本文選題：錐狀斯氏藻 + 休眠孢囊��； 參考：《中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院海洋研究所)》2017年博士論文

【摘要】：甲藻是引起海洋有害藻華形成的主要種類,生活史研究是研究甲藻種群結(jié)構(gòu)和種群動(dòng)態(tài)及基本生物學(xué)特征的重要基礎(chǔ),從而也是甲藻藻華生態(tài)學(xué)的重要研究?jī)?nèi)容。在產(chǎn)孢囊類甲藻的生活史中,休眠孢囊是甲藻渡過不利環(huán)境條件如季節(jié)性低溫、缺氧、黑暗和營(yíng)養(yǎng)限制等的重要階段。研究甲藻孢囊的形成、休眠和萌發(fā)過程,特別是在亞細(xì)胞及分子水平研究休眠孢囊形成、休眠和萌發(fā)的基因調(diào)控機(jī)制,對(duì)從基本生物學(xué)機(jī)理上認(rèn)識(shí)甲藻藻華的動(dòng)力學(xué)過程和進(jìn)一步認(rèn)識(shí)藻華的地理擴(kuò)散具有重要理論和實(shí)際意義。然而,迄今為止,文獻(xiàn)中關(guān)于這一關(guān)鍵生物學(xué)過程的分子生物學(xué)研究還幾乎空白。為此,本論文以世界性分布、經(jīng)常性形成藻華、較易形成休眠孢囊的甲藻-錐狀斯氏藻為模式研究對(duì)象,研究了甲藻休眠孢囊與營(yíng)養(yǎng)細(xì)胞間差異性表達(dá)的基因和孢囊休眠過程中的生物化學(xué)過程對(duì)休眠條件的響應(yīng),包括以下研究:(1)建立獲取大量休眠孢囊的方法:通過添加天然的細(xì)沙與錐狀斯氏藻營(yíng)養(yǎng)細(xì)胞共培養(yǎng)的方式,實(shí)現(xiàn)了錐狀斯氏藻休眠孢囊的產(chǎn)量和產(chǎn)速大幅提升。實(shí)驗(yàn)數(shù)據(jù)表明,添加細(xì)沙的處理組休眠孢囊數(shù)量最多時(shí)為同期對(duì)照組的107倍,該方法有效地解決了實(shí)驗(yàn)過程對(duì)休眠孢囊大量需要的問題。同時(shí)還發(fā)現(xiàn)此方法同樣可以提高短溝別什萊藻、Levanderina fissa等產(chǎn)孢囊類甲藻的休眠孢囊產(chǎn)量,但對(duì)一些正常培養(yǎng)條件下不易產(chǎn)生休眠孢囊的種類如多環(huán)旋溝藻、紅色赤潮藻、哈曼褐色多溝藻等則效果不大。(2)成功構(gòu)建了休眠孢囊的cDNA消減文庫:采用抑制性消減雜交技術(shù),以休眠孢囊的cDNA作為測(cè)試方(tester),營(yíng)養(yǎng)細(xì)胞的cDNA作為驅(qū)動(dòng)方(driver),經(jīng)過消減雜交、二輪PCR擴(kuò)增產(chǎn)物克隆轉(zhuǎn)化,再去掉空載、多克隆后,從1314個(gè)單菌落中獲得陽性克隆1247個(gè),陽性率為94.9%,插入片段大小主要分布在250~1000 bp之間。隨后,經(jīng)過斑點(diǎn)雜交進(jìn)一步篩選和驗(yàn)證,共得到1075個(gè)顯著差異性表達(dá)的基因片段,篩選效率為86.2%。(3)對(duì)消減文庫進(jìn)行序列分析及驗(yàn)證:消減文庫中長(zhǎng)度大于100 bp的序列有925條,總堿基數(shù)為390,746 bp,序列長(zhǎng)度在102-1806 bp之間,平均長(zhǎng)度為422 bp。經(jīng)拼接組裝后,共得到280條序列,其中44條為contigs。在280條序列中,74.3%的序列均有功能注釋。對(duì)于未有注釋信息的序列,可能主要由于序列較短不足以判斷其功能或功能未知(因?yàn)榧自逯幸阎δ艿幕蜓芯可袠O少)。對(duì)具有功能注釋的基因,187條序列注釋到生物學(xué)過程,其中55條為代謝過程;142條注釋到分子功能,主要是催化活動(dòng)(61條);57條基因序列注釋到代謝通路,占KEGG代謝通路總數(shù)的70%。通過采用甲藻中特異存在的剪接前導(dǎo)序列(Spliced Leader,SL)為上游引物和基于所獲得的序列設(shè)計(jì)的特異性下游引物對(duì)注釋為能量代謝的若干序列進(jìn)行PCR擴(kuò)增、克隆、測(cè)序,結(jié)果證明篩選到的消減序列確實(shí)來自于甲藻錐狀斯氏藻。(4)錐狀斯氏藻熒光定量PCR內(nèi)參基因的篩選:在進(jìn)行基因表達(dá)差異研究之前,以營(yíng)養(yǎng)細(xì)胞cDNA為模板,用三個(gè)生物平行及三個(gè)技術(shù)平行,根據(jù)Ct值和溶解曲線篩選內(nèi)參基因。6個(gè)候選基因的平均Ct值均在30以內(nèi),GeNorm軟件通過計(jì)算平均變異度M得出候選基因的穩(wěn)定性由高到低依次為:CYCUBCPEPCKACTGAPDHCOX1。根據(jù)V_(2/3)0.15確定需要內(nèi)參基因的最佳數(shù)目為2。通過Normfinder軟件計(jì)算各候選內(nèi)參基因在細(xì)胞中的表達(dá)穩(wěn)定值SV,得出穩(wěn)定性從高到低依次為PEPCKACTCYCGAPDHCOX1UBC。Bestkeeper軟件通過計(jì)算各候選基因Ct值的相關(guān)系數(shù)r,得出6個(gè)基因穩(wěn)定性由高到低為:CYCUBCPEPCKGAPDHACTCOX1。綜合上述三個(gè)常用內(nèi)參基因穩(wěn)定性分析軟件的分析結(jié)果,后續(xù)的基因表達(dá)定量分析研究選用CYC和PEPCK作為內(nèi)參基因。(5)能量代謝相關(guān)基因在營(yíng)養(yǎng)細(xì)胞和休眠孢囊及處于不同休眠條件下的休眠孢囊內(nèi)的差異表達(dá)研究:從消減文庫與錐狀斯氏藻轉(zhuǎn)錄組文庫相似性大于90%且有功能注釋的65條序列中,根據(jù)所注釋的功能在孢囊休眠過程中可能的作用,綜合序列長(zhǎng)度、擴(kuò)增曲線Ct值及溶解曲線,選定ATP合成酶β亞基、細(xì)胞色素c氧化酶亞基1及一個(gè)翻譯延伸因子,以CYC(Cyclophilin,親環(huán)素)和PEPCK(Phosphoenolpyruvate carboxykinase,磷酸烯醇式丙酮酸激酶)為內(nèi)參基因,進(jìn)行后續(xù)差異表達(dá)分析,即定量檢測(cè)所選基因在處于不同休眠狀態(tài)的休眠孢囊中的表達(dá)狀況。結(jié)果表明,上述三個(gè)基因在休眠孢囊中的表達(dá)水平均明顯低于營(yíng)養(yǎng)細(xì)胞中的表達(dá)水平,而且,表達(dá)水平總體上隨著休眠孢囊在黑暗、低溫、無氧或低氧環(huán)境下的延長(zhǎng)而有所降低,表明休眠孢囊在休眠狀態(tài)下對(duì)環(huán)境條件變化仍保持著快速的應(yīng)激反應(yīng),且通過降低能量代謝水平以保存能量?jī)?chǔ)存從而延長(zhǎng)存活時(shí)間或者為萌發(fā)儲(chǔ)備能量。這一結(jié)果為從分子生物學(xué)水平解釋休眠孢囊在海洋沉積物中渡過長(zhǎng)期黑暗、低溫而保持存活提供了重要基礎(chǔ)。
