鯉魚Intersex基因之選殖與定性之研究

Abstract

中文摘要 在過去的研究中，對Drosophila melanogaster性別決定機制已瞭解得很清楚，其中一個名為intersex (ix)的基因，在雌性個體會與doublesex (dsx)共同作用於此調節層級的最底層。Drosophila的ix在雌雄個體上皆會表現，但對於體細胞性別發展只作用於雌性，在雄性個體上並無影響。其胺基酸序列並沒有可辨識的DNA結合區能直接與DNA結合，但是有類似轉錄活化結構功能區，可與其他蛋白質產生交互作用，與DSXF共同作用時，可能扮演了相當於DSXM C端的功能。 Mediator complex發現於酵母菌，為由20個以上的分子所構成的蛋白複合體，對轉錄的調控扮演著相當重要的角色，其中Brower等人(2002)與Wang等人(2004)皆選殖到一個為哺乳類Mediator complex次分子的新基因，是Drosophila ix的同源體，北方墨點中驗證了在許多組織中皆有表現，且蛋白主要分佈於細胞核內，推測其功能為轉錄調節者。 本實驗室同樣也在鯉魚的睪丸中發現了類似Drosophila intersex的基因(cIX)，鑒於intersex在果蠅中所扮演性別決定的重要功能，對於此基因在鯉魚中所擔任的功能而感到興趣，為了進一步探討cIX的功能，本論文完成cIX全長cDNA，設計並建構cIX的重組DNA，再利用純化之重組蛋白誘導抗體。根據PCR以及西方墨點的實驗結果，顯示cIX是一個廣泛存在於各個鯉魚組織的蛋白質，並沒有組織特異性。然而也發現cIX有主要兩種不同的形式存在，另外在與其他物種的同源蛋白質的胺基酸序列比對中，顯示此類蛋白質在C端的保守性很高，N端的變異性則較大，至於這些資訊所代表的意義則需要再進一步的研究。

Abstract The sex-determination of Drosophila melanogaster has been known clearly in previous studies. There is a gene called intersex (ix), which functions together with doublesex (dsx) at the bottom of the hierarchy in the sex deternination females. Drosophila ix is expressed in both sex, but only functions in female somatic cell. It has no effect on male. There is no recognizable DNA-binding domain on the amino acid sequence of ix for binding to DNA, but it contains a region like transcriptional activation domains to interact with other proteins, and ix might play a similar role as the C-terminal of DSXM when acts together with DSXF. Mediator complex was found in yeast, it is a protein complex composed over 20 subunits, and plays an essential role in the regulation of transcription. Brower et al. (2002) and Wang et al. (2004) also have cloned a navel gene of mammalian Mediator complex subunit, which is the homolog of Drosophila ix. Northern blot analysis demonstrated that ix is expressed in various tissues, and the protein is localized mainly to nucleus, supporting its function as a transcriptional regulator. Our laboratory also found a gene like Drosophila intersex in the testis of Cyprinus carpio (cIX). According to the significant function played by Drosophila ix in sex-determination, we are interested in the function of this gene in carp. To study the functions of cIX further, I cloned the full-length cDNA of cIX. The recombinant protein is produced and purified to serve as the antigen to induce antibody. The results of PCR and western blot analysis demonstrated that cIX is broadly existing in various tissue of carp, when the tissue specificity is not distinct. I also found that cIX has two mainly form, and the alignment of the homologs of other species indicates that IX proteins are conserved in C-terminal, and shows variation in N-terminal. The meaning of these information needs further study.