發(fā)布時間：2018-05-07 10:35

  本文選題：大菱鲆 + 鹽度　； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：本文首先闡述了鹽度脅迫對魚類的影響,分別從魚類生長、存活,營養(yǎng)組成,魚類抗氧化酶,魚類免疫能力,魚類生理代謝等五個方向分析了鹽度對魚類的影響,從而揭示了研究鹽度脅迫對魚類影響的重要性。其后對大菱鲆微衛(wèi)星遺傳標(biāo)記研究進(jìn)展以及魚類鹽度相關(guān)基因的研究進(jìn)展和大菱鲆轉(zhuǎn)錄組研究進(jìn)展進(jìn)行了綜述,希望能將先輩的研究結(jié)果和本實驗研究相結(jié)合,期望為大菱鲆滲透壓研究的提供理論依據(jù)。本研究采用熒光定量PCR技術(shù)對不同鹽度脅迫下各時間點大菱鲆幼魚腸、鰓中催乳素(PRL)基因和Na+-K+-ATPaseα1兩種基因的表達(dá)量進(jìn)行檢測。以鹽度30數(shù)據(jù)為對照組,5、10、40和50鹽度為實驗組,進(jìn)行數(shù)據(jù)分析,結(jié)果顯示:兩種基因在兩種組織中均有表達(dá),且基因的表達(dá)量具有組織和時間特異性。腸組織PRL、Na+-K+-ATPaseα1基因的表達(dá)量,在鹽度50和5條件下,隨脅迫時間的積累呈先升高后降低的變化趨勢;鰓組織PRL基因表達(dá)量,在鹽度50和鹽度5條件下,隨脅迫時間積累先升高后降低;而Na+-K+-ATPaseα1基因表達(dá)量在低鹽5條件下沒有顯著變化,在高鹽50條件下Na+-K+-ATPaseα1基因表達(dá)量隨時間積累呈現(xiàn)先降低后升高的變化趨勢。在腸組織中,兩基因存在極顯著的協(xié)同作用,隨著鹽度的升高,兩基因的表達(dá)量都呈現(xiàn)先升高后降低的趨勢,且相關(guān)系數(shù)均接近于1;在鰓組織中,在鹽度10-40區(qū)間內(nèi),兩種基因的表達(dá)存在明顯的拮抗作用,當(dāng)PRL基因的表達(dá)量呈現(xiàn)升高(下降)趨勢時,Na+-K+-ATPaseα1基因的表達(dá)量呈現(xiàn)下降(升高)趨勢,且兩基因的相關(guān)系數(shù)均為負(fù)值,證實了催乳素具有抑制Na+/K+-ATP酶活性的作用,為今后鹽度脅迫分子調(diào)控機(jī)理研究提供理論依據(jù)。利用高通量測序技術(shù),通過對鹽度50海水脅迫后和正常鹽度的腎組織進(jìn)行轉(zhuǎn)錄組測序,構(gòu)建了大菱鲆幼魚鹽度相關(guān)的轉(zhuǎn)錄組數(shù)據(jù)庫,全面了解大菱鲆鹽度脅迫后基因的表達(dá)情況,篩選了與鹽度相關(guān)的候選基因,鑒定了與鹽度相關(guān)的代謝通路,檢測到大量的分子標(biāo)記,并對部分鹽度基因進(jìn)行熒光定量表達(dá)分析,得到的結(jié)果如下:1.得到干凈閱讀子后拼接得到182225個unigenes,拼接得到的Unigenes可全部注釋到7個數(shù)據(jù)庫中。全部基因的功能聚類成43個GO范疇,聚類基因最多的依次是“細(xì)胞過程”,“連接”,“代謝過程”,“單一生物過程”;將KOG注釋成功的基因按KOG的group進(jìn)行分類,聚類基因最多的依次是“信號轉(zhuǎn)導(dǎo)機(jī)制”,“一般功能”,“翻譯修飾、蛋白轉(zhuǎn)換、伴侶”;根據(jù)KEGG代謝通路進(jìn)行分類,聚類基因最多的依次是“信號轉(zhuǎn)導(dǎo)”、“內(nèi)分泌系統(tǒng)”、“免疫系統(tǒng)”。2.通過與其他魚類代謝通路和基因功能分析,鑒定出與大菱鲆鹽度相關(guān)的通路分別是:膽汁分泌,礦物質(zhì)吸收,鈣信號通路,收集管酸分泌;還鑒定出基因顯著(P0.05)富集的通路有16個。3.采用DESeq進(jìn)行分析,篩選閾值為padj0.05,得到262個上調(diào)基因和506個下調(diào)基因,對比其他魚類鹽度轉(zhuǎn)錄組數(shù)據(jù)鑒定出59個與大菱鲆鹽度相關(guān)的基因;既有與Cl-相關(guān)的基因,如:NACC2、PAT1、PRLR等;也有和Na+相關(guān)的基因,如:NHE-3、SGLT1、ASBT等;還有與H2O相關(guān)的基因,如:AQP1、AQP4、AQP8、CA等;還有和能量有關(guān)的基因,如,NKA、ATP6V1E1等;還有代謝相關(guān)的基因,如:CHST6、INO1、ANXA11、IDH2、ECH1、ECH8等;還有既與抗原處理相關(guān)又與應(yīng)激相關(guān)的HSP70基因;還有既與生長相關(guān)又與滲透壓調(diào)節(jié)相關(guān)的PRLR基因。4、根據(jù)基因的作用以及前人的研究結(jié)果,選取10個基因進(jìn)行熒光定量分析發(fā)現(xiàn),除PAT1基因外等9個基因在低鹽度(鹽度5)脅迫下的腎中均有較高的表達(dá);10個基因在腸中的表達(dá)量較少,尤其PAT1基因、APOC1基因、APOM基因、CALM基因、FABP6基因、HMDH基因等6個基因在腸中的表達(dá)量顯著(P0.05)低于其他組織。本文利用MISA軟件對大菱鲆轉(zhuǎn)錄組中的182225條Unigenes序列進(jìn)行搜索,共檢測到81314個SSR位點,SSR發(fā)生頻率為31.17%;EST-SSR的長度在12-20bp的有78247條,其中二核苷酸重復(fù)SSR最多,共計34783條;共檢測到335種重復(fù)基元,其中以單核苷酸的A/T和二核苷酸的AC/GT最多,分別占總SSR的31.17%和31.05%。選擇30對擴(kuò)增引物中,共有3對EST-SSR引物表現(xiàn)出個體多態(tài)性,多態(tài)性比率為10%,SSR的等位基因數(shù)目為2個,平均有效基因數(shù)為1.8141,平均觀測雜合度為0.5514,平均期望雜合度為0.4486,多態(tài)性信息含量分別為0.6897、0.6474、0.5713,平均為0.6361。本研究利用本實驗室前期篩選出特異性較好的151對微衛(wèi)星標(biāo)記對大菱鲆鹽度性狀展開研究。結(jié)合分群分離分析法,初步篩選出4個可以在正常鹽度組和低鹽脅迫組出現(xiàn)差異條帶的微衛(wèi)星位點,然后對4個微衛(wèi)星位點進(jìn)行單個樣本的微衛(wèi)星分析驗證,應(yīng)用SPSS軟件分析,得到1個極顯著(P0.01)相關(guān)位點,2個顯著(P0.05)相關(guān)位點。
[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.

【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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相關(guān)期刊論文 前10條

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本文編號：1856585