[Abstract]:The study of life history is the important basis for the study of the structure and population dynamics and basic biological characteristics of the diatom population, and it is also an important research content of the ecology of the alga blooms. The important stages of sexual hypothermia, hypoxia, darkness, and nutritional constraints. Study the formation, dormancy and germination of the cystis cysts, especially the gene regulation mechanism of the formation of dormant cysts, dormancy and germination at the subcellular and molecular levels, and the understanding of the kinetics of the alga blooms and the further understanding of the algal bloom from the basic biological mechanism. Geographical diffusion has important theoretical and practical significance. However, so far, the molecular biology of this key biological process is still almost blank in the literature. For this reason, this paper is based on the worldwide distribution, the formation of algal blooms frequently, and the formation of the dormant spores of the alga ticerospora, which is easier to form, and studies the dormancy of the alga. The differentially expressed genes between the cysts and the vegetative cells and the response of the biochemical process to the dormancy of the cysts include the following studies: (1) the establishment of a method for obtaining a large number of dormant spores: the production of the dormant cysts of the cones by adding natural fine sand and the common culture of the vegetative cells of the cones. The experimental data showed that the number of dormant spores in the treated group with fine sand was 107 times as much as that of the control group. This method effectively solved the problem of the large demand for the dormant cysts in the experimental process. It also found that this method could also improve the short gully algae, Levanderina fissa and other cyclosporin. The yield of dormant spores, but not easy to produce dormant cysts in some normal conditions, such as polycyclic alga, red red tide algae, Haman brown polygutter, and so on. (2) the subtractive library of dormant spores was successfully constructed: using inhibitory subtractive hybridization technique, the cDNA of dormant spores as the test party (tester), nutrition (tester). The cDNA of the cell was used as the driving force (driver). After subtractive hybridization, two rounds of PCR amplification products were cloned and transformed, and then no load was removed. After polyclonal, 1247 positive clones were obtained from 1314 single colonies. The positive rate was 94.9% and the size of the inserted fragments was mainly distributed between 250~1000 BP. Then, a total of 1075 were screened and verified by dot blot. A total of 1075 were obtained. A significant difference expression gene fragment, the screening efficiency is 86.2%. (3) sequence analysis and verification of Subtractive Library: there are 925 sequences in the subtractive library with length more than 100 bp, the total base number is 390746 BP, the sequence length is 102-1806 BP, the average length is 422 bp., and the 280 sequences are obtained, 44 of them are cont. In the 280 sequence, 74.3% of the 280 sequences have functional annotations. For a sequence with no annotated information, it may be mainly because the sequence is short enough to judge the function or function of the unknown (because the gene for the known function in the dinoflagellate is very few). For the genes with functional annotation, 187 sequences are annotated to the biological process, of which 55 are substituted. Xie Guocheng; the 142 is annotated to molecular function, mainly catalytic activity (61); 57 gene sequences are annotated to metabolic pathways, accounting for 70%. of the