学校编码 :10384 分类号密级学号 : UDC 硕士学位论文高静水压对葡萄牙牡蛎胚胎和幼体发育的影响 The effects of high hydrostatic pressure on the development of embryos and larvae

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1 学校编码 :10384 分类号密级学号 : UDC 硕士学位论文高静水压对葡萄牙牡蛎胚胎和幼体发育的影响 The effects of high hydrostatic pressure on the development of embryos and larvae of Crassostrea angulata 梁翠指导教师姓名 : 王德祥副教授专业名称 : 海洋生物学论文提交日期 :2011 年 08 月论文答辩时间 :2011 年 09 月学位授予日期 :2011 年 04 月答辩委员会主席 : 评阅人 : 2011 年 09 月

2 厦门大学学位论文原创性声明本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中以适当方式明确标明, 并符合法律规范和厦门大学研究生学术活动规范 ( 试行 ) 另外, 该学位论文为 ( ) 课题 ( 组 ) 的研究成果, 获得 ( ) 课题 ( 组 ) 经费或实验室的资助, 在 ( ) 实验室完成 ( 请在以上括号内填写课题或课题组负责人或实验室名称, 未有此项声明内容的, 可以不作特别声明 ) 声明人 ( 签名 ): 年月日

3 厦门大学学位论文著作权使用声明本人同意厦门大学根据中华人民共和国学位条例暂行实施办法等规定保留和使用此学位论文, 并向主管部门或其指定机构送交学位论文 ( 包括纸质版和电子版 ), 允许学位论文进入厦门大学图书馆及其数据库被查阅借阅本人同意厦门大学将学位论文加入全国博士硕士学位论文共建单位数据库进行检索, 将学位论文的标题和摘要汇编出版, 采用影印缩印或者其它方式合理复制学位论文本学位论文属于 : ( )1. 经厦门大学保密委员会审查核定的保密学位论文, 于 2013 年 12 月 1 日解密, 解密后适用上述授权 ( )2. 不保密, 适用上述授权 ( 请在以上相应括号内打或填上相应内容保密学位论文应是已经厦门大学保密委员会审定过的学位论文, 未经厦门大学保密委员会审定的学位论文均为公开学位论文此声明栏不填写的, 默认为公开学位论文, 均适用上述授权 ) 声明人 ( 签名 ): 年月日

4 目录目录缩略词表... I 摘要...III Abstract...V 第 1 章绪论高压生物学的发展及研究现状高压对生物大分子的作用高压对细胞的影响高压对生物有机体的影响高压生物技术及应用生物的遗传变异生物的变异表观遗传变异转录组学的研究 DNA 微阵列 cdna-aflp EST 表达序列标签 SAGE 基因表达系列分析 RNA-Seq 技术本课题的研究材料目的和意义研究材料研究目的和意义研究内容和方法...18 第 2 章高静水压作用下葡萄牙牡蛎幼体的形态学分析材料与方法仪器试剂与溶液配制生物材料来源...20 I

5 目录牡蛎受精卵的获得正交试验设计以及压力实验取样幼体培育和观察牡蛎幼体染色体制片幼体的扫描电镜观察结果和分析葡萄牙牡蛎早期发育特征观察牡蛎幼体在不同压力处理条件下的成活率牡蛎幼体在不同压力处理条件下生长大小的测量牡蛎幼体染色体制片结果牡蛎幼体的扫描电镜观察讨论正交试验设计方法高静水压对牡蛎幼体生长成活率的影响高静水压对染色体变异的影响...35 第 3 章高静水压作用下葡萄牙牡蛎幼体基因组的甲基化分析材料与方法仪器试剂与溶液配制取样牡蛎幼体基因组 DNA 的提取及其纯度与含量检测甲基化敏感的扩增多态性分析 (MSAP) 结果和分析牡蛎幼体基因组 DNA 的提取 DNA 的预扩增效果检测 DNA 的选择性扩增效果检测多态性引物的筛选高静水压处理前后的牡蛎幼体基因组 DNA 的 MSAP 分析讨论...50 II

