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香港牡蛎 ( Crassostrea hongkongensis ) Akt-FoxO信号通路相关基因的分子克隆与功能鉴定; Molecular cloning and functional characterization of the related genes involved in the Akt-FoxO signaling pathway from the Hong Kong oyster, Crassostrea hongkongensis
王富轩
Subtype硕士
Thesis Advisor严岩,喻子牛
2016
Degree Discipline生物工程
Keyword香港牡蛎 Akt1蛋白激酶 叉头框转录因子 先天免疫 压力响应
Abstract香港牡蛎 ( Crassostrea hongkongensis ) 是华南沿海重要的养殖贝类,生活环境复杂多变,长期受到温度、盐度、pH等天然生态环境因子的影响。在长期演化过程中,香港牡蛎形成了很多独特的优良性状,很好地适应了其生存环境,这是基因与环境长期相互作用的结果。解析基因与环境的相互作用,从分子和基因水平上来认识生物适应环境改变的遗传基础,不但是生物学研究中的核心问题之一,而且就香港牡蛎来说,也是对开发保护地区特色的基因资源和良种创制都有深远意义的工作。 通过搜索香港牡蛎血淋巴EST数据库,发现一个Akt1基因的EST序列,然后利用RACE技术克隆获得香港牡蛎Akt1全长cDNA序列 ( ChAkt1 )。香港牡蛎Akt1 cDNA 长度为2223 bp,编码493个氨基酸。该蛋白具有三个保守的结构域:氨基末端的PH结构域,中间的激酶结构域和羧基末端的调节区。多重序列比对分析显示,香港牡蛎Akt1与来自其他物种的同源蛋白高度保守。实时荧光定量PCR分析表明,ChAkt1在香港牡蛎各成体组织和不同胚胎发育时期均有表达,且其mRNA水平在受到病原菌(溶藻弧菌、酿酒酵母)感染和高温刺激后显著上调。亚细胞定位分析显示,ChAkt1蛋白在主要分布于HEK293T细胞的胞质中,少量存在于细胞核中。进一步研究发现,ChAkt1的过表达能够显著激活NF-κB信号通路而抑制p53信号通路。 利用RACE技术,我们从香港牡蛎克隆获得FoxO基因的全长cDNA序列。该序列长度为2367 bp,包括249 bp的5'非编码区,117 bp的3'非编码区以及2001 bp的开放阅读框,它编码666个氨基酸,其中保守的叉头框结构域位于第66位到155位氨基酸,三个高度保守的Akt磷酸化位点分别为T23、S163和S218。组织表达模式分析显示,ChFoxO在消化腺和鳃中分布最多,而在心脏中分布最少。在三种胁迫因子(病原菌、热激和干露)刺激下,ChFoxO的转录水平均明显上升。另外,ChFoxO蛋白主要分布于HEK293T细胞的胞质中,其过表达能够明显激活NF-κB和L8G5报告基因的转录活性。 总而言之,本研究首次克隆Akt-FoxO信号通路中的两个关键基因,并对其功能进行了初步分析。我们的结果不仅丰富了河口软体动物适应多变环境的分子特征、调控与演化机制等相关研究,而且为其优良品种创制等开发利用提供了理论基础和科学根据。
Other AbstractHong Kong oyster, Crassostrea hongkongensis, is an economically important marine aquaculture animal along the coastal waters of the South China Sea. They live a physically harsh and dynamic environment with tremendous exposure, such as daily changes in temperature, salinity and pH. However, the improved oyster varieties with many fine properties have been formed during the long term process of natural evolvement and evolved to acquire powerful defense mechanisms to withstand the environmental stress, which is the result of the interaction of genetic and environmental factors. Understanding the interactions between environments and gene expressions and its molecular basis by which organisms adapt to environmental conditions is one of the fundamental issues of biological research and has far-reaching practical significance for the development and protection of gene resources and the breeding of good strains starting from the Hong Kong oyster. A BLAST analysis of all EST sequences from a C. hongkongensis hemocyte EST library revealed that one EST was homologous to Crassostrea gigas Akt1. By RACE amplification, the full-length cDNA sequence of ChAkt1 was obtained from oysters. The full-length cDNA is 2,223 bp and encodes a putative protein of 493 amino acids that contains an amino-terminal pleckstin homology ( PH ) domain, a central catalytic domain and a carboxy-terminal regulatory domain. Quantitative real-time PCR ( qRT-PCR ) analysis showed that ChAkt1 mRNA is broadly expressed in various tissues and during different stages of the oyster’s embryonic and larval development. Upon exposure to two stressors ( microbial infection and heat shock ), the expression level of ChAkt1 mRNA increases significantly. Furthermore, ChAkt1 is located mainly in the cytoplasm in HEK293T cells, where the over-expression of ChAkt1 regulates the transcriptional activities of NF-κB and p53 reporter genes. ChFoxO, another gene in the Akt-FoxO signaling pathway, was cloned by RACE amplification from C. hongkongensis. The full length cDNA is 2,367 bp containing a 5'-untranslated region ( UTR ) of 249 bp, a 3'-UTR of 117 bp and an open reading frame ( ORF ) of 2,001 bp. The ORF encodes a predicted protein of 666 amino acid residues with a conserved forkhead ( FH ) domain from aa 66 to 155, three highly conserved Akt phosphorylation sites ( T23; S163; S218 ). ChFoxO mRNA could be detected in the above tissues, with poor expression in heart and positive expression in digestive gland and gill. Furthermore, upon exposure to three stressors ( microbial infection, heat shock and aerial exposure ), the expression levels of ChFoxO transcripts had a significant improvement. More importantly, ChFoxO protein is mostly deposited in the cytoplasm in HEK293T cells, while over-expression of the protein enhances the transcriptional activity of NF-κB and L8G5 reporter genes. All in all, we first reported the molecular cloning of two key genes in the Akt-FoxO signaling pathway in the present study from the Hong Kong oyster, C. hongkongensis. Hopefully, these results not only enrich the related study on the molecular characteristics, regulatory and evolutionary mechanisms under which estuarine mollusk adapts to the climate of constant change, but also provide a theoretical and applied foundation for utilizing and creating new germplasm resources.
Document Type学位论文
Identifierhttp://ir.scsio.ac.cn/handle/344004/14747
Collection学位论文(硕士)
Recommended Citation
GB/T 7714
王富轩. 香港牡蛎 ( Crassostrea hongkongensis ) Akt-FoxO信号通路相关基因的分子克隆与功能鉴定, Molecular cloning and functional characterization of the related genes involved in the Akt-FoxO signaling pathway from the Hong Kong oyster, Crassostrea hongkongensis[D],2016.
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