Dhar Arun K, Kaizer Krista N, Betz Yelena M, Harvey Thomas N, Lakshman Dilip K
Viracine Therapeutics Corporation, Columbia, MD 21046, USA.
Virus Genes. 2011 Dec;43(3):367-75. doi: 10.1007/s11262-011-0648-y. Epub 2011 Aug 3.
In silico analysis of three Penaeus stylirostris densovirus (PstDNV) promoters, designated P2, P11, and P61, revealed sequence motifs including the TATA box, downstream promoter element (DPE), GC- and A-rich regions, inverted repeat, activation sequence-1 like (ASL) box, and a conserved guanosine (G) at +24. To delineate the regulatory role of these motifs on promoter activity, deletion constructs were made in a promoter assay vector, pGL3 Basic, that contains a luciferase reporter gene. Luciferase assay showed that P2 had the highest promoter activity followed by P11 and P61 in Sf9 cells. The deletions of inverted repeat, DPE, and GC-rich regions in P2 had the highest negative impact on this promoter. Deletions of DPE, G at the +24, and ASL box in P11 had the highest negative impact on this promoter activity. In P61, DPE and G at +24 are the two key regulators of transcriptional activity. Identification of the key transcriptional regulators is important in understanding the PstDNV pathogenesis in shrimp. This information is also valuable in constructing shrimp viral promoter-based vectors for protein expression in insect cell culture system as well as in shrimp.
对三种斑节对虾浓核病毒（PstDNV）启动子（分别命名为P2、P11和P61）进行的电子分析揭示了一些序列基序，包括TATA框、下游启动子元件（DPE）、富含GC和A的区域、反向重复序列、类激活序列-1（ASL）框以及位于+24处的保守鸟苷（G）。为了阐明这些基序对启动子活性的调控作用，在一个含有荧光素酶报告基因的启动子检测载体pGL3 Basic中构建了缺失构建体。荧光素酶检测表明，在Sf9细胞中，P2的启动子活性最高，其次是P11和P61。P2中反向重复序列、DPE和富含GC区域的缺失对该启动子的负面影响最大。P11中DPE、位于+24处的G和ASL框的缺失对该启动子活性的负面影响最大。在P61中，DPE和位于+24处的G是转录活性的两个关键调节因子。鉴定关键转录调节因子对于理解对虾中PstDNV的发病机制很重要。该信息对于构建基于虾病毒启动子的载体以在昆虫细胞培养系统以及对虾中进行蛋白质表达也很有价值。