6 目录第 4 章高静水压作用下葡萄牙牡蛎幼体转录组学分析材料仪器试剂与溶液配制生物样品方法葡萄牙牡蛎幼体总 RNA 的提取 mrna 的制备 cdna 第一链的合成 ds cdna 的合成标签文库的构建结果和分析葡萄牙牡蛎幼体 RNA 的提取双链 cdna 破碎时间尝试 PCR 检测接头连接测序结果分析讨论利用转录组数据进行基因表达差异分析的方法利用 Gene Ontology 对差异表达的基因进行注释与功能分类高静水压诱导处理下葡萄牙牡蛎幼体的适应机制...69 结论...72 参考文献...74 在学期间发表的文章和成果...85 致谢...86 附录 I-III...87 III

7 Contents Contents Abbreviations... I Abstract in Chinese...III Abstract in English...V Chapter 1 Introduction Development of high pressure biology The effect of HHP on the stucture of molecules The effect of HHP on the behavior of cell The effect of HHP on the role of organism HHP technic and application Genetic variation Biological variation Epigenetic variation Method of transcriptomics DNA microarray cdna-aflp Expressed sequence tag Serial analysis of gene expression RNA-Seq technology Brief introduction to this study Materials Purpose and meaning Content and methods...18 Chapter 2 Morphology analysis of C. angulata after HHP treatment Materials and methods Equipment, reagents and solution Materials Acquiring the fertilized eggs of C. angulata...20 IV

8 Contents Orthogonal experiment design and pressure conduct Sample collection Cultivation and observation of larvae Chromosome preparation Scanning electron microscope of larval shells Results Early developmental character of C. angulata Survival rate of larvae after different HHP conduct Growth rate of oyster larvae after different HHP conduct Karyotype of C. angulata larvae Scanning electron microscope of larvae Discussion Orthogonal experiment design Effect of HHP on growth and survival rate Effect of HHP on oyster karyotype...35 Chapter 3 DNA methylation of C. angulata larvae after HHP treatment Materials and methods Equipment, reagents and solution Sample collection DNA extraction and detection Analysis of methylation sensitive amplified polymorphism Results DNA extraction DNA amplification and detection Selective amplification Selection of polymorphic primers MSAP analysis Discussion...50 Chapter 4 Transcriptomics analysis of C. angulata larvae after HHP V

9 Contents treatment Materials Equipment, reagents and solution Sample source Methods Total RNA extraction of larval C. angulata Preparation of mrna First strand cdna synthesis ds cdna synthesis Construction of 454 barcoded library Results and analysis RNA extraction of C. angulata The optimal duration of ds cdna broken by sonication Detection of the adapters Result analysis of 454 sequencing Discussion Method of 454 data analysis Annotation of differentially expressed genes using Gene Ontology Induced adaptation mechanism of larval C. angulata in HHP...69 Summary...72 References...74 Achievements...85 Acknowledgements...86 Appendix I-III...87 VI

10 缩略词表缩略词表英文缩写英文名称中文名称 APS ammonium persulfate 过硫酸胺 bp base pair 碱基对 BSA bovine serum albumin 牛血清白蛋白 cdna complementary DNA 互补 DNA cdna-aflp cdna amplified restriction fragment length polymorphism cdna 扩增限制性片段长度多态性 ddh 2 O double distilled H 2 O 双蒸水 DEPC diethypyrocabonate 焦乙酸二乙酸 dntp deoxy-ribonucleoside triphosphate 三磷酸脱氧核糖核苷 dsdna double-stranded DNA 双链 DNA DTT diethyl pyrocarbonate 二硫苏糖醇 EDTA ethylene diamine tetraacetic acid 乙二胺四乙酸 EST expressed sequence tag 表达序列标签 fs cdna First-stranded cdna 第一条链 cdna GO gene ontology 基因本体数据库 HHP high hydrostatic pressure 高静水压 HSP heat shock proteins 热休克蛋白 IPTG Isopropyl β-d-1-thiogalactopyranoside 异丙基 -β-d- 硫代吡喃半乳糖 KEGG Kyoto Encyclopedia of Genes and Genomes 京都基因与基因组百科全书 MID Multiplex Identifier 多样标识符苷 MIPS the Munich Information Center for Protein Sequences 慕尼黑蛋白质序列信息中心 mirna microrna 小 RNA mrna messenger RNA 信使 RNA I

