白蝦 Kruppel-like factor於白點症病毒感染時之功能性探討

Role of the Kruppel-like factor in Litopenaeus vannamei after white spot syndrome virus infection

楊舒涵(Shu-Han Yang)

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(2011年)

https://doi.org/10.6342/NTU.2011.01155

摘要

白點症病毒 (white spot syndrome virus, WSSV) 感染蝦會導致高度的致死率，而本研究室先前在草蝦 (Penaeus monodon) 中成功選殖了第一個Kruppel-like factor (KLF)，若將KLF抑制則會增加被WSSV感染的蝦體存活率。經由基因體資料庫分析比對，發現草蝦和白蝦 (Litopenaeus vannamei) 彼此具有高度的基因相似性，因此根據草蝦KLF (PmKLF) 的序列，本研究在白蝦組織中也成功的選殖出KLF，並命名為LvKLF。LvKLF全長可以轉譯出360個胺基酸，經由序列分析發現草蝦和白蝦KLF胺基酸具有93%相似性，且鋅指區域完全相同，而利用抗KLF抗體能偵測到大小約為40 kDa的LvKLF。利用螢光顯微鏡觀察LvKLF在Sf9細胞表現時會在細胞核中聚集成點狀分布。之後進行病毒極早期基因整體分析，篩選了在白點症病毒台灣分離株基因體上編號分別為108、126、135、136、137、140、150、154、155、156、157、159、164、234以及304號的開放譯讀區 (open reading frame)，在白蝦體中利用siRNA抑制LvKLF基因表現後，再感染白點症病毒，於不同時間點收取蝦體組織直到感染病毒48小時，之後利用反轉錄聚合酶鏈反應分析，結果發現這十五個白點症病毒極早期基因表現均被抑制，另外以即時聚合酶鏈反應定量分析也得到相同的抑制結果。最後，進一步的在昆蟲細胞Sf9中，以預測的WSSV極早期基因啟動子進行報導基因分析，發現LvKLF會活化108、126、140、164、234、304號基因啟動子的活性。因此，本研究發現了白蝦的KLF對於蝦體在被WSSV感染的過程中扮演很重要的角色。

關鍵字

白蝦 ； 白點症病毒 ； 白點症病毒極早期基因 ； Kruppel-like factor蛋白質 ； KLF蛋白質

並列摘要

White spot syndrome virus (WSSV) infection can cause a significant mortality to shrimp cultivation. Our previous study identified the first kruppel-like factor (PmKLF) in Penaeus monodon. Inhibition of PmKLF by double-stranded RNAs decreased the mortality in WSSV-infected shrimps. According to genomic database, the sequences of P. monodon and Litopenaeus vanname display highly identity. In this study, a KLF from L. vannamei was cloned and characterized. The full-length LvKLF gene is 1080 bp, encoding a polypeptide of 360 amino acids. Sequence analysis showed that the identity of LvKLF and PmKLF amino acids is 93% and the zinc-finger DNA binding domain are the same. Immunoblotting using the anti-KLF antibody indicated that the apparent molecular mass of LvKLF protein is about 40 kDa. Moreover, immunofluorescence analysis revealed that LvKLF is located in the nucleus as dots after overexpressing LvKLF in Sf9 insect cells. So far, fifteen WSSV candidate immediate early (IE) genes including wssv108、wssv126、wssv135、wssv136、wssv137、wssv140、wssv150、wssv154、wssv155、wssv156、wssv157、wssv159、wssv164、wssv234 and wssv304 in the Taiwan isolate had been identified. This study indicated that these IE genes were inhibited in WSSV-challenged shrimps that had been injected with double-stranded RNAs of LvKLF. Furthermore, the reporter assay by using predictive WSSV IE promoters revealed that LvKLF enhanced the promoter activities of wssv108, wssv126, wssv140, wssv164, wssv234 and wssv304. Hence, this study demonstrates that the kruppel-like factor in Litopenaeus vannamei plays a crucial role in WSSV infection.

並列關鍵字

Litopenaeus vannamei ； WSSV ； WSSV immediate early gene ； Kruppel-like factor

參考文獻

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