大菱鲆（Scophthalmus maximus）盐度胁迫转录组构建及相关功能基因研究

发布时间：2018-05-07 10:35

本文选题：大菱鲆 + 盐度　；参考：《上海海洋大学》2017年硕士论文

【摘要】：本文首先阐述了盐度胁迫对鱼类的影响,分别从鱼类生长、存活,营养组成,鱼类抗氧化酶,鱼类免疫能力,鱼类生理代谢等五个方向分析了盐度对鱼类的影响,从而揭示了研究盐度胁迫对鱼类影响的重要性。其后对大菱鲆微卫星遗传标记研究进展以及鱼类盐度相关基因的研究进展和大菱鲆转录组研究进展进行了综述,希望能将先辈的研究结果和本实验研究相结合,期望为大菱鲆渗透压研究的提供理论依据。本研究采用荧光定量PCR技术对不同盐度胁迫下各时间点大菱鲆幼鱼肠、鳃中催乳素(PRL)基因和Na+-K+-ATPaseα1两种基因的表达量进行检测。以盐度30数据为对照组,5、10、40和50盐度为实验组,进行数据分析,结果显示:两种基因在两种组织中均有表达,且基因的表达量具有组织和时间特异性。肠组织PRL、Na+-K+-ATPaseα1基因的表达量,在盐度50和5条件下,随胁迫时间的积累呈先升高后降低的变化趋势;鳃组织PRL基因表达量,在盐度50和盐度5条件下,随胁迫时间积累先升高后降低;而Na+-K+-ATPaseα1基因表达量在低盐5条件下没有显著变化,在高盐50条件下Na+-K+-ATPaseα1基因表达量随时间积累呈现先降低后升高的变化趋势。在肠组织中,两基因存在极显著的协同作用,随着盐度的升高,两基因的表达量都呈现先升高后降低的趋势,且相关系数均接近于1;在鳃组织中,在盐度10-40区间内,两种基因的表达存在明显的拮抗作用,当PRL基因的表达量呈现升高(下降)趋势时,Na+-K+-ATPaseα1基因的表达量呈现下降(升高)趋势,且两基因的相关系数均为负值,证实了催乳素具有抑制Na+/K+-ATP酶活性的作用,为今后盐度胁迫分子调控机理研究提供理论依据。利用高通量测序技术,通过对盐度50海水胁迫后和正常盐度的肾组织进行转录组测序,构建了大菱鲆幼鱼盐度相关的转录组数据库,全面了解大菱鲆盐度胁迫后基因的表达情况,筛选了与盐度相关的候选基因,鉴定了与盐度相关的代谢通路,检测到大量的分子标记,并对部分盐度基因进行荧光定量表达分析,得到的结果如下:1.得到干净阅读子后拼接得到182225个unigenes,拼接得到的Unigenes可全部注释到7个数据库中。全部基因的功能聚类成43个GO范畴,聚类基因最多的依次是“细胞过程”,“连接”,“代谢过程”,“单一生物过程”;将KOG注释成功的基因按KOG的group进行分类,聚类基因最多的依次是“信号转导机制”,“一般功能”,“翻译修饰、蛋白转换、伴侣”;根据KEGG代谢通路进行分类,聚类基因最多的依次是“信号转导”、“内分泌系统”、“免疫系统”。2.通过与其他鱼类代谢通路和基因功能分析,鉴定出与大菱鲆盐度相关的通路分别是:胆汁分泌,矿物质吸收,钙信号通路,收集管酸分泌;还鉴定出基因显著(P0.05)富集的通路有16个。3.采用DESeq进行分析,筛选阈值为padj0.05,得到262个上调基因和506个下调基因,对比其他鱼类盐度转录组数据鉴定出59个与大菱鲆盐度相关的基因;既有与Cl-相关的基因,如:NACC2、PAT1、PRLR等;也有和Na+相关的基因,如:NHE-3、SGLT1、ASBT等;还有与H2O相关的基因,如:AQP1、AQP4、AQP8、CA等;还有和能量有关的基因,如,NKA、ATP6V1E1等;还有代谢相关的基因,如:CHST6、INO1、ANXA11、IDH2、ECH1、ECH8等;还有既与抗原处理相关又与应激相关的HSP70基因;还有既与生长相关又与渗透压调节相关的PRLR基因。4、根据基因的作用以及前人的研究结果,选取10个基因进行荧光定量分析发现,除PAT1基因外等9个基因在低盐度(盐度5)胁迫下的肾中均有较高的表达;10个基因在肠中的表达量较少,尤其PAT1基因、APOC1基因、APOM基因、CALM基因、FABP6基因、HMDH基因等6个基因在肠中的表达量显著(P0.05)低于其他组织。本文利用MISA软件对大菱鲆转录组中的182225条Unigenes序列进行搜索,共检测到81314个SSR位点,SSR发生频率为31.17%;EST-SSR的长度在12-20bp的有78247条,其中二核苷酸重复SSR最多,共计34783条;共检测到335种重复基元,其中以单核苷酸的A/T和二核苷酸的AC/GT最多,分别占总SSR的31.17%和31.05%。选择30对扩增引物中,共有3对EST-SSR引物表现出个体多态性,多态性比率为10%,SSR的等位基因数目为2个,平均有效基因数为1.8141,平均观测杂合度为0.5514,平均期望杂合度为0.4486,多态性信息含量分别为0.6897、0.6474、0.5713,平均为0.6361。本研究利用本实验室前期筛选出特异性较好的151对微卫星标记对大菱鲆盐度性状展开研究。结合分群分离分析法,初步筛选出4个可以在正常盐度组和低盐胁迫组出现差异条带的微卫星位点,然后对4个微卫星位点进行单个样本的微卫星分析验证,应用SPSS软件分析,得到1个极显著(P0.01)相关位点,2个显著(P0.05)相关位点。
[Abstract]:In this paper, the effects of salinity stress on fish were first expounded. The effects of salinity on fish were analyzed from five directions, such as fish growth, survival, nutrient composition, fish antioxidant enzymes, fish immunity and fish physiological metabolism, and the importance of salinity stress on fish was revealed. Research progress and advances in research progress of fish salinity related genes and research progress of turbot transcriptome are reviewed. We hope to provide theoretical basis for the study of the osmotic pressure of turbot. This study uses fluorescence quantitative PCR technology to increase the time points of different salinity stress. The expression of prolactin (PRL) and Na+-K+-ATPase alpha 1 genes in the sausages of turbot and gills were detected. The salinity 30 data as the control group, 5,10,40 and 50 salinity as the experimental group were analyzed. The results showed that the two genes were expressed in two tissues, and the expression of the gene was organized and time specific. The intestinal tissue was PRL, The expression of Na+-K+-ATPase alpha 1 gene, under the condition of salinity 50 and 5, increased first and then decreased with the accumulation of stress time. The expression of PRL gene in the gill tissue, under the condition of salinity 50 and salinity 5, increased first and then decreased with the accumulation of stress time, while the expression of Na+-K+-ATPase alpha 1 was not significantly changed under the condition of low salt 5. Under salt 50, the expression of Na+-K+-ATPase alpha 1 gene expression decreased first and then increased. In the intestinal tissue, the two gene had a very significant synergistic effect. With the increase of salinity, the expression of the two gene showed a tendency to increase first and then decrease, and the correlation coefficient was close to 1 in the gill tissue, in the salinity 10-40 area. In the interval, the expression of the two genes was obviously antagonistic. When