11 缩略词表英文缩写英文名称中文名称 MSAP Methylation sensitive amplified polymorphism 甲基敏感扩增片段多态性 OD optical density 光密度 PA polyacrylamide 聚丙烯酰胺 PBS phosphate-buffered saline 磷酸盐缓冲液 rdna ribosomal DNA 核糖体 DNA RNA-seq RAN sequencing RNA 测序 RT-PCR reverse transcription polymerase chain reaction 逆转录聚合酶链式反应 SAGE serial analysis of gene expression 基因表达系列分析 SAM S-acid armour sulfur acid atp S - 腺苷酰甲硫氨酸 SDS sodium dodecyl sulfate 十二烷基磺酸钠 SEM scanning electron microscope 扫描电镜 sirna small interfering RNA 小的干扰 RNA TAE Tris-acetic- EDTA(buffer) Tris/ 醋酸 /EDTA 缓冲液 TBE Tris-borate-EDTA(buffer) Tris/ 硼酸 /EDTA 缓冲液 TEMED N,N,N N -tetramethylethylenediamine N,N,N N - 四甲基乙二胺 Tris Tris(hydroxylmethyl) amino-methane 三羟甲基氨基甲烷 II

12 摘要摘要高静水压 (High Hydrostatic Pressure,HHP) 是一种极端的物理压力效应, 存在于压强超过 10MPa 的液体中, 它相当于 1000m 水深所产生的压强, 可以对细胞产生多种影响, 包括染色体的结构膜的整合性 DNA 和蛋白的相互作用及改变基因的表达等除深海生物外, 高压也会影响常压生物的正常生活在高压环境下, 生物体会产生相应的对应机制, 以适应极端环境, 有关高静水压对生物生长影响的研究目前仅限于从海洋中分离的细菌真菌以及部分陆生动植物在水产动物方面, 高静水压更多的被运用于鱼类及贝类的遗传育种, 如三倍体育种等在本文, 我们研究了高静水压对葡萄牙牡蛎胚胎及幼体发育的影响, 这些信息能够帮助我们更好的理解高压环境下生物体适应和生存机制主要研究结果如下 : 1 本文应用正交试验的方法设计了 3 因素 3 水平的 9 组试验, 同时设立在常压下生长的对照组, 利用高静水压仪在牡蛎发育的几个关键时期实施压力诱导, 研究静水压对于牡蛎胚胎和幼体发育的影响在水温 25, 盐度 30 的条件下,3 个实验因子及 3 个实验水平分别是 : 牡蛎早期不同的发育阶段 ( 囊胚期担轮幼虫期 D 型幼虫 ); 高压的持续时间 ( min); 静水压强的大小 ( MPa) 以牡蛎受精后第 12 天的成活率和第 19 天的平均体长为指标, 分析了 9 个实验组牡蛎幼体的成活率及体长结果显示, 以牡蛎受精后第 12 天的成活率为指标, 三个因子重要性顺序是 : 压力大小 > 高压持续时间 > 开始处理时间 ; 最佳处理条件是 : 在牡蛎幼体发育至 D 型幼虫时, 施以 10MPa 的压力 50min, 有望获得较高成活率的幼体若以受精后第 19 天的平均体长为指标, 三个因子重要性顺序是 : 开始处理时间 > 高压持续时间 > 压力大小 ; 最佳处理条件是 : 在牡蛎幼体发育至担轮幼虫时, 施以 10MPa 的压力 30min, 有望获得生长较快的幼体对高静水压处理前后的葡萄牙牡蛎胚胎和幼体进行染色体制片及倍性分析, 结果显示, 各个压力实验组与对照组在染色体数目及结构均无显著差异, 说明在本实验的静水压处理条件下, 静水压对染色体畸变的影响较小对牡蛎幼体原生和次生贝壳进行扫描电镜观察, 结果显示, 受精后 36h, 第 2 组 ( 囊胚 III

