【摘要】：金錢魚(Scatophagus argus),是一種廣鹽性海水魚,可以適應(yīng)不同的環(huán)境鹽度變化,尤其是對(duì)鹽度波動(dòng)具有極強(qiáng)耐受能力。鑒于該特性,我們認(rèn)為金錢魚是一個(gè)研究鹽度適應(yīng)性調(diào)控過程中分子機(jī)制的良好模型。由于其具有良好的經(jīng)濟(jì)價(jià)值,近年來該品種成為我國南方新興的海水養(yǎng)殖對(duì)象。鹽度是重要的環(huán)境因子之一,對(duì)海洋生物的生長(zhǎng)、繁殖等有重要影響。目前,關(guān)于金錢魚人工繁殖的研究仍處于起步階段,在繁殖過程中,由于鹽度變化而引起的分子調(diào)控機(jī)制仍不清楚。1.在本章中,我們旨在研究金錢魚腦多巴胺及其受體的鹽度耐受性調(diào)節(jié)機(jī)制。多巴胺主要由位于中樞神經(jīng)系統(tǒng)的多巴胺細(xì)胞合成和分泌。我們利用LC-MS/MS技術(shù)對(duì)腦以及血漿中的DA,DA前體物(L-Dopa)及其代謝產(chǎn)物的含量進(jìn)行檢測(cè)。在低鹽馴化過程中,腦中的L-Dopa、DA以及血漿中的HVA含量顯著上升(*p0.05;**p0.01)。在高鹽脅迫時(shí),腦中的L-Dopa含量顯著下降,而DA并未檢測(cè)到。此外,我們用灌流法固定了正常鹽度下飼養(yǎng)的金錢魚腦組織,進(jìn)行免疫組化定位實(shí)驗(yàn)。用RT-q PCR法對(duì)急性淡水脅迫下的腦組織中的多巴胺1類受體的表達(dá)變化進(jìn)行檢測(cè)。通過免疫熒光染色實(shí)驗(yàn)我們發(fā)現(xiàn)SaDRD1和SaDRD5在金錢魚腦紋狀體中表達(dá)。通過熒光定量實(shí)驗(yàn),我們發(fā)現(xiàn)SaDRD1和SaDRD5的表達(dá)量在急性淡水脅迫下均出現(xiàn)顯著增加,在脅迫7d時(shí)并未檢測(cè)到SaDRD5表達(dá)。利用Western Blotting技術(shù)對(duì)D1類受體的蛋白表達(dá)水平進(jìn)行檢測(cè),發(fā)現(xiàn)其變化趨勢(shì)與RTqPCR結(jié)果相一致。低鹽脅迫引起金錢魚腦中SaDRD1和SaDRD5的表達(dá)增加說明鈉離子/滲透因子的外流可以引起兩種受體亞型的上調(diào)。最終,這種上調(diào)引起的功能變化反映在NKA蛋白水平的表達(dá)下降。綜上所述,我們證明了環(huán)境鹽度的變化可以影響金錢魚腦中的DA合成和分泌,而多巴胺系統(tǒng)對(duì)淡水脅迫的應(yīng)答主要是通過激活D1類受體,從而抑制NKA的活性或蛋白表達(dá),達(dá)到維持腦滲透平衡狀態(tài)。我們的結(jié)果為研究硬骨魚類的鹽度耐受的分子機(jī)制提供了新的思路。2.研究金錢魚鹽度調(diào)節(jié)器官中的滲透壓調(diào)節(jié)相關(guān)基因的表達(dá)調(diào)控特征對(duì)水產(chǎn)養(yǎng)殖業(yè)具有重要意義。我們利用比較基因組學(xué)的手段來研究長(zhǎng)期鹽度馴化下的金錢魚腎臟滲透壓的適應(yīng)性調(diào)節(jié)機(jī)制。我們提取了腎臟總RNA,來自三個(gè)實(shí)驗(yàn)組的RNA等量混合,構(gòu)建出兩個(gè)cDNA文庫(低鹽馴化組與高鹽馴化組)。利用Illumina雙末端測(cè)序技術(shù),測(cè)序得到超過5.75億的長(zhǎng)度為150bp的clean data,裝配得到186,397條鄰接片段。去除冗余數(shù)據(jù)后,得到平均長(zhǎng)度為2,022 bp的基因片段43,933條。在這些數(shù)據(jù)中,14,259條(32.45%)轉(zhuǎn)錄本序列成功注釋到NCBI-NR庫。通過GO,KEGG注釋以及KOG分析來注釋潛在的基因并預(yù)測(cè)其功能。根據(jù)序列分析結(jié)果以及已發(fā)表的研究成果,我們鑒定得到金錢魚腎臟中與長(zhǎng)期鹽度適應(yīng)性調(diào)節(jié)相關(guān)的多巴胺系統(tǒng)的候選基因。這些轉(zhuǎn)錄組數(shù)據(jù)為金錢魚生理學(xué)研究提供了有價(jià)值的新數(shù)據(jù)。相關(guān)基因功能的深入研究對(duì)金錢魚繁殖、生長(zhǎng)以及疾病的控制具有重要意義。我們的結(jié)果為廣鹽性魚類的滲透壓調(diào)節(jié)機(jī)制的深入研究提供了可靠的基礎(chǔ)。3.多巴胺是腎臟尿鈉排泄的重要調(diào)控因子,對(duì)動(dòng)物適應(yīng)環(huán)境鹽度變化極為關(guān)鍵。然而,多巴胺通過哪種分子機(jī)制來促進(jìn)這種適應(yīng)性調(diào)節(jié)并不是很清楚。本章節(jié)我們研究了金錢魚遭受急性淡水脅迫時(shí),腎臟多巴胺如何影響其低滲適應(yīng)性調(diào)節(jié)。海水轉(zhuǎn)淡水后,血清中多巴胺含量降低。腎臟中的多巴胺激活多巴胺受體1的表達(dá)(命名為SaDRD1),觸發(fā)滲透壓調(diào)節(jié)信號(hào)級(jí)聯(lián)反應(yīng)。體內(nèi)實(shí)驗(yàn)證明,SaDRD1主要在腎臟近端小管中表達(dá)。而在體外培養(yǎng)的腎原代細(xì)胞中,SaDRD1在細(xì)胞膜上表達(dá)。利用siRNA干擾技術(shù)對(duì)腎原代細(xì)胞中的SaDRD1mRNA進(jìn)行敲降后發(fā)現(xiàn)Na+/K+-ATP酶(NKA)的活性出現(xiàn)顯著增加(*p0.05),這表明腎臟中SaDRD1的表達(dá)可能抑制了NKA的活性。將原代腎細(xì)胞從等滲培養(yǎng)基轉(zhuǎn)至低滲培養(yǎng)基中培養(yǎng)后,適當(dāng)劑量的外源性多巴胺的給予提早了NKA對(duì)于低鹽脅迫的應(yīng)答。我們的結(jié)果表明,金錢魚腎臟分泌的多巴胺通過SaDRD1激活多巴胺系統(tǒng),調(diào)控下游的信號(hào)通路,最終抑制NKA活性。因此,我們認(rèn)為金錢魚腎多巴胺系統(tǒng)的激活促進(jìn)了有機(jī)體對(duì)低滲環(huán)境的適應(yīng),為研究魚類低鹽應(yīng)答機(jī)制的研究提供了新的視野。4.金錢魚雌雄個(gè)體擁有相同的基因組。然而,與雄魚相比,金錢魚雌魚長(zhǎng)得更快、體型更大。這種表型的差異可能受基因的差異表達(dá)以及性別特異調(diào)控影響。我們通過大規(guī)模轉(zhuǎn)錄組測(cè)序來全面分析金錢魚雌雄性腺發(fā)育過程中分子調(diào)控機(jī)制的差異性。利用Illumina雙末端測(cè)序技術(shù),對(duì)金錢魚II、III以及IV期雌雄性腺進(jìn)行高通量測(cè)序,獲得9.44×108條clean reads。通過NCBI-NR數(shù)據(jù)庫注釋,得到33,520條unigenes。通過對(duì)轉(zhuǎn)錄組數(shù)據(jù)的分析發(fā)現(xiàn)多巴胺作用相關(guān)基因的表達(dá)存在差異。這些數(shù)據(jù)有助于我們篩選性別決定基因,確定性別差異表達(dá)基因并驗(yàn)證這些基因的表達(dá)模式。
