes. The study shows that both of them have different regulatory roles in the hypoxia response, and the QTL analysis of genomic.3. for low oxygen tolerance of tilapia. Two high throughput sequencing methods of QTLseq and ddRADseq were used to analyze the genetic regulation network of hypoxic traits of tilapia. The two methods were all in LG3 and in LG3. The LG14 chain identified a QTL. with a significant association with hypoxic traits to verify the accuracy of the identified QTLs. 4 microsatellite markers were developed within two QTL intervals. The study confirmed that there was a significant correlation between the two QTL interval markers / hypoxia tolerance phenotypes. Further segregation of two hypoxia candidate genes was found in the QTL interval. The GPR132 gene in the LG3 QTL interval and the ABCG4 gene in the LG14QTL interval. The structural and functional analysis of the two genes showed that there was a significant correlation between the SNPs and the hypoxia in the partial exons of the two genes. The fluorescence quantitative PCR test showed that the expression of the two genes was related to the hypoxia tolerance of the tilapia. The results showed that the expression of the two genes was related to the low oxygen tolerance of the tilapia. The two genes are likely to be related to the low oxygen tolerance of tilapia. By using a variety of methods to analyze the low oxygen genetic network of tilapia, we identified the important QTL interval and candidate functional genes related to the hypoxic traits of the tilapia, and obtained the molecular markers for the tight linkage of the traits. Molecular marker (or gene) assisted breeding technology was used to breed new varieties of tilapia resistant to hypoxia.
【學(xué)位授予單位】：中山大學(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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