中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究

IOCAS-IR > 海洋环流与波动重点实验室

	中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究
其他题名	The proteomic analysis of diapause embryo development and heavy metal stress response in Artemia sinica
	周茜
学位类型	博士
	2008-05-31
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中华卤虫滞育卵激活卵胚胎发育硫酸铜过氧化物还原酶蛋白质组学双向电泳质谱鉴定
摘要	卤虫(Artemia)是一种广温、耐高盐的小型甲壳动物，广泛分布于内陆盐湖和沿海盐田中。卤虫的无节幼体作为重要的蛋白优质饵料，被广泛的应用于水产养殖生产。卤虫具有特殊的生物学特性，是研究甲壳动物胚胎发育的良好的实验材料，同时也是一种研究动物抗逆机制的模式动物。卤虫有卵生和卵胎生两种繁殖后代的方式，当环境条件适宜时，卤虫倾向于采取卵胎生方式，即直接产生无节幼体；而在恶劣的环境条件下，卵生方式占主要地位，产生处于滞育状态的、具有复杂外壳的休眠卵。卤虫的滞育卵具有独特的生物学特性和特殊的生理生化特点。其发育停滞，细胞分裂停止，酶活力下降，代谢活动受到抑制并可耐受各种极端恶劣环境，如缺氧、低温、紫外线、干燥等。即使在最适的环境中滞育卵的孵化率也很低，只有受到某些特定的非生物信号的刺激才自能终止这种滞育状态，恢复生理代谢；当环境条件适宜时，能够继续发育孵化成无节幼体。因此，卤虫的滞育卵在卤虫的整个生活史中占有重要的地位。另一方面，卤虫是极端环境生物，能够抵抗各种恶劣环境胁迫刺激，因此是研究抗逆机理的良好的实验动物。本论文利用蛋白质组学技术，研究了卤虫滞育卵及滞育卵发育过程中的蛋白质组表达情况，并研究了卤虫幼体在重金属刺激后蛋白表达的变化情况。得到如下结果：建立了中华卤虫滞育卵可溶性总蛋白的双向凝胶电泳对照图谱。在pH 4–7、分子量10-100 kDa范围内，检测到约 233个蛋白点，并利用高效液相色谱-质谱联用（LC-ESI-MS/MS）技术鉴定了其中的48个丰度较大及感兴趣的蛋白点，根据这些蛋白的生物学功能进行分类，功能类别包括细胞防御蛋白、抗氧化蛋白、细胞骨架蛋白、代谢相关蛋白等。在卤虫滞育卵中共分离鉴定到6个分子量和等电点存在差异的小热休克蛋白p26的异构体，生物信息学分析表明该蛋白有三种不同的功能位点，分别是蛋白激酶C磷酸化位点，Casein 激酶II磷酸化位点及 N-myristoylation 位点。采用低温脱水的方法对滞育卵进行激活刺激，并对活化卵和滞育卵蛋白表达图谱进行了对比分析。结果表明对卤虫滞育卵的激活刺激引起了其蛋白表达的明显变化。活化卵图谱中蛋白点总数比滞育卵中明显增多，特别是在pI<5.5范围内。约70个蛋白点在激活刺激后上调表达，包括部分只在激活卵中表达的蛋白；25个下调表达，包括部分只在滞育卵中表达的蛋白；其余约60％（占滞育卵蛋白点数目百分比）的蛋白点表达量基本恒定。热休克蛋白家族、抗氧化蛋白家族成员等蛋白变化明显，小热休克蛋白p26、小热休克蛋白ArHsp21蛋白以及过氧化物还原酶异构体在激活卵中特异表达。活化卵孵化过程中不同发育时期的蛋白表达又呈现出不同的特点，分别在孵化后6h、12h、18h和24h的蛋白质组学图谱上检测到267、285、195和210个蛋白点。孵化后6h和12h休眠卵蛋白表达个数相对较多，与胚胎发育过程中的器官发生和剧烈的形态变化相适应；孵化后18h和24h休眠卵蛋白表达明显下降，部分蛋白的表达关闭，部分蛋白开始富集表达。利用双向凝胶电泳技术分析了中华卤虫幼体受到急性硫酸铜刺激后的蛋白表达变化情况。通过图谱对比分析，检测到了5mM硫酸铜刺激24h后，卤虫幼体中14个差异表达的蛋白点。利用LC-ESI-MS/MS技术鉴定了其中的7个蛋白，其中3个蛋白上调表达，分别是热休克蛋白70（7.5倍）, 肌动蛋白（2.3倍）和伴侣分子亚基1（3.0倍）。3个蛋白下调表达，分别是：精氨酸激酶（2.8倍）, 延伸因子2 (2.0倍）和富含甘氨酸蛋白(2.0倍）。硫酸铜刺激后特异表达的一个蛋白被鉴定为过氧化物还原酶(Peroxiredoxin，Prx)。根据质谱检测提供的蛋白肽段信息和其他生物过氧化物还原酶保守氨基酸序列设计简并引物，结合RACE技术，从中华卤虫幼体中克隆到了过氧化物还原酶基因，该基因的cDNA全长为756个碱基，其中开放阅读框为594个碱基，编码198个氨基酸，其蛋白理论分子量为22.0 kDa，理论等电点为6.98。多序列比对结果显示中华卤虫Prx基因的推导氨基酸序列与美国卤虫和中国对虾的同源性高达98%和94%。实时荧光定量PCR结果显示，硫酸铜刺激后，该基因在卤虫无节幼体中的转录水平明显升高，在24h达到正常水平的3.0倍。
其他摘要	Brine shrimp (Artemia) is a worldwide-distributed crustacean, widely used as a main food resource in aquaculture and applied in basic research areas ranging from developmental biology to evolution and ecology. Artemia presents interesting features in development and depending on the environmental condition, two developmental pathways can be taken namely ovoviviparous and oviparous development. In the former case, free-swimming nauplii are released from females and in another style encysted gastrulae (cysts) are produced under unfavorable environmental conditions. The cysts enter diapause, a state at which development arrests, metabolic activity reduces greatly, and the resistance to severe physiological stress is high. Upon breakage of diapause and placed in suitable conditions, the embryo is activated and resumes its development, releasing swimming nauplii. This is a special strategy of the brine shrimp to cope with harsh environment and to breed offspring. Therefore, the encysted diapause state of the brine shrimp embryo is a crucial feature and of major importance in Artemia’s life history. Moreover, Artemia can withstand great physiological stresses. They can survive long term anoxia, temperature extremes, repeated hydration and dehydration, desiccation, and exposure to pollutants. Therefore, the brine shrimp is a good model for studies