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草鱼胸腺cDNA文库的构建、EST分析及两个趋化因子的特征分析
Alternative TitleConstruction, EST analysis of thymus cDNA library and characterization of two chemokines in grass carp (Ctenopharyngodon idellus)
李洋洋
Subtype硕士
Thesis Advisor桂建芳
2008-05-28
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword草鱼 胸腺 Cdna 文库 Est 免疫相关基因 趋化因子 表达
Abstract胸腺是大多数鱼类最早发育的中枢淋巴器官,也是产生功能性T淋巴细胞的主要免疫器官。目前对于草鱼胸腺的认识仅是对组织结构的了解,还没有胸腺组织的基因表达的相关研究。为了更清晰认识草鱼胸腺在病毒免疫中的作用,本研究构建了感染草鱼出血病病毒(GCHV)的草鱼胸腺SMART cDNA文库。筛选文库得到了1933条有效EST序列。BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E10-3,Identities≥30%),另外1350条序列则找不到显著同源性。583个EST可以代表327个不同基因,其中78个为斑马鱼,河豚等基因组中预测的未知功能基因,249个为已知基因。已知基因按具体功能可分为6类,大部分与细胞内的各种生理过程,细胞结构以及免疫防御相关。与免疫相关的基因有59个,在免疫反应中发挥不同作用。本研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作用,同时也表明胸腺组织在病毒感染后可能表达很多目前还不清楚功能的新基因。 趋化因子是一类可以调控白细胞迁移至感染和损伤部位,在机体免疫系统和免疫反应中发挥重要作用的细胞因子。从胸腺文库中获得了代表两个趋化因子的全长cDNA。草鱼CC趋化因子cDNA长883bp,ORF为306bp,编码101个氨基酸,4个保守Cys符合CC趋化因子的结构特征,N端的前29个氨基酸为预测的信号肽。遗传进化分析发现该CC趋化因子与人CCL25聚成一枝。该CC趋化因子的DNA由4个外显子和3个内含子组成。RT-PCR检测到该CC趋化因子在健康草鱼各组织普遍表达,且在PolyI:C诱导后的肝,头肾和鳃中的表达发生上调。草鱼CXC趋化因子cDNA长617bp,编码117个氨基酸,预测N端的前25个氨基酸为信号肽。系统进化分析显示草鱼CXC趋化因子不与任何已知的鱼类CXC趋化因子聚成一类,可能是一个新的鱼类趋化因子。该趋化因子的DNA也由4个外显子和3个内含子组成。该基因在正常草鱼组织中的表达相对特异,而且Poly I:C的刺激会引起该基因在皮肤,肝,头肾和脾中表达的改变。两个趋化因子在正常草鱼中的组成型表达说明它们参与着维持自身的稳定,在受到外源刺激后表达的改变则暗示它们在鱼体的免疫反应中发挥了作用。
Other AbstractThe thymus is the primary tissues for production of functional T lymphocytes in vertebrate. Histological studies showed that grass carp (Ctenopharyngodon idellus) display thymus tissue. In order to obtain much more molecular evidence for the physiological function of grass carp thymus, a SMART cDNA library was constructed with mRNAs derived from thymus of grass carp infected with grass carp hemorrhage virus (GCHV), and then EST (expressed sequence tags) analysis was performed. A total of 1933 ESTs longer than 220bp were yielded and sequenced. BLASTX analysis showed that 583 ESTs represent grass carp genes which are homologous to known genes in mammals (E-value≤1.00E 10-3,Identities≥30%), while the other 1350 ESTs share low or no similarity by search of public protein databases in NCBI. Further analyses revealed that these genes, based on the functions of the homologous mammalian genes, could be classified into 6 categories, including immune-related, transcription and translation, metabolism, cell structure, signal transduction, cell proliferation, differentiation and apoptosis. 100 ESTs representing 59 different immune-related genes have been identified from the cDNA library. These genes encoding proteins involved in antigen processing and presentation, cell recognition, adhesion and activation, innate immune and so on. Many of the immune related genes have various immune functions. This study reveals from molecular level that thymus is the important immune tissue grass carp and might play an important role in the immune response to virus infection. In addition, this result also showed that grass carp thymus express many novel genes that are functionally unknown so far. Chemokines are a superfamily of chemotactic cytokines that coordinate the migration of leukocytes and play crucial role in host defenses and the development of immune system. Two full-length cDNA encoding a CC and a CXC chemokine were identified from the thymus cDNA library. The CC chemokine cDNA consists of 883 bp in full length and encodes a putative 101-amino-acid protein with a predicted signal peptide of 29 aa. The deduced protein has four cysteines that are also found in mammalian and fish CC chemokines in the same positions. The grass carp CC chemokine genocmic gene is composed of four exons and three introns. Homology and phylogenetic analysis revealed that the grass carp CC chemokine is most similar to human CCL25. Constitutive expression was observed in the 11 tissues of the healthy fish. When induced by poly I:C, the mRNA transcripts were upregulated in liver, gill and head kidney. The CXC chemokine cDNA is 618 bp encoding a putative 117-amino-acid protein. The deduced protein has a predicted 25 aa signal peptide at the N terminus and owns all the four cysteines in conserved positions as found in other CXC chemokines. The grass carp CXC chemokine genomic gene is composed of four exons and three introns. Homology and phylogenetic analysis revealed that the grass carp CXC chemokine is different from the known fish CXC chemokines. Constitutive expression was not detected in heart, brain and musle of healthy fish. Upon poly I:C induction, the chemokine transcripts were upregulated in skin, liver, spleen and head kidney. These results collectively suggested that both chemokines might be in relation to homeostatic functions and play an important role in response to dsRNA stimulation.
Pages75
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12354
Collection学位论文
Recommended Citation
GB/T 7714
李洋洋. 草鱼胸腺cDNA文库的构建、EST分析及两个趋化因子的特征分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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