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原生动物四膜虫金属硫蛋白与细胞色素P450基因的克隆鉴定、表达特征与进化分析
Alternative TitleCLONING,EXPRESSION CHARACTERIZATION AND EVOLUTIONARY ANALYSIS OF METALLOTHIONEIN AND CYTOCHROME P450 GENES IN THE PROTOZOAN TETRAHYMENA
傅诚杰
Subtype博士
Thesis Advisor沈韫芬 ; 缪炜
2008-06-12
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Degree Discipline水生生物学
Keyword四膜虫 金属硫蛋白 细胞色素p450 基因复制 进化分析 Microarray
Abstract作为单细胞真核生物,四膜虫对于外源性化学物质的响应灵敏和迅速;其基因组中含有众多与细胞感知和应对环境变化的生理过程密切相关的基因家族,因此是研究真核生物基因家族的起源、进化及功能演化的良好模式生物之一。在本研究中,我们以四膜虫中金属硫蛋白和细胞色素P450这两类与环境胁迫密切相关、高度保守的基因为研究对象,通过克隆鉴定、表达特征与进化分析的方法开展了在基因的结构、功能及进化关系方面的研究: 一、 通过RACE的方法得到梨形四膜虫的一个金属硫蛋白新基因型TpMT-2,发现其具有特殊的基因内重复结构。利用实时定量PCR法考察了TpMT-1与TpMT-2对多重处理条件下的转录响应模式,其中镉的转录诱导效应作用最为显著;并首次发现锌可诱导四膜虫金属硫蛋白基因的表达。推测TpMT-1与TpMT-2基因可能具有不同的细胞内功能。同时进一步探讨了四膜虫属金属硫蛋白7a亚家族基因序列的结构特征和可能的基因进化方式; 二、在进行生物信息学分析的基础上,采用RT-PCR亚克隆cDNA序列进行确认,鉴定得到44个具有完整开放阅读框的P450基因型。基因内含子位点与时相与系统树各分枝的拓扑结构具有非常好的一致性,提示P450各亚家族内近期存在着基因复制事件。利用半定量RT-PCR与Microarray 的方法分析了P450基因家族在三个重要的细胞生理阶段内的各个时期的表达丰度与波动情况。P450高表达基因型与密码子偏好之间不存在简单的直接对应关系。此外,对CYP5005与CYP5010家族的序列的位点特异性选择分析表明了不同的进化选择压力的存在,并进一步探讨了进化分析对推测P450基因相关功能的作用与局限性。
Other AbstractAs a unicellular eukaryotic organism, Tetrahymena behaves a rapid and sensitive response to xenobiotics. A large number of gene families that are involved in some core processes associated with the sensing of and responding to environmental changes are presented within its genome. Thus, Tetrahymena can serve as an ideal model system to study the origin, evolutionary and functional diversity of eukaryotic gene families. In the current study, two well-conserved gene families — Metallothionein and Cytochrome P450, which are closely associated with the cellular physiological process under environmental stress, were investigated. Analyses were carried out on the aspects such as gene structure, gene function and evolutionary relationships by cloning and identification, expression characterization and evolutionary calculations: 1. A new multi-stress inducible metallothionein gene isoform (TpMT-2) was cloned and characterized from T. pyriformis. The putative protein sequence of TpMT-2 contains typical characteristics of the Tetrahymena Cd-inducible MT genes, but the sequence has a special feature of four intragenic tandem repeats within its first half. To investigate the transcriptional activities of both TpMT-2 and TpMT-1 genes toward heavy metals and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Cd was the most effective metal inducer of both two isoforms, but the relative expression level of TpMT-2 was much lower than that of TpMT-1. Zn was found to have the transcriptional inducing effect on both Tetrahymena MT genes. It was suggested that TpMT-1 and TpMT-2 have different physiological roles. The putative unique structural characterization and evolutionary mode of the MT 7a subfamily in the Tetrahymena genus are discussed; 2. 44 putative cytochrome P450 genes with full-length ORFs in the T. thermophila genome were identified by computer analyses coupled with cDNA sequence cloning methods. A good correlation between the conservation of intron position and phylogenetic relationships of T. thermophila P450 subfamily members was indicated; therefore, it can be inferred that the enlargement of several P450 families in T. thermophila resulted from recent separate small duplication events. The gene expression patterns during three important cell physiological cycles (vegetative growth, starvation and conjugation) were analyzed based on the EST and microarray data. For the T. thermophila P450 genes, there is no simple correlation between the expression level and the use of a preferred set of codons. The site-specific selection test showed that different rate of substitutions were estimated to occur within the coding regions among the CYP5005 and CYP5010 family members. We further discussed both the significance and limitation of evolutionary analysis for understanding the characteristics and functional diversities of the T. thermophila P450 isoforms.
Pages125
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12258
Collection学位论文
Recommended Citation
GB/T 7714
傅诚杰. 原生动物四膜虫金属硫蛋白与细胞色素P450基因的克隆鉴定、表达特征与进化分析[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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