on the mechanism of stress resistance. In this thesis, the protein expression of the diapause, activated cyst and cysts at different postdiapause developmental stages were studied using proteomic methods. The changes in protein expression in response to CuSO4 in Artemia nauplii were also analyzed. A key genes of antioxidant enzymes: peroxiredoxin has been clone and analyzed. The main results are: A proteomic reference map for the diapause embryo of Artemia sinica was obtained using two-dimensional gel electrophoresis with a pH range of 4–7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC–MS/MS). Of these, 48 spots were identified, which are categorized into 9 functional groups including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. The protein expression profiles in the different postdiapause embryo development stages were detected and analyzed. The result showed that the activation stimulation induces apparent changes in protein expression in the activated cysts. Compared to diapause cysts, more protein spots were detected in the activated cysts proteome map, especially in the pH<5.5 range. There were approximately 70 protein spots up-regulated, including proteins unique in activated cysts; 25 protein spots down-regulated, including proteins unique in diapause cysts; and others, about 60％(in diapause cysts) expressed constantly after activation. The regulated proteins include heat shock proteins, anti-oxidant proteins and many other proteins. Isoforms of small heat shock protein p26, small heat shock protein ArHsp21 and peroxiredoxin expressed in activated cysts specifically. The protein expression at different stages in the hatching process showed different characteristics. We found 267、285、195 and 210 protein spots in the proteome map of 6h、12h、18h and 24h post hatching cysts. The cysts at 6h and 12h contain more protein spots than that of 18h and 24h. These changes could be attributed to the morphogenetic events and development process during the embryo development. Proteomic approaches were also applied to reveal the differential protein expression profiles of the acute responses to copper sulfate exposure in the larvae of Artemia sinica, which is a representative of high saline ecosystem. Fourteen significantly differentially-expressed proteins were detected and seven of these were determined through MS analyses. Three spots were up-regulated and identified as actin, heat shock protein 70 and chaperone subunit 1. Three down-regulated proteins were identified to be arginine kinase, elongation factor-2, and glycine-rich protein. An induced protein was identified as Peroxiredoxin (Prx). A Prx gene was cloned from nauplii of Artemia sinica by RACE and degenerate primers designed according to the peptide sequence noted by MS analysis and sequence homolog with Prx genes from other species. The full-length cDNA of Prx consists of 756 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22.0 Da with an estimated pI of 6.98. Sequence comparison showed that Prx of Artemia sinica shares 98% and 94% identity with that of A. franciscana and F. chinensis, respectively. Real-time PCR analysis indicated that the Prx gene transcripts increased to 3.0 fold of nomal level in nauplii of brine shrimp after exposed to CuSO4 for 24h.
页数	108
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/979
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	周茜. 中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20051800681204（4899KB）			限制开放	--	浏览