【作者】 刘永健；

【导师】 戴继勋； 杨官品；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 东海原甲藻(Prorocentrum donghaiense)是近期在我国东海区出现的大规模赤潮的引发种,使当地经济蒙受巨大损失,环境受到极大破坏。目前,东海原甲藻的研究已成为赤潮生物学的研究热点之一。近几年来,关于东海原甲藻的分类、生活史、生理生态的研究很是广泛,然而,对东海原甲藻赤潮形成和消亡的机制却知之甚少。我们构建了对数生长期东海原甲藻细胞cDNA文库。我们使用mRNA Purification Kit MagExtractor? - mRNA-试剂盒抽提纯化对数生长期东海原甲藻细胞poly(A)+ RNA,Super SMART? PCR cDNA Synthesis Kit试剂盒逆转录poly(A)+ RNA为单链cDNA并进行PCR扩增,然后将双链cDNA连接到质粒pMD 18-T vector载体上,并将连接产物电转化至感受态细胞E. coli JM109。结果显示,电转化转化效率大于109,并且,文库中绝大部分插入片断大小在700-1500bp之间。使用此种方法,我们能够对微量东海原甲藻细胞(细胞数小于105个细胞)构建cDNA文库。以正常磷酸盐营养浓度培养条件下的东海原甲藻为对照组,使用cDNA-AFLP技术对磷酸盐胁迫下的东海原甲藻细胞的表达情况进行了分析。通过AFLP分析,我们共分离了50条表达差异条带,经过克隆测序分析,最后得到43条有效序列。网上BLAST分析结果显示,其中只有8条序列搜寻到匹配序列,而剩余的35条序列未搜寻到任何匹配序列。以β-actin基因为内参,我们对其中的三个基因片断(TDF68、TDF72和TDF85)进行了表达差异分析。结果表明:东海原甲藻细胞在受到磷酸盐胁迫时,三个片断的表达量全部增加,并且与对照相比,差异显著。其中,基因片段TDF72(antioxidant,AhpC/Tsa family)的表达量是对照组的1.24倍(p=0.04) ;TDF68(dual-specificity protein phosphatase )是对照组的1.60倍( p=0.002 ) TDF85(Phosphoglycerate/bisphosphoglycerate mutase)是对照组的2.30倍(p=0.0001)。通常认为,单细胞浮游藻是“不死”的,除非是被捕食、或者被病毒或细菌感染致死、或者是受到物理损伤、或者是永久的、不可逆转的沉降到海洋深处。然而,研究资料显示,受到环境胁迫时,单细胞真核浮游植物也存在类似细胞程序化死亡的现象。通过显微观察,我们观测到了东海原甲藻衰老细胞破裂死亡现象,衰老阶段的东海原甲藻细胞死亡是否也是一种主动地死亡呢?我们在东海原甲藻衰老细胞表达序列标签(EST)分析中发现了部分半胱氨酸基天冬氨酸特异性蛋白酶(cysteinyl aspartate specific proteinase, caspase,caspase)编码基因序列,在本文中,我们克隆了东海原甲藻caspase编码基因的全长cDNA序列,序列分析发现,其核苷酸全长520bp,包含一个258bp的开放阅读框(open reading frame,ORF),编码一个85氨基酸多肽,其5′非翻译区长135 bp,3′非翻译区长127 bp,并在其3′非翻译区发现一个poly(A)+加尾信号(AATAA)。以β-actin基因为内参,我们对Caspase基因在东海原甲藻衰老细胞中的表达情况进行了分析。结果表明:在处于各个衰老阶段的东海原甲藻细胞中都存在Caspase基因表达,并且各阶段表达存在显著差异。在开始阶段,Caspase基因的表达量逐渐增加,到第6天时,达到最大值0.689(是第0天的19倍);之后,其表达量逐渐降低,到第9天时,不能检测到任何基因的表达。表明衰老东海原甲藻细胞的破裂死亡可能一个主动过程,而Caspase基因在这一过程可能起着重要作用。这一结果为揭示东海原甲藻死亡的分子机制以及东海原甲藻赤潮消亡的分子机制,提供了理论基础。更多还原

【Abstract】 Prorocentrum donghaiense, one of dinoflagellates, has caused most frequent large-scale red tides at Changjiang River Estuary and Zhoushan Mountain Archipelago in recent years, bringing us tremendous economic losses and local environments serious impacts. Currently, there are much more knowledge concerning P. donghaiense’s life history, ecology and taxonomy. However, we know little about how the bloom forming and declineA cDNA library was constructed using P. donghaiense at exponential growth phase. Poly(A)+ RNA was extracted and purified using mRNA Purification Kit MagExtractor? - mRNA- and cDNAs were synthesized and amplified by Super SMART? PCR cDNA Synthesis Kit. Finally, the ligated cDNAs with pMD 18-T vector were transformed into E. coli JM109. Results showed that transform efficiency high above 109 and almost all the insert cDNA were between 500bp and 1500bp. Using this method, it is possible to constructed a cDNA library from micro-quantities of P. donghaiense (ca. <105 cells).The cDNA-amplified fragment length polymorphism (cDNA-AFLP) profiling technique was used to analysis early gene expression associated with inorganic phosphate-stress of dinoflagellate P. donghaiense. And the resulting difference cDNA-AFLP fragments were sequenced. Homology searches revealed that 8 of 43 transcript-derived fragments (TDFs) induced by phosphate stress were significantly homologous to genes in GenBank. We subsequently analyzed 3 of those TDFs and the results indicated that all of them are up-regulated when subjected to phosphate-stress, and the differences are significant compare to the control: of TDF72 (antioxidant, AhpC/Tsa family), TDF68(dual-specificity protein phosphatase) and TDF85(Phosphoglycerate/bisphosphoglycerate mutase), it is 1.24(p=0.04), 1.60(p=0.002) and 2.30(p=0.0001) folds higher than that of the control respectively.Usually, phytoplankton are considered immortal unless the cells are eaten by heterotrophic zooplankton or infected by viruses, or sink irreversibly into the deep ocean. However over the past decade, it has become clear that these organisms can undergo an autogenic control process in response to environmental stress, which shows similarity with programmed cell death (PCD) as commonly known in multicellular organisms. We have observed cell-lysis of senescence P. donghaiense and we have retrieved partial capsase encoding gene sequences in aging P. donghaiense EST analysis. In this study, we cloned the full-length cDNA sequence of caspase encoding gene of P. donghaiense which was 520bp in length, containing a 258bp open reading frame encoding 85 amino acids and two stretches of 135 and 127bp in length for the 5′and 3′untranslated regions, respectively. We also detected the expression level of caspase encoding gene usingβ-actin as a reference during culture senescence and found that, an increase at beginning and then a decrease of expression level was observed. On Day 6, the value reached the maximum, 0.689 (19 folds higher than that of Day 0). The transcripts of both genes were not detectable on Day 9. Our results suggested that caspase may play a role in the molecular mechanism of self-lysis in aging P. donghaiense. This may provide us some clues of how the bloom of Prorocentrum donghaiense crashed.更多还原