total number of KEGG metabolic pathways, as upstream primers and specific downstream primers designed based on the sequences obtained by using specific existing splicing preamble (Spliced Leader, SL) in dinoflagellate Several sequences of quantitative metabolism were amplified, cloned, and sequenced. The results showed that the selected subtractive sequences were indeed derived from PCR. (4) screening of PCR internal reference genes in the fluorescence quantitation of spina coniformis: prior to the study of gene expression differences, the nutrient cell cDNA was used as a template and parallel to the three biological parallel and three techniques. The average Ct values of.6 candidate genes screened by Ct and dissolving curves were within 30. GeNorm software calculated the stability of candidate genes from high to low by calculating the average variability M: CYCUBCPEPCKACTGAPDHCOX1. based on V_ (2/3) 0.15 to determine the optimal number of the required internal reference genes 2. through Normfinder software. The stability value of the expression of the internal reference gene in the cells was SV, and the results showed that the stability from high to low was PEPCKACTCYCGAPDHCOX1UBC.Bestkeeper software by calculating the correlation coefficient r of the Ct value of each candidate gene, and the stability of 6 genes from high to low was obtained: CYCUBCPEPCKGAPDHACTCOX1. comprehensive analysis software for the stability of the three commonly used internal parameter genes. Analysis results, followed by quantitative analysis of gene expression, CYC and PEPCK were selected as the internal reference genes. (5) the differential expression of energy metabolism related genes in the dormant spores of the vegetative and dormant cysts and under different dormancy conditions: the similarity between the subtractive library and the taper leaf algae transcriptome is greater than 90% and has functional annotation. In the 65 sequence, the function of the annotated function during the spore dormancy process, the length of the sequence, the Ct value of the amplification curve and the dissolution curve, the ATP synthase beta subunit, the cytochrome c oxidase subunit 1 and a translation elongation factor, with CYC (Cyclophilin, procyclin) and PEPCK (Phosphoenolpyruvate carboxykinase, phosphoryl phosphate). Alcohol pyruvate kinase (PK) was an internal reference gene, followed by differential expression analysis, which was used to quantify the expression of the selected genes in the dormant spores of different dormant states. The results showed that the expression level of the three genes in the dormant cysts was significantly lower than that in the nutrient cells, and the expression level was generally associated with the expression level. The dormant spores are reduced in the dark, low temperature, anaerobic, or hypoxic environment, indicating that the dormant cysts still maintain a rapid stress response to the changes in the environment under the dormant state, and save energy storage by lowering the energy metabolism level to prolong the survival time or to reserve the energy of the germination. The subbiological level explains that resting spores play an important role in the long dark and low temperature survival of marine sediments.
【學(xué)位授予單位】：中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院海洋研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：X173;Q943.2

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