College of Life Sciences, Shaanxi Normal University, Xi'an, China.
School of Life Science and Technology, Xidian University, Xi'an, China.
Protein Pept Lett. 2022;29(2):143-155. doi: 10.2174/0929866528666211125105627.
For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear.
The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes.
Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter.
The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter.
In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.
对于两栖动物来说，抗菌肽是抵抗外界环境刺激的先天免疫分子。然而，青蛙抗菌肽基因表达的调控机制尚不清楚。
两种抗菌肽，palustrin-2CE2 和 brevinin-2CE3，在中华大蟾蜍受到外界刺激时产生。利用这一模型，我们分析了两种抗菌肽基因的结构和调控元件，并探讨了相关转录因子对两种基因的调控作用。
将不同的刺激物，如大肠杆菌、金黄色葡萄球菌和化学物质脂多糖（LPS）应用于不同发育阶段的中华大蟾蜍蝌蚪，通过 RT-PCR 检测抗菌肽的表达水平。采用生物信息学分析、5'-RACE 和基因组步移技术，分析抗菌肽基因的基因组结构和启动子区。通过双荧光素酶报告基因检测、酵母单杂交实验和 EMSA 实验，评估细胞内内源性调节因子对抗菌肽启动子的调控作用。
不同刺激物处理后，前原 palustrin-2CE2 和前原 brevinin-2CE3 的转录水平均显著上调。基因组结构分析表明，这两个基因均含有三个外显子和两个内含子。启动子分析表明，在启动子区域存在 NF-κB 家族调控因子的结合位点，实验表明，蛙细胞内源性 NF-κB 家族调控因子激活抗菌肽基因的启动子。酵母单杂交实验和 EMSA 实验表明，RelA 和 NF-κB1 可能与前原 palustrin-2CE2 启动子的特定基序相互作用。
本文发现中华大蟾蜍抗菌肽 palustrin-2CE2 和 brevinin-2CE3 的基因表达水平在环境刺激下会增加，并验证了基因表达水平的变化受转录因子 RelA 和 NF-κB1 的影响。酵母单杂交实验和 EMSA 实验证实，RelA 和 NF-κB1 可直接与蛙抗菌肽基因启动子相互作用，为抗菌肽对环境刺激的响应调控机制提供了新的数据。