[Abstract]:Scatophagus Argus, a widely salt marine fish, can adapt to different environmental salinity changes, especially the strong tolerance to salinity fluctuations. In view of this characteristic, we think that money fish is a good model of molecular mechanism in the study of salinity adaptation and regulation. This variety has become a new marine culture object in the south of China. Salinity is one of the important environmental factors, and it has an important influence on the growth and reproduction of marine organisms. At present, the research on the artificial reproduction of money fish is still in its infancy. In the breeding process, the molecular regulation mechanism caused by the salinity change is still not clear about.1. in this process. In this chapter, we aim to study the salinity tolerance regulation mechanism of dopamine and its receptors in the money fish brain. Dopamine is mainly composed and secreted by dopamine cells located in the central nervous system. We use LC-MS/MS technology to detect the content of DA, DA precursor (L-Dopa) and its metabolites in brain and plasma. In low salt domestication process In the brain, the content of HVA in L-Dopa, DA and plasma increased significantly (*p0.05; **p0.01). When high salt stress, the content of L-Dopa in the brain decreased significantly, but DA was not detected. In addition, we immobilized the brain tissue of the money fish under normal salinity by perfusion method and carried out the immunohistochemical localization experiment. The RT-q PCR method was used for the acute fresh water stress. The expression of 1 kinds of dopamine receptors in the brain tissue was detected. By immunofluorescence staining, we found that SaDRD1 and SaDRD5 were expressed in the striatum of the money fish. By fluorescence quantitative experiments, we found that the expression of SaDRD1 and SaDRD5 increased significantly under acute fresh water stress and did not detect SaDR at the time of stress 7d. D5 expression. Western Blotting technique was used to detect the protein expression level of D1 receptor, and it was found that the change trend was in accordance with the RTqPCR results. Low salt stress caused the increase of SaDRD1 and SaDRD5 in the brain of money fish indicating that the exodus of sodium ion / osmotic factor could cause the up regulation of two types of subtypes. Functional changes are reflected in the decline in the expression of NKA protein. In summary, we have shown that the changes in environmental salinity can affect the DA synthesis and secretion in the brain of the money fish, and the response of the dopamine system to fresh water stress is mainly by activating the D1 receptor, thus inhibiting the activity of NKA or the expression of protein to maintain the state of brain osmotic balance. Our results provide a new approach to study the molecular mechanism of salinity tolerance in hard fishes..2. studies are of great significance for the regulation of osmotic pressure related genes in the regulation organs of money fish salinity. We use comparative genomics to study the money fish kidney under long-term salinity domestication. We extracted the total RNA of the renal osmotic pressure. We extracted the total RNA of the kidney from the RNA isometric mixing of three experimental groups and constructed two cDNA libraries (low salt domestication and high salt domestication group). Using Illumina double terminal sequencing technology, the clean data of more than 575 million of the length of 150bp was sequenced, and 186397 adjacent segments were removed. After redundant data, 43933 genes with an average length