English  |  正體中文  |  简体中文  |  Items with full text/Total items : 28611/40652
Visitors : 778561      Online Users : 42
RC Version 4.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
Scope Adv. Search
LoginUploadHelpAboutAdminister

Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53571

Title: 南美白對蝦造血相關基因 Lvbgb 之選殖與特性分析
Molecular cloning and characterization of hematopoiesis-related gene, Lvbgb, in white shrimp (Litopenaeus vannamei)
Authors: Tang, Wei-Chi
唐偉琦
Contributors: NTOU:Institute of Marine Biology
國立臺灣海洋大學:海洋生物研究所
Keywords: 南美白對蝦;核結合因子 (Core Binding Factor, CBF);Big Brother (Bgb)
Litopenaeus vannamei;Core Binding Factor (CBF);Big Brother (Bgb)
Date: 2019
Issue Date: 2020-07-03T08:34:03Z
Abstract: 核結合因子 (Core Binding Factor, CBF) 為二聚體轉錄因子複合物,由會和 DNA 結合的 CBF與不和DNA 結合的CBF兩個次單元所組成,在造血作用的調節中扮演重要的角色。本實驗室先前從南美白對蝦 (Litopenaeus vannamei) 血細胞中選殖出與果蠅 (Drosophila melanogaster) CBF中的Lozenge (lz) 具有高相似性的基因,命名為 Lvlz,並對其進行初步研究。本研究接著以南美白對蝦CBF基因為主題,初探 CBF 與造血作用的關聯性。實驗室先前已在南美白對蝦的血細胞轉錄體資料庫中發現一基因片段是與果蠅 CBF中的Big Brother (Bgb) 基因具有高相似性,因此命名為 Lvbgb。透過 RACE-PCR 自南美白對蝦的血細胞得到 Lvbgb 的全長 cDNA 共 2724 bp,包含 167 bp 的 5’ 端非轉譯區 (5’ UTR),1873 bp 的 3’ 端非轉譯區 (3’ UTR) 以及 684 bp 的開放譯讀區,可轉譯出 228 個胺基酸序列。利用 qRT-PCR 檢測發現 Lvbgb 基因主要表現在肝胰腺、神經及泳足。以脂多醣 (lipopolysaccharide, LPS) 刺激造血反應,血細胞及造血組織中的 Lvbgb 的表現在注射後 3 小時 (hour post injection, hpi) 開始被誘導表現,在 6 hpi 的表現量達到最高,48 hpi 後恢復正常。經由注射 Lvbgb dsRNA 干擾 Lvbgb 的表現後,發現血細胞免疫基因 prophenoloxidase (proPO)、penaeidin 3a 及astakine 在不同時間點的表現於統計上有顯著上升的現象 (p0.05)。將 pDHSP70-Lvbgb-Flag 及 pDHSP70-Lvlz-V5 質體分別轉染至秋夜盜蛾 (Spodoptera frugiperda) Sf9 細胞株,經熱誘導後 48 小時,其蛋白質產量最高,並發現 Lvlz-V5 在 24 小時後開始有轉譯後修飾 (post-translational modification) 發生。pDHSP70-Lvbgb-Flag 及 pDHSP70-Lvlz-V5 共轉染至 Sf9 細胞,誘導 48 小時後利用 anti-V5 agarose beads 進行共免疫沉澱,結果顯示共表現的 Lvbgb-Flag 存在於 anti-V5 agarose beads的沉澱物中,顯示Lvlz-V5與Lvbgb-Flag之間有交互作用。研究結論推測,Lvbgb 及 Lvlz有結合的現象,並且參與了造血作用。
Core Binding Factor (CBF), a heterodimer composed of a DNA binding α-subunit and a non-DNA binding β subunit, plays a vital role in hematopoiesis regulation. We had identified the Litopenaeus vannamei -subunit gene, L. vannamei lozenge (Lvlz), and had a preliminary research on it. This study then aimed at unveiling the possible role of L. vannamei -subunit gene, L. vannamei CBFLvCBF, in shrimp hematopoiesis. We had identified from our Lv hemocyte transcriptome a cDNA fragment, which had high similarity with Drosophila big brother (Dmbgb) gene and was named L. vannamei bgb (Lvbgb), We used RACE-PCR to obtain the full-length cDNA of Lvbgb from white shrimp hemocyte. The full-length Lvbgb transcript had 2724 bp, consisting of a 5’ untranslated region of 167 bp, and a 3’ untranslated region of 1873 bp. The open reading frame (ORF) had 684 bp, which encoded a protein of 228 amino acid residues. qRT-PCR showed that Lvbgb could be detected in all assayed tissues and was mainly expressed in the hepatopancreas, nerves and pleopod. After lipopolysaccharide (LPS) injection, the expression of Lvbgb in hemocyte and hematopoietic tissues were increased, peaking at 6 hour post injection (hpi), decreasing to unstimulated level at 48 hpi. In the RNA interference (RNAi) experiments, Lvbgb expression could be silenced by injection of Lvbgb dsRNA at 6, 24 and 48 hpi. In the Lvbgb-silenced shrimp, the expression levels of prophenoloxidase, penaeidin 3a and astakine were increased with statistical significance (p0.05) in hemocyte. In cell transfection experiments, we separately transfected pDHSP70-Lvbgb-Flag or pDHSP70-Lvlz-V5 plasmids to Spodoptera frugiperda Sf9 cells. After heat shock to induce protein expression, the amounts of induced proteins were highest at 48 hpi. We also found that Lvlz had post-translational modification after 24 hpi. Lvbgb-Flag and Lvlz-V5 co-immunoprecipitation experiments by using anti-V5 agarose beads revealed that co-expressed Lvbgb-Flag could be precipitated by anti-V5 agarose beads, suggesting that Lvbgb-Flag might interact with Lvlz-V5. We concluded that Lvbgb and Lvlz could interact with each other and might be involved in hematopoiesis.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010534012.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53571
Appears in Collections:[海洋生物研究所] 博碩士論文

Files in This Item:

File SizeFormat
index.html0KbHTML21View/Open


All items in NTOUR are protected by copyright, with all rights reserved.

 


著作權政策宣告: 本網站之內容為國立臺灣海洋大學所收錄之機構典藏,無償提供學術研究與公眾教育等公益性使用,請合理使用本網站之內容,以尊重著作權人之權益。
網站維護: 海大圖資處 圖書系統組
DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - Feedback