13 摘要期 30min 20MPa), 第 3 组 ( 囊胚期 50min 40MPa) 和第 4 组 ( 担轮幼虫期 10min 40MPa) 的个体看不到明显的 D 型壳, 大小明显小于其他 7 组的幼体个体大小, 可见高静水压能够改变牡蛎幼体从受精卵到 D 形幼虫的发育快慢 ; 实验组与对照组受精后 3 天和眼点期幼虫的形态比较结果, 未发现有显著的差异综上, 不同的高静水压处理条件不仅能够提高牡蛎幼体成活率和生长速度, 也能导致幼体发育滞后甚至死亡在实际生产中, 该实验结果可以用于指导水产养殖生产实践, 有利于提高幼体的成活率和生长速率 2 利用甲基化敏感扩增多态性技术(methylation sensitive amplified polymorphism, MSAP), 对葡萄牙牡蛎幼体高静水压处理前后的基因组 DNA 5 -CCGG-3 位点进行全基因组甲基化扫描检测聚丙烯酰胺凝胶电泳及银染结果表明, 与对照组相比, 虽然在静水压处理前后相同遗传背景材料中获得了多个甲基化位点的差异, 但是经过高静水压处理后的 DNA 甲基化条带比率仅由对照组的 9.4% 增加至 10%, 差异不显著我们尚不能判断甲基化是否参与静水压对牡蛎幼体的互作, 结果尚需进一步的验证 3 利用转录组数据进行基因表达差异分析构建高静水压处理组第 9 组和对照组的 cdna 文库, 经过 454 测序拼接和验证, 拼接后共获得条序列, 并对这些序列进行 GO 注释选取在测序中被表达的次数在 20 以上的片段, 共获得序列 275 条, 其中被 GO 注释的有 170 条, 为总序列的 61.8%,2 倍以上的上调序列有 26 条 ( 占 15.3%),2 倍以上的下调序列有 31 条 ( 占 18.2%) 根据 GO 功能注释,2 倍以上的上调或下调基因主要涉及一些能量和代谢基因转录和翻译调节基因细胞的运输调节和信号转导基因细胞防御和内环境的调节基因细胞结构成分基因以及一些功能未知的基因这些差异基因的上调或抑制表达可能对牡蛎幼体在高静水压下的适应和生存具有重要的作用本研究首次对海洋贝类在高静水压诱导处理下的基因表达进行分析, 基因组表达类型的功能分析揭示了高压特异的压力反应类型, 提供了关于逆境环境下生物体适应和生存机制的丰富信息关键词 : 高静水压 ; 葡萄牙牡蛎 ; 胚胎和幼体 ;DNA 甲基化分析 ; 转录组分析 IV

14 Abstract Abstract High hydrostatic pressure (HHP) is an extreme thermal physical pressure effect, and exists in more than 10MPa pressure of liquid. It is equivalent to 1000m depth of sea water. High hydrostatic pressure have different effects on cells, including the structure of the chromosomes, the integration of membrane, the interaction of DNA and proteins, and the changes in gene expression, etc. In addition, HHP can also affect the organisms in atmosphere pressure. At the same time, the organisms can form corresponding defense mechanism to adapt to high pressue. Many adaptation mechanism to HHP has been well studied in bacterias and fungus: isolated from the deep sea, and some terrestrial organisms. In aquatic animals, HHP is mainly used in the genetic breeding of fish and shellfish, such as the triploid breeding. The different effects of HHP on the development of embryos and larvae of C. angulata were studied. These informations could help to understand the pressure-adapataion mechanism for C. angulata. 1 Three factors and three levels of nine groups orthogonal test were designed. We designed the HHP instrument to carry out the pressure-induced experiment in several key stages of oyster development, and explored the effect of HHP on the development of embryos and larvae of C. angulata. Under the condition of water temperature 25 and the salinity 30, three factors and three levels were: different early development stages of C. angulata (blastocysts stage, trochosphere stage and D shape larvae); The duration of HHP (10, 30 and 50 min); The magnitude of HHP (10, 20 and 40 MPa). The osyter survival rates of the 12 th day and the shell sizes of the 19 th day after fertilization as indexes, the survival rates and shell sizes of larvae were analysed. The results showed that, 12 th day after fertilization as the index, the magnitude was the most important; The optimal condition of treatment: the higher survival rate of the oysters was required in the stage of D shape larvae, under the pressure of 10MPa and duration of 50min. Nineteenth day after fertilization as the index, the stage of treatment was the most important; The optimal condition of V