of 2022 BP were obtained. In these data, 14259 (32.45%) transcripts were successfully annotated to the NCBI-NR library. The potential genes were annotated and predicted by GO, KEGG annotation and KOG analysis. We identified the results based on the sequence analysis and published research results. A candidate gene for the dopamine system associated with long-term salinity adaptation in the money fish kidney. These transcriptional data provide valuable new data for the physiological studies of the fish. The in-depth study of related gene functions is important for the breeding, growth, and disease control of money fish. Our results are widely salting fish. The in-depth study of osmotic pressure regulation provides a reliable basis for.3. dopamine as an important regulator of renal urinary sodium excretion, which is crucial to the adaptation to environmental salinity changes in animals. However, the molecular mechanism of dopamine to promote this adaptive regulation is not very clear. When fresh water stresses, how does dopamine affect its hypotonic adaptation regulation. After the sea water turns to fresh water, the content of dopamine in the serum is reduced. The expression of dopamine receptor 1 in the kidneys (named SaDRD1) triggers the cascade reaction of osmotic pressure regulation. In vivo experimental evidence shows that SaDRD1 is mainly expressed in the proximal tubules of the kidney. In the cultured renal primary cells, SaDRD1 was expressed on the cell membrane. The activity of Na+/K+-ATP enzyme (NKA) increased significantly (*p0.05) after the knockdown of SaDRD1mRNA in the primary renal cells by siRNA interference technique, which indicated that the expression of SaDRD1 in the kidney might inhibit the activity of NKA. The primary renal cells were transferred from the isosotic medium to low level. After culture in the infiltration medium, the appropriate dose of exogenous dopamine gives NKA response to low salt stress early. Our results show that the dopamine secreted by the money fish kidneys activates the dopamine system through SaDRD1, regulates downstream signaling pathways and ultimately inhibits NKA activity. Therefore, we think the excitation of the dopamine system in the money fish kidney. It promotes the adaptation of the organism to the low permeability environment, and provides a new vision for the study of the mechanism of low salt response in fish..4. money fish and male individuals have the same genome. However, compared with the male, the female fish grow faster and larger. The difference in this phenotype can be regulated by gene differential expression and sex specific regulation. The difference of molecular regulation mechanism in the development of male and female sex gland of money fish was analyzed by large scale transcriptome sequencing. Using Illumina double terminal sequencing technology, high flux sequencing was carried out for the II, III and IV male and female gonads of money fish, and 9.44 x 108 clean reads. were annotated by NCBI-NR database to obtain 33520 u. By analyzing the data of the transcriptome, nigenes. found that there was a difference in the expression of the genes related to dopamine. These data help us to screen the sex determining genes, determine the sex differentially expressed genes and verify the expression patterns of these genes.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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