锥状斯氏藻休眠孢囊差异表达基因的筛选及能量代谢相关基因在不同休眠条件下的表达研究

发布时间：2018-05-21 02:42

本文选题：锥状斯氏藻 + 休眠孢囊　；参考：《中国科学院大学(中国科学院海洋研究所)》2017年博士论文

【摘要】：甲藻是引起海洋有害藻华形成的主要种类,生活史研究是研究甲藻种群结构和种群动态及基本生物学特征的重要基础,从而也是甲藻藻华生态学的重要研究内容。在产孢囊类甲藻的生活史中,休眠孢囊是甲藻渡过不利环境条件如季节性低温、缺氧、黑暗和营养限制等的重要阶段。研究甲藻孢囊的形成、休眠和萌发过程,特别是在亚细胞及分子水平研究休眠孢囊形成、休眠和萌发的基因调控机制,对从基本生物学机理上认识甲藻藻华的动力学过程和进一步认识藻华的地理扩散具有重要理论和实际意义。然而,迄今为止,文献中关于这一关键生物学过程的分子生物学研究还几乎空白。为此,本论文以世界性分布、经常性形成藻华、较易形成休眠孢囊的甲藻-锥状斯氏藻为模式研究对象,研究了甲藻休眠孢囊与营养细胞间差异性表达的基因和孢囊休眠过程中的生物化学过程对休眠条件的响应,包括以下研究:(1)建立获取大量休眠孢囊的方法:通过添加天然的细沙与锥状斯氏藻营养细胞共培养的方式,实现了锥状斯氏藻休眠孢囊的产量和产速大幅提升。实验数据表明,添加细沙的处理组休眠孢囊数量最多时为同期对照组的107倍,该方法有效地解决了实验过程对休眠孢囊大量需要的问题。同时还发现此方法同样可以提高短沟别什莱藻、Levanderina fissa等产孢囊类甲藻的休眠孢囊产量,但对一些正常培养条件下不易产生休眠孢囊的种类如多环旋沟藻、红色赤潮藻、哈曼褐色多沟藻等则效果不大。(2)成功构建了休眠孢囊的cDNA消减文库:采用抑制性消减杂交技术,以休眠孢囊的cDNA作为测试方(tester),营养细胞的cDNA作为驱动方(driver),经过消减杂交、二轮PCR扩增产物克隆转化,再去掉空载、多克隆后,从1314个单菌落中获得阳性克隆1247个,阳性率为94.9%,插入片段大小主要分布在250~1000 bp之间。随后,经过斑点杂交进一步筛选和验证,共得到1075个显著差异性表达的基因片段,筛选效率为86.2%。(3)对消减文库进行序列分析及验证:消减文库中长度大于100 bp的序列有925条,总碱基数为390,746 bp,序列长度在102-1806 bp之间,平均长度为422 bp。经拼接组装后,共得到280条序列,其中44条为contigs。在280条序列中,74.3%的序列均有功能注释。对于未有注释信息的序列,可能主要由于序列较短不足以判断其功能或功能未知(因为甲藻中已知功能的基因研究尚极少)。对具有功能注释的基因,187条序列注释到生物学过程,其中55条为代谢过程;142条注释到分子功能,主要是催化活动(61条);57条基因序列注释到代谢通路,占KEGG代谢通路总数的70%。通过采用甲藻中特异存在的剪接前导序列(Spliced Leader,SL)为上游引物和基于所获得的序列设计的特异性下游引物对注释为能量代谢的若干序列进行PCR扩增、克隆、测序,结果证明筛选到的消减序列确实来自于甲藻锥状斯氏藻。(4)锥状斯氏藻荧光定量PCR内参基因的筛选:在进行基因表达差异研究之前,以营养细胞cDNA为模板,用三个生物平行及三个技术平行,根据Ct值和溶解曲线筛选内参基因。6个候选基因的平均Ct值均在30以内,GeNorm软件通过计算平均变异度M得出候选基因的稳定性由高到低依次为:CYCUBCPEPCKACTGAPDHCOX1。根据V_(2/3)0.15确定需要内参基因的最佳数目为2。通过Normfinder软件计算各候选内参基因在细胞中的表达稳定值SV,得出稳定性从高到低依次为PEPCKACTCYCGAPDHCOX1UBC。Bestkeeper软件通过计算各候选基因Ct值的相关系数r,得出6个基因稳定性由高到低为:CYCUBCPEPCKGAPDHACTCOX1。综合上述三个常用内参基因稳定性分析软件的分析结果,后续的基因表达定量分析研究选用CYC和PEPCK作为内参基因。(5)能量代谢相关基因在营养细胞和休眠孢囊及处于不同休眠条件下的休眠孢囊内的差异表达研究:从消减文库与锥状斯氏藻转录组文库相似性大于90%且有功能注释的65条序列中,根据所注释的功能在孢囊休眠过程中可能的作用,综合序列长度、扩增曲线Ct值及溶解曲线,选定ATP合成酶β亚基、细胞色素c氧化酶亚基1及一个翻译延伸因子,以CYC(Cyclophilin,亲环素)和PEPCK(Phosphoenolpyruvate carboxykinase,磷酸烯醇式丙酮酸激酶)为内参基因,进行后续差异表达分析,即定量检测所选基因在处于不同休眠状态的休眠孢囊中的表达状况。结果表明,上述三个基因在休眠孢囊中的表达水平均明显低于营养细胞中的表达水平,而且,表达水平总体上随着休眠孢囊在黑暗、低温、无氧或低氧环境下的延长而有所降低,表明休眠孢囊在休眠状态下对环境条件变化仍保持着快速的应激反应,且通过降低能量代谢水平以保存能量储存从而延长存活时间或者为萌发储备能量。这一结果为从分子生物学水平解释休眠孢囊在海洋沉积物中渡过长期黑暗、低温而保持存活提供了重要基础。
[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.
【学位授予单位】：中国科学院大学(中国科学院海洋研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X173;Q943.2

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