15 Abstract treatment: the faster grow rate of the oysters was required in the stage of trochosphere, under the pressure of 10MPa and duration of 30min. Comparision of the chromosome of embryos and larvae of C. angulata between HHP and control groups, there were no significant differences. This indicated that HHP had little influence on the distortion of chromosome. By using scanning electron microscope for the primary and secondary shell of larval C. angulata, the results showed that the D shape shells of the larvae of group 2 (blastocysts stage, 30min and 20MPa), group 3 (blastocysts stage, 50min and 40MPa) and group 4 (trochophore, 10 min and 40 MPa) were not obvious, and the sizes of larvae were much smaller than other senven groups. It was indicated that HHP could affect the growth rate of larval C. angulata development from fertilized eggs to D shape larvae. There was no distinction in the comparison of morphology of larvae of the third day after fertilization and eye spot between HHP and control groups. In conclusion, different conditions of HHP could not only improve the survival and growth rate of C. angulata, but also induce development lag and even death. In practice, the results can be used to guide aquaculture and improve the survival and growth rate of C. angulata. 2 We scanned and deteced the whole genome DNA 5 -CCGG-3 methylation of larval C. angulata of HHP and control groups by using the method of methylation sensitive amplified polymorphism(msap). Compared with the control group, the ratio of DNA methylation of larvae after HHP treatment was only increased from 9.4% to 10% and the differences were not significant, although the differences of multiple methylation sites in the same genetic background materials of before and after HHP treatment. Whether the methylation participated in the interaction of HHP with larval C. angulata was unclear, and need further research. 3 We used transcriptome data to analyse the differences of gene expression, and constructed the cdna librarys of HHP and control groups. We analysed GO annotation by the obtained sequences. Selecting the sequences which were showed more than 20 times in sequencing and 275 were acquired. Among these sequences, 170 were annotated and represented 61.8% of total. Twenty six genes (15.3% of total) were >2-fold induced while 31 (18.2% of total) suffered a > 2-fold VI

16 Abstract downregulation. According to GO annotation, the majority of the > 2-fold upregulated and repressed genes were involved in some energy and metabolic genes, transcription and translation regulated genes, cell transportation, signal path genes, cell defense and regulation of cellular environment, cell organization and some unknown genes. These differential genes may play an important role in the adaption and survival of C. angulata under HHP. The gene expression of the marine shellfish afer HHP induction treatment was firstly analysed. The functional analysis of genomic expression revealed a hydrostatic pressure-specific stress response pattern and provided abundant informations of the mechanisms involed in the adaptation and survival of organisms under extreme environment. These changes caused by HHP indicated that HHP treatment could change the expression of genes and induce the adaptive response of C. angulata. Key Words: high hydrostatic pressue; Crassostred angulata; embryos and larvae; DNA methylation analysis; transcriptome analysis VII

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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10384 -------- 9601008 UDC---------------------- 1999 1999 1999 ------------------------ ------------------------ 1 2 .4...4 7 14.14..18..22..22..24 3 4.27.32.39 1 2 3 4 5 6 7 8 9 10 11 5 12 6 1 2 3 4

学校编码 :10384 分类号密级 学号 : UDC 硕士学位论文 高静水压对葡萄牙牡蛎胚胎和幼体发育的影响 The effects of high hydrostatic pressure on the development of embryos and larvae

学校编码 :10384 分类号密级学号 : UDC 硕士学位论文高静水压对葡萄牙牡蛎胚胎和幼体发育的影响 The effects of high hydrostatic pressure on the development of embryos and larvae