Kluebsoongnoen Jakkapong, Panyim Sakol, Sarnowski Tomasz J, Udomkit Apinunt
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand.
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Comp Biochem Physiol A Mol Integr Physiol. 2021 Apr;254:110877. doi: 10.1016/j.cbpa.2020.110877. Epub 2020 Dec 20.
Effective inducing of ovarian maturation in female shrimp broodstock is important for successful breeding programs. Vitellogenesis is a biochemical process during which a yolk protein precursor vitellogenin (Vg) is synthesized and thus, can be used to indicate ovarian maturation stage. In this study, transcriptional regulation of Vg synthesis in the black tiger shrimp, Penaeus monodon was investigated. Genome walking on 5' upstream sequence of Vg gene revealed several putative binding sites of lipophilic retinoic acid response elements (RARE), and nuclear hormone responsive elements. Deletion of RARE significantly reduced the promoter activity to drive the expression of luciferase reporter gene in Sf-9 cells. To validate the trans-factor that potentially controls Vg expression through RARE, a cDNA encoding retinoid X receptor (PmRXR), one of the RARE-bound transcription factors was cloned from P. monodon's ovary. PmRXR expression was detected in various shrimp tissues, and was up-regulated during ovary development in a similar way to Vg expression. The DNA-binding domain of PmRXR protein showed specific binding to RARE-containing region on Vg 5' upstream sequence as determined by Electrophoretic Mobility Shift Assay (EMSA). Furthermore, dsRNA-mediated PmRXR silencing in previtellogenic and vitellogenic shrimp revealed that suppression of PmRXR could reduce Vg transcript in both stages. Taken together, the results presented in this study indicate that RXR is possibly an activator protein that modulates Vg expression in shrimp ovary through the binding to RARE.
有效诱导雌虾亲体卵巢成熟对于成功的育种计划至关重要。卵黄发生是一个生化过程，在此过程中卵黄蛋白前体卵黄原蛋白（Vg）被合成，因此可用于指示卵巢成熟阶段。在本研究中，对黑虎虾（凡纳滨对虾）Vg合成的转录调控进行了研究。对Vg基因5'上游序列进行基因组步移，发现了几个推定的亲脂性视黄酸反应元件（RARE）和核激素反应元件的结合位点。缺失RARE显著降低了在Sf-9细胞中驱动荧光素酶报告基因表达的启动子活性。为了验证可能通过RARE控制Vg表达的转录因子，从凡纳滨对虾卵巢中克隆了一种编码视黄酸X受体（PmRXR）的cDNA，它是与RARE结合的转录因子之一。在对虾的各种组织中检测到了PmRXR的表达，并且在卵巢发育过程中其表达上调，上调方式与Vg表达相似。通过电泳迁移率变动分析（EMSA）确定，PmRXR蛋白的DNA结合结构域与Vg 5'上游序列上含RARE的区域具有特异性结合。此外，在卵黄发生前期和卵黄发生期的对虾中，通过dsRNA介导的PmRXR沉默显示，抑制PmRXR可在两个阶段降低Vg转录本。综上所述，本研究结果表明，RXR可能是一种激活蛋白，通过与RARE结合来调节对虾卵巢中的Vg表达。
