白蝦YY1蛋白質調控白點症病毒極早期基因wssv126 (ie1) 的表現

Regulation of the wssv126 (ie1) of white spot syndrome virus by Litopenaeus vannamei YY1

黃庭儀(Ting-Yi Huang)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10789

摘要

Yin-yang 1 (YY1) 為一種廣泛表現於各組織之GLI-Krüppel家族的鋅指蛋白質 (zinc finger protein)，其特色為在蛋白質C端 (C-terminal) 具有一個Krüppel type與三個GLI type，共四個的鋅指元素，並會結合於保守性序列ATGG上，調節基因表現，在細胞中並扮演重要的調控角色。日前中國研究團隊自中國對蝦 (Fenneropenaeus chinensis) 中成功選殖出一轉錄抑制調控子基因，本研究根據序列比較與 RACE 分析，成功從白蝦 (Litopenaeus vannamei) 選殖出與其相似的基因序列。將該基因鋅指區胺基酸進行序列比對後發現與人類 (Homo sapiens)、斑馬魚 (Danio rerio)、果蠅 (Drosophila melanogaster) 的類YY1蛋白質鋅指區有96%的相似性，因此命名此基因為LvYY1。LvYY1的開放譯讀區全長1065核苷酸，並可轉譯出含有354個胺基酸的蛋白質產物。使用抗LvYY1抗體可以觀察到白蝦YY1蛋白質約位在65 kDa。透過免疫螢光分析可發現V5-LvYY1融合蛋白質在秋夜盜蛾細胞 (Spodoptera frugiperdam, Sf9) 中主要分布於細胞核內。進一步在果蠅細胞 (Schneider 2 cells, S2) 進行冷光報導分析 (reporter assay)，結果發現LvYY1會活化白點症病毒極早期基因ie1轉錄活性 (WSSV immediate early gene 1, ie1)，並以電泳流動性轉移分析 (EMSA) 證實LvYY1會結合在ie1啟動子上的ATGG序列形成蛋白質-DNA複合體。而在白蝦死亡率實驗中，LvYY1基因的表現若受到抑制，會使WSSV感染的白蝦呈現較低的死亡率。由本研究發現，LvYY1可結合於極早期基因ie1的啟動子上調控基因表現並影響白點症病毒的感染。

關鍵字

Yin-yang 1蛋白質 ； 白點症病毒 ； 南美白對蝦

並列摘要

Yin-yang 1 (YY1) is an ubiquitously expressed GLI-Krüppel family of zinc finger protein. The protein has one KLF-type and three GLI-type zinc finger motifs at the C-terminal, binding to the consensus sequence ATGG, and regulating gene expression. China researcher recently cloned a transcriptional repressor gene from Fenneropenaeus chinensis. According to the gene sequence blast and RACE analysis, a similar gene sequence was cloned from Litopenaeus vannamei. The zinc finger domain of the gene showed 96% identical to human, zebrafish, and flies YY1-like genes, thus it was named LvYY1. The ORF sequence of LvYY1 is 1065 bps, encoding a polypeptide of 354 amino acids. Western blot analysis showed that L. vannamei endogenous YY1 protein migrated as 65 kDa and was recognized by anti-YY1 antibody. Immunofluorescence analysis showed that LvYY1-V5 was located mainly in the nucleus in Sf9 cells. Additionally, LvYY1 activated the expression of white spot syndrome virus immediate early gene wssv126 (ie1) in S2 cells by reporter assay. Moreover, electrophoretic mobility shift assay (EMSA) demonstrated that LvYY1 binds to the sequence ATGG in the ie1 promoter. Knockdown of the expression of LvYY1 transcript in WSSV-infected shrimp caused delayed cumulative mortality. Thus, LvYY1 can bind to WSSV ie1 promoter, which is critical to WSSV infection.

並列關鍵字

Yin-yang 1 ； white spot syndrome virus (WSSV) ； Litopenaeus vannamei

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