the expression of the PRL gene showed a rising (decline) trend, the expression of the Na+-K+-ATPase alpha 1 gene showed a downward trend, and the correlation coefficient of the two gene was negative. It proved that the prolactin had the effect of inhibiting the activity of Na+/K+-ATP enzyme and was a salt stress molecule in the future. The study provided a theoretical basis for the study of regulation mechanism. By using high throughput sequencing technology, the transcriptional database of juvenile salty of turbot (turbot) was constructed by sequencing the kidney tissue of salinity 50 and normal salinity. The gene expression of turbot was fully understood and the candidate base related to salinity was screened. A large number of molecular markers were identified and some of the salt genes were detected by fluorescence quantitative analysis. The results were as follows: 1. 182225 unigenes were obtained after clean reading, and the spliced Unigenes could be completely released into 7 databases. The function clustering of all genes was 43. In the category of GO, the most clustering genes are "cell process", "connection", "metabolic process", "single biological process". The gene of KOG annotated by KOG is classified according to the group of KOG. The most clustering genes are "signal transduction mechanism", "general work energy", "translation modification, protein conversion, partner"; according to the KEGG generation. The categories of the metabolic pathways are "signal transduction," "signal transduction", "endocrine system", "immune system".2., through the analysis of metabolic pathways and gene functions of other fish, identified the pathways related to the salinity of turbot, respectively: bile secretion, mineral absorption, calcium signaling pathway, collection of acid secretion, and identification. The gene significant (P0.05) enrichment pathway was analyzed by 16.3. using DESeq, the threshold was padj0.05, 262 up-regulated genes and 506 down-regulation genes were obtained. Compared with other fish salinity transcripts, 59 genes related to the salinity of turbot were identified; there were genes related to Cl-, such as NACC2, PAT1, PRLR, etc., and also associated with Na+. Genes, such as NHE-3, SGLT1, ASBT, and other genes related to H2O, such as AQP1, AQP4, AQP8, CA, and other genes related to energy, such as NKA, ATP6V1E1, etc. The PRLR gene.4 related to pressure regulation, according to the role of the gene and the results of previous studies, 10 genes were selected for quantitative fluorescence analysis. The expression of 9 genes except PAT1 gene was high in the kidney under low salinity (5) stress, and the expression of 10 genes in the intestine was less, especially the PAT1 gene, APOC1 gene, and APOM gene. The expression of 6 genes, such as CALM gene, FABP6 gene and HMDH gene, was significantly lower than that of other tissues (P0.05). In this paper, 182225 Unigenes sequences in turbot transcription group were searched by MISA software, 81314 SSR loci were detected, the frequency of SSR was 31.17%, and the length of EST-SSR was 78247 in 12-20bp, of which dinucleotide was found. The maximum number of repeated SSR was 34783, and 335 repeated primers were detected, of which the single nucleotide A/T and the dinucleotide AC/GT were the most, and 31.17% of the total SSR and 31.05%. selected 30 pairs of primers respectively, and 3 pairs of EST-SSR primers showed individual polymorphism, the polymorphism ratio was 10%, the number of SSR alleles was 2, and the average effective group was available. The average heterozygosity is 1.8141, the average heterozygosity is 0.5514, the average heterozygosity is 0.4486, the polymorphism information content is 0.6897,0.6474,0.5713, and the average is 0.6361.. This study uses the early screening of 151 pairs of microsatellite markers to study the salinity of turbot. 4 microsatellite loci could be screened in normal salinity group and low salt stress group, and then 4 microsatellite loci were analyzed by microsatellite analysis. 1 significant (P0.01) related loci and 2 significant (P0.05) related sites were obtained by SPSS software analysis.

【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

【参考文献】