多巴胺系统在金钱鱼环境盐度适应及性腺发育中的调节机制

发布时间：2018-08-02 18:25

【摘要】：金钱鱼(Scatophagus argus),是一种广盐性海水鱼,可以适应不同的环境盐度变化,尤其是对盐度波动具有极强耐受能力。鉴于该特性,我们认为金钱鱼是一个研究盐度适应性调控过程中分子机制的良好模型。由于其具有良好的经济价值,近年来该品种成为我国南方新兴的海水养殖对象。盐度是重要的环境因子之一,对海洋生物的生长、繁殖等有重要影响。目前,关于金钱鱼人工繁殖的研究仍处于起步阶段,在繁殖过程中,由于盐度变化而引起的分子调控机制仍不清楚。1.在本章中,我们旨在研究金钱鱼脑多巴胺及其受体的盐度耐受性调节机制。多巴胺主要由位于中枢神经系统的多巴胺细胞合成和分泌。我们利用LC-MS/MS技术对脑以及血浆中的DA,DA前体物(L-Dopa)及其代谢产物的含量进行检测。在低盐驯化过程中,脑中的L-Dopa、DA以及血浆中的HVA含量显著上升(*p0.05;**p0.01)。在高盐胁迫时,脑中的L-Dopa含量显著下降,而DA并未检测到。此外,我们用灌流法固定了正常盐度下饲养的金钱鱼脑组织,进行免疫组化定位实验。用RT-q PCR法对急性淡水胁迫下的脑组织中的多巴胺1类受体的表达变化进行检测。通过免疫荧光染色实验我们发现SaDRD1和SaDRD5在金钱鱼脑纹状体中表达。通过荧光定量实验,我们发现SaDRD1和SaDRD5的表达量在急性淡水胁迫下均出现显著增加,在胁迫7d时并未检测到SaDRD5表达。利用Western Blotting技术对D1类受体的蛋白表达水平进行检测,发现其变化趋势与RTqPCR结果相一致。低盐胁迫引起金钱鱼脑中SaDRD1和SaDRD5的表达增加说明钠离子/渗透因子的外流可以引起两种受体亚型的上调。最终,这种上调引起的功能变化反映在NKA蛋白水平的表达下降。综上所述,我们证明了环境盐度的变化可以影响金钱鱼脑中的DA合成和分泌,而多巴胺系统对淡水胁迫的应答主要是通过激活D1类受体,从而抑制NKA的活性或蛋白表达,达到维持脑渗透平衡状态。我们的结果为研究硬骨鱼类的盐度耐受的分子机制提供了新的思路。2.研究金钱鱼盐度调节器官中的渗透压调节相关基因的表达调控特征对水产养殖业具有重要意义。我们利用比较基因组学的手段来研究长期盐度驯化下的金钱鱼肾脏渗透压的适应性调节机制。我们提取了肾脏总RNA,来自三个实验组的RNA等量混合,构建出两个cDNA文库(低盐驯化组与高盐驯化组)。利用Illumina双末端测序技术,测序得到超过5.75亿的长度为150bp的clean data,装配得到186,397条邻接片段。去除冗余数据后,得到平均长度为2,022 bp的基因片段43,933条。在这些数据中,14,259条(32.45%)转录本序列成功注释到NCBI-NR库。通过GO,KEGG注释以及KOG分析来注释潜在的基因并预测其功能。根据序列分析结果以及已发表的研究成果,我们鉴定得到金钱鱼肾脏中与长期盐度适应性调节相关的多巴胺系统的候选基因。这些转录组数据为金钱鱼生理学研究提供了有价值的新数据。相关基因功能的深入研究对金钱鱼繁殖、生长以及疾病的控制具有重要意义。我们的结果为广盐性鱼类的渗透压调节机制的深入研究提供了可靠的基础。3.多巴胺是肾脏尿钠排泄的重要调控因子,对动物适应环境盐度变化极为关键。然而,多巴胺通过哪种分子机制来促进这种适应性调节并不是很清楚。本章节我们研究了金钱鱼遭受急性淡水胁迫时,肾脏多巴胺如何影响其低渗适应性调节。海水转淡水后,血清中多巴胺含量降低。肾脏中的多巴胺激活多巴胺受体1的表达(命名为SaDRD1),触发渗透压调节信号级联反应。体内实验证明,SaDRD1主要在肾脏近端小管中表达。而在体外培养的肾原代细胞中,SaDRD1在细胞膜上表达。利用siRNA干扰技术对肾原代细胞中的SaDRD1mRNA进行敲降后发现Na+/K+-ATP酶(NKA)的活性出现显著增加(*p0.05),这表明肾脏中SaDRD1的表达可能抑制了NKA的活性。将原代肾细胞从等渗培养基转至低渗培养基中培养后,适当剂量的外源性多巴胺的给予提早了NKA对于低盐胁迫的应答。我们的结果表明,金钱鱼肾脏分泌的多巴胺通过SaDRD1激活多巴胺系统,调控下游的信号通路,最终抑制NKA活性。因此,我们认为金钱鱼肾多巴胺系统的激活促进了有机体对低渗环境的适应,为研究鱼类低盐应答机制的研究提供了新的视野。4.金钱鱼雌雄个体拥有相同的基因组。然而,与雄鱼相比,金钱鱼雌鱼长得更快、体型更大。这种表型的差异可能受基因的差异表达以及性别特异调控影响。我们通过大规模转录组测序来全面分析金钱鱼雌雄性腺发育过程中分子调控机制的差异性。利用Illumina双末端测序技术,对金钱鱼II、III以及IV期雌雄性腺进行高通量测序,获得9.44×108条clean reads。通过NCBI-NR数据库注释,得到33,520条unigenes。通过对转录组数据的分析发现多巴胺作用相关基因的表达存在差异。这些数据有助于我们筛选性别决定基因,确定性别差异表达基因并验证这些基因的表达模式。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4

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