State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan Center for Developmental Biology, Institute of Hydrobiology, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Wuhan, China.
PLoS One. 2010 Dec 20;5(12):e12883. doi: 10.1371/journal.pone.0012883.
Defensins are a group of cationic peptides that exhibit broad-spectrum antimicrobial activity. In this study, we cloned and characterized a β-defensin from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides). Interestingly, the β-defensin was shown to be dominantly expressed in pituitary and testis by RT-PCR and Western blot analysis, and its transcript level is significantly upregulated in reproduction organs from intersexual gonad to testis during the natural and artificial sex reversal. Promoter sequence and the responsible activity region analyses revealed the pituitary-specific POU1F1a transcription binding site and testis-specific SRY responsible site, and demonstrated that the pituitary-specific POU1F1a transcription binding site that locates between -180 and -208 bp is the major responsible region of grouper β-defensin promoter activity. Immunofluorescence localization observed its pituicyte expression in pituitary and spermatogonic cell expression in testis. Moreover, both in vitro antibacterial activity assay of the recombinant β-defensin and in vivo embryo microinjection of the β-defensin mRNA were shown to be effective in killing gram-negative bacteria. And, its antiviral role was also demonstrated in EPC cells transfected with the β-defensin construct. Additionally, the antibacterial activity was sensitive to concentrations of Na(+), K(+), Ca(2+) and Mg(2+). The above intriguing findings strongly suggest that the fish β-defensin might play significant roles in both innate immunity defense and reproduction endocrine regulation.
防御素是一类阳离子肽，具有广谱抗菌活性。本研究从雌雄同体的斜带石斑鱼（Epinephelus coioides）脑 cDNA 文库中克隆并鉴定了一种β-防御素。有趣的是，通过 RT-PCR 和 Western blot 分析表明，β-防御素主要在脑和睪丸中表达，其转录水平在自然和人工性反转过程中从两性性腺到睪丸的生殖器官中显著上调。启动子序列和负责活性区域分析揭示了脑特异性 POU1F1a 转录结合位点和睪丸特异性 SRY 负责位点，并表明位于-180 至-208 bp 之间的脑特异性 POU1F1a 转录结合位点是斜带石斑鱼 β-防御素启动子活性的主要负责区域。免疫荧光定位观察到其在脑中的垂体细胞表达和在睪丸中的精原细胞表达。此外，重组β-防御素的体外抗菌活性测定和β-防御素 mRNA 的体内胚胎微注射均显示出有效杀死革兰氏阴性菌的作用。并且，在转染β-防御素构建体的 EPC 细胞中也证明了其抗病毒作用。此外，抗菌活性对 Na(+)、K(+)、Ca(2+)和 Mg(2+)的浓度敏感。这些有趣的发现强烈表明，鱼类β-防御素可能在先天免疫防御和生殖内分泌调节中发挥重要作用。
