草蝦Kruppel-Like Factor蛋白質的功能性分析

Functional Analysis of the Kruppel-Like Factor in Penaeus monodon

沈維道(Wei-Tao Shen)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00437

摘要

Sp1-like蛋白質和Kruppel-like factors (KLFs)均具有高相關性的鋅指 (zinc finger)區域，在細胞轉錄過程中扮演重要角色。利用NCBI資料庫比對發現一個草蝦 (Penaeus monodon)的表現序列標誌 (EST, HPA-N-S01-EST0038)和Sp1序列具有高度同源性。因此本研究利用5’端cDNA快速放大法 (5’RACE)成功地選殖出草蝦的KLF，並命名為PmKLF。PmKLF全長可轉譯出498個胺基酸。序列分析發現PmKLF和人類，老鼠及斑馬魚的KLF11相似。另外，演化分析此鋅指DNA結合區域在不同物種間具有高度相同性。利用RT-PCR分析PmKLF mRNA發現在蝦的中腸、肌肉、心臟、神經、鰓、肝胰腺和胃中均會表現。利用大腸桿菌重組蛋白質當作抗PmKLF蛋白質的抗原並生產抗PmKLF抗體得知PmKLF在草蝦中大小約為60 kDa。利用螢光顯微鏡觀察發現當過度表現PmKLF於昆蟲細胞Sf9時，PmKLF以點狀聚集在細胞核。另外，以PmKLF的刪除突變株分析發現PmKLF的鋅指區域含有進核訊號。而在未感染白點症病毒 (White spot syndrome virus, WSSV)及感染病毒的白蝦中，在其血球細胞中也觀察到PmKLF在細胞核內聚集成點狀。由於WSSV的極早期基因ie1在昆蟲細胞Sf9具有高度轉錄活性，將PmKLF的突變株及報導基因pIE1-Luc共同轉染入Sf9中，發現PmKLF的第166到381個胺基酸可能會影響ie1的轉錄。最後，在蝦體中以siRNA抑制PmKLF的表現後，再感染WSSV病毒，發現可以延長蝦的存活時間。綜合以上的結果，本研究首先分離出蝦的第一個KLF，並證實PmKLF在蝦被WSSV感染時可能扮演重要的角色。

關鍵字

草蝦 ； Kruppel-like factor蛋白質 ； KLF蛋白質 ； 鋅指 ； 白點症病毒

並列摘要

Sp1-like proteins and Kruppel-like factors (KLFs) are zinc-finger proteins and important to transcriptional regulation. One expressed sequence tag (EST, HPA-N-S01-EST0038) from shrimps is homologous to Sp1. In this study, a KLF from Penaeus monodon was cloned and characterized by 5’RACE. The full-length PmKLF encodes a polypeptide of 498 amino acids. Sequence analysis showed that PmKLF has a sequence that of similar to KLF11 in human, mouse and zebrafish. A phylogenetic analysis indicates that the zinc-finger DNA binding domain of PmKLF has high degree of sequences identities with KLF in other species. RT-PCR analysis indicates that PmKLF mRNA is expressed in midguts, muscles, hearts, nerves, gills, hepatopancreas and stomaches of shrimps. The antibody against PmKLF was raised using E. coli recombinant protein as an antigen. Immunoblotting using the antibody reveals that the molecular mass of PmKLF protein is about 60 kDa. Immunofluorescence analysis revealed that PmKLF is located in the nucleus as dots after overexpressing in a Spodoptera frugiperda cell line, Sf9. Moreover, analysis of PmKLF deletion mutants indicates that the zinc finger domain of PmKLF contains a nuclear localizaton signal (NLS). Localization of PmKLF in the nucleus is detected in the hemocytes from white spot syndrome virus (WSSV)-infected and WSSV-uninfected Litopenaeus vannamei. Moreover, it is well known that the immediate-early gene ie1 of WSSV displays high transcription activity in Sf9 cells. Cotransfection of PmKLF deletion mutants and the ie1 reporter plasmid into Sf9 cells suggested that the region between 166 and 381 amino acid residues of PmKLF might affect the ie1 transcription. Furthermore, inhibiting the expression of PmKLF by siRNA in WSSV-challenged shrimps prolongs the survival time. Taken together, this study identifies the first Kruppel-like factor in shrimps and reveals the importance of PmKLF to WSSV infection.

並列關鍵字

Penaeus monodon ； Kruppel-like factor ； KLF ； zinc finger ； White spot Syndrome Virus ； WSSV

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