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六种四膜虫的镉金属硫蛋白基因的克隆及分子进化研究
Alternative TitleCLONING AND EVOLUTIONARY ANALYSIS OF METALLOTHIONEIN GENES IN SIX TETRAHYMENA SPECIES
郭利娜
Subtype硕士
Thesis Advisor缪炜
2009-05-26
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四膜虫 金属硫蛋白 基因复制 进化
Abstract原生动物四膜虫的基因组中与细胞感知和响应环境变化密切相关的基因家族表现出其成员大量倍增的特点,是开展相应基因家族分子进化研究的良好模式生物。金属硫蛋白正是一类与环境胁迫密切相关、高度保守的超基因家族蛋白,本研究通过对六种四膜虫镉金属硫蛋白(Cd-MT)基因的克隆、鉴定和进化分析得到了以下研究结果: 一, 从Tetrahymena pigmentosa(HG2株系)中克隆得到了一个新的镉金属硫蛋白基因Tpig-MT1的全长序列,其具有四膜虫Cd-MT典型的CCC和CC簇;但不同于在T. pyriformis、T. thermophila和T. pigmentosa中已报道的Cd-MT的结构,其含有两个重复结构(Tpig-MT1A1和Tpig-MT1A2),相似度达72.9%。利用实时定量PCR方法考察了不同重金属(Cd, Cu, Zn, Hg, Pb)及H2O2诱导下Tpig-MT1的转录情况,结果显示五种重金属诱导1小时后,Tpig-MT1的表达水平为Hg> Pb> Cd> Cu>Zn;而H2O2只在15分钟短暂处理下Tpig-MT1表达调高5倍,30分钟后表达水平下降到不显著。基于Tpig-MT1基因独特的结构和已报道的四膜虫Cd-MT异构型的遗传多样性,我们推测四膜虫Cd-MT基因中的模块“A”和模块“B”两个大的结构单元可能作为四膜虫MTs 7a亚家族的祖先模块而分别独立存在,同时也推测可能会有仅由模块“B”组成的四膜虫MTs 7a亚家族基因的存在。 二, 分别从T. elliotti、T. vorax、T. mobilis、T. malaccensis和T. hegewischi中克隆得到了镉金属硫蛋白基因(Tell-MT1、Tvor-MT1、Tmob-MT1、Tmal-MT1、Tmal-MT2)和MT ESTs(Tmal-MT3,Theg-MT1和 Theg-MT3)及一个基因的3’端cDNA末端全长(Theg-MT2)。它们均具有四膜虫MTs 7a 亚家族的典型特征,并且每个基因均存在由若干个模块“A” 或“B”组成的基因内重复现象:Tell-MT1 (AA),Tvor-MT1 (AAB),Tmob-MT1(AAA),Tmal-MT1(AA),Tmal-MT2(BBBB),Tmal-MT3(AAB),Theg-MT1(AA)和Theg-MT3(BBBB)。基于这6种四膜虫和已报道的四膜虫Cd-MT异构型的结构特点进行的进化分析得出,四膜虫Cd-MT基因中的模块“A”和模块“B”很可能早在“borealis”和“australis”两大四膜虫进化分枝分歧前就已独立存在。
Other AbstractAs a unicellular eukaryotic ciliated protozoan, Tetrahymena showed the special feature in its genome: the expansion of large gene families associated with the sensing of and responding to environmental changes. Thus it is a good model organism to study the evolution of these gene families. Metallothioneins (MTs) is a well-conserved gene superfamily which is closely associated with the cellular physiological process under environmental stress. Based on the cloning, identification and evolutionary analysis of cadmium-inducible MT genes from six Tetrahymena species, this study showed: 1.A novel Cd-inducible metallothionein (MT) gene (Tpig-MT1) was cloned and sequenced from the ciliate Tetrahymena pigmentosa. The deduced polypeptide possesses CCC and CC clusters with characteristics of typical Tetrahymena Cd-MTs. The structure of Tpig-MT1 is different from the known Cd-MTs in T. pyriformis, T. thermophila and T. pigmentosa. Tpig-MT1 contains two intragenic tandem repeats with 72.9% identity, which were designated as Tpig-MT1 (repeat A1) and Tpig-MT1 (repeat A2). The transcriptional response of Tpig-MT1 gene to different heavy metals (Cd, Cu, Zn, Hg, Pb) and oxidative stress (H2O2) was measured using real-time quantitative PCR. The results showed that the gene was quickly induced (1 h) by the five heavy metals and the order of expression level was Hg> Pb> Cd> Cu>Zn. The induction effect of H2O2 was 5-fold after about 15 min, but soon decreased to a non-significant level (30 min). Based on the genetic diversity of Tetrahymena Cd-MT isoforms, We hypothesize that the module A and B of Tetrahymena subfamily 7a MTs genes may already exist respectively as the ancestral subfamily 7a MT component, and Tetrahymena subfamily 7a MT gene composed of only module B could exist. 2.Several novel Cd-MTs (Tell-MT1, Tvor-MT1, Tmob-MT1, Tmal-MT1, Tmal-MT2)and MT ESTs(Tmal-MT3, Theg-MT1和 Theg-MT3)and a cDNA sequence with full 3’terminus(Theg-MT2)were obtained from T. elliotti, T. vorax, T. mobilis, T. malaccensis and T. hegewischi. All of them showed typical characteristic of Tetrahymena MTS 7a subfamily (Cd-MTs), and composed of module “A” or “B” by intragenic tandem repeats: Tell-MT1 (AA), Tvor-MT1 (AAB), Tmob-MT1 (AAA), Tmal-MT1 (AA), Tmal-MT2 (BBBB), Tmal-MT3 (AAB), Theg-MT1 (AA) and Theg-MT3 (BBBB). The analysis of gene structures and evolution of these Cd-MT isoforms and known Tetrahymena Cd-MT isoforms revealed that, Tetrahymena Cd-MT modules A and B may already exist respectively before the divergence of two major clades (T. borealis and T. australis) within the Tetrahymena genus.
Pages77
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12452
Collection学位论文
Recommended Citation
GB/T 7714
郭利娜. 六种四膜虫的镉金属硫蛋白基因的克隆及分子进化研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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