Identification and functional analysis of Relish homologs in the silkworm, Bombyx mori
Identification and functional analysis of Relish homologs in the silkworm, Bombyx mori
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DOI：
10.1016/j.bbaexp.2007.07.001
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发表时间：
2007-09-01
期刊：
影响因子：
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通讯作者：
Yamakawa, Minoru
中科院分区：
文献类型：
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作者：
Tanaka, Hirornitsu;Matsuki, Hiroyuki;Yamakawa, Minoru
从家蚕中克隆了两个编码味觉同源物的基因，命名为BmRelish1和BmRelish2。BmRelish1有一个类似I kappa B的结构域，除了Rel同源结构域(RHD)、核定位信号(NLS)和富含酸性和疏水氨基酸(AHAA)的区域外，还有5个Ankyrin重复序列。另一方面，BmRelish2缺乏AHAA和Ankyrin重复序列(ANK)。转基因家蚕中BmRelish基因的敲除导致大肠杆菌不能激活抗菌肽基因，提示BmRelish基因在抗菌肽基因表达中起重要作用。BmRelish1和2在MBN-2细胞中的功能分析表明，这两个ARISE同系物都不能激活编码天蚕素B1、阿特辛、Lebocin 3和Lebocin 4的家蚕抗菌肽基因的启动子。但BmRelish1-D2基因构建的BmRelish1-D2缺乏这些基因的ANK强激活启动子。另一个缺乏AHAA和ANK的基因结构未能激活这些基因，这表明BmRelish通过去除ANK而变得活跃，富含AHAA的区域是一个反式激活区域。BmRelish2可抑制BmRelish1-D2对Cecropin B1基因表达的激活，提示BmRelish2是BmRelish1活性形式的显性负性因子。BmRelish1-D2对Cecropin B1、Attakin和Lebocin 4基因的kappa B位点的充分激活是必要的。BmRelish1活性形式和BmRe1A对Lebocin 4基因表达的必需kappa B位点的要求不同，提示kappa B位点在不同转录因子激活抗菌肽基因中的作用不同。BmRelish I融合蛋白的RHD部分与抗菌肽B1和Attakin基因的kappa B位点结合。(C)2007爱思唯尔B.V保留所有权利。
Two cDNAs designated BmRelish1 and 2, that encode Relish homologs, were cloned from the silkworm, Bombyxmori. BmRelish1 had an I kappa B-like domain with 5 ankyrin repeats in addition to Rel homology domain (RHD), nuclear localization signal (NLS), and acidic and hydrophobic amino acids (AHAA) rich regions. On the other hand, BmRelish2 lacked the AHAA and ankyrin repeats (ANK). Knockdown of the BmRelish gene in transgenic silkworms resulted in failure of the activation of antimicrobial peptide genes by Escherichia coli, suggesting that BmRelish plays an important role in antimicrobial peptide gene expression. Functional analysis of BmRelish1 and 2 in mbn-2 cells showed that both Relish homologs do not activate promoters of B. mori antimicrobial peptide genes encoding cecropin B1, attacin, lebocin 3 and lebocin 4. However, a gene construct BmRelish1-d2 lacking the ANK strongly activated promoters of these genes. Another gene construct lacking AHAA and ANK failed to activate these genes, suggesting that BmRelish becomes active by removal of the ANK and that the AHAA-rich region is a transactivation domain. BmRelish2 was shown to repress activation of Cecropin B1 gene expression by BmRelish1-d2, suggesting that BmRelish2 plays a role as a dominant negative factor against the BmRelish1 active form. Necessity of kappa B sites of Cecropin B1, Attacin and Lebocin 4 genes for the full activation of these genes by BmRelish1-d2 was confirmed. The requirement of the mandatory kappa B sites for Lebocin 4 gene expression was different between BmRelish1 active form and BmRe1A, suggesting differential roles for kappa B sites in antimicrobial peptide gene activation by different transcription factors. The binding of the RHD portion of BmRelish I fusion protein to the kappa B sites of Cecropin B1 and Attacin genes was also confirmed. (c) 2007 Elsevier B.V All rights reserved.