草蝦Kruppel-like factor 在白點症病毒感染時所扮演之角色

Abstract

Sp1-like proteins /Kruppel-like factors (Sp/KLF)轉錄因子家族，為相似度極高的鋅指蛋白質 (zinc finger protein)，在細胞內調控多種轉錄活性。Sp/KLF轉錄因子於胺基酸序列C端皆具有三組Cys2-His2 (C2H2)鋅指元素 (zinc-finger DNA binding motif)，並且會結合在GC-Box、CACCC-Box以及GT-Box序列上調節轉錄活性。本研究成功地自草蝦 (Penaeus monodon)選殖出Kruppel-like factor (PmKLF)，PmKLF cDNA全長為1702 bps，並且可以轉譯出由360個胺基酸組成的蛋白質產物。胺基酸序列比對後發現，PmKLF和人類 (Homo sapiens)、小鼠(Mus musculus)、斑馬魚 (Danio rerio)以及水蚤的KLF11具有較高的相似度。此外，免疫螢光分析顯示，GFP-KLF融合蛋白質可於秋夜盜蛾細胞 (Spodoptera frugiperda, Sf9)細胞核中形成點狀聚集。冷光報導分析 (reporter assay)也指出 PmKLF促進白點症病毒(White spot syndrome virus, WSSV)啟動子ie1之轉錄活性。接著以電泳流動性轉移分析 (EMSA)發現PmKLF會結合於ie1啟動子GC- box序列形成蛋白質-DNA複合體。另外，若抑制白蝦PmKLF基因表現，能降低白點症病毒感染造成的高死亡率，因此推論PmKLF於白點症病毒感染時應扮演重要角色。而且當PmKLF表現受到抑制時，白點症病毒的複製數以及極早期基因ie1的表現量也會受到抑制，因此PmKLF可能藉由調節ie1基因表現影響白點症病毒的感染。

Sp1-like proteins and Kruppel-like factors (Sp/KLF) are highly related zincfinger proteins that have crucial roles in transcription. The characteristic feature of this fami- ly is the presence of three zinc fingers in the C-terminal domain, which bind to GC- box, CACCC-box and GT-box sequence, to mediate transcription. This study reports the cloning and characteristics of a Kruppel-like factor (PmKLF) from shrimp, Penae- usmonodon (PmKLF). The full-length PmKLF cDNA is 1702 bps, encoding a poly- peptide of 360 amino acids. Sequence analysis revealed that the sequence of PmKLF is similar to that of KLF11 in humans, mice and zebrafish. Additionally, immunofluo- rescence analysis revealed that GFP-fusion KLF protein is located in the nucleus as dots in an insect cell line, Sf9. Reporter assay revealed that PmKLF enhances the ie1 promoter activity. Moreover, a specific protein-DNA complex binds to the GC-box sequences in the ie1 by electrophoresis mobility shift assays (EMSA). Knockdown of the expression of PmKLF transcript in WSSV-infected shrimp resulted in delayed cu- mulative mortalities, suggesting that PmKLF is important to WSSV infection. Additi- onally, inhibition of PmKLF expression reduced the copy number of WSSV and ie1 expression,revealing that PmKLF affects WSSV infection via interfering with ie1 expression.