Department of Biology and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, China; School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, 521041, China.
Department of Biology and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, China.
Fish Shellfish Immunol. 2019 Jan;84:639-647. doi: 10.1016/j.fsi.2018.10.054. Epub 2018 Oct 23.
Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5'-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression.
血蓝蛋白（HMC）是一种呼吸糖蛋白，在虾类中也具有多功能非特异性先天免疫防御功能。然而，血蓝蛋白基因表达的转录调控机制尚未报道。在本研究中，我们通过基因组步移法从凡纳滨对虾（Litopenaeus vannamei）中克隆了一个 4324bp 的小亚基血蓝蛋白（HMCs）基因片段，包括 5'-侧翼区，上游 2475bp 至下游 1849bp（外显子 1-内含子 1-外显子 2）。然后生成了四个缺失构建体，并使用荧光素酶报告系统评估了它们的启动子活性。有趣的是，我们鉴定了一个位于外显子 2 中的替代启动子（+1516/+1849bp），其启动子活性比全长或其他构建体更强。生物信息学分析显示，替代启动子区域包含转录因子 c-Jun 的两个保守结合位点。突变分析和电泳迁移率变动分析表明，凡纳滨对虾 c-Jun（Lvc-Jun）结合到替代启动子的+1582/+1589bp 和+1831/+1837bp 区域。此外，Lvc-Jun 的过表达显著增加了替代启动子的活性，而 dsRNA-Lvc-Jun 的共转染则显著降低了 HMCs 的替代启动子活性。总之，我们目前的数据表明，转录因子 Lvc-Jun 是 HMCs 基因表达转录调控所必需的。
