Molecular evolutionary analysis of the immune-related genes in insects ; in the case of Drosophila Andropin gene

在以前的研究中，我们发现雄激素具有快速的进化模式，并且氨基酸序列在密切相关的物种之间不是保守的。如果某种正向选择操作了雄激素的进化，人们认为它的功能可能会因每个物种而略有不同。在本研究中，我们试图对雄激素进行一系列的功能分析，以比较不同物种之间的基因表达模式和蛋白质活性。首先，我们测定了不同物种之间雄激素基因51个上游调控区的序列。目前还不清楚哪些转录因子结合基序是组织和阶段特异性表达雄激素所必需的。然而，预测的转录因子结合基序在所研究的物种之间非常保守。有趣的是，我们在这些区域发现了两个TCTAA基序，它们可能对果蝇雄性射精管中的组织特异性表达模式具有调节作用，参考…更多的是葡萄糖脱氢酶(GLD)基因。这一主题在物种之间也是保守的。可以认为，雄激素的表达模式在物种间没有差异，在调控区域获得这样的基序加速了雄激素从祖先基因分化的过程。其次，我们通过RT-PCR分析了雄激素在物种间的表达模式。值得注意的是，雄激素基因在所有与黑素密切相关的物种中都有表达，仅限于成年男性生殖器官。在成年女性中，没有观察到雄激素的表达。接下来，为了检验雄激素在物种之间是否具有保守的功能，进行了抗菌活性测定。人工合成的雄激素多肽用于黑腹果蝇和粉纹夜蛾的雄激素，其氨基酸序列与黑腹果蝇雄激素的氨基酸同源性最低，预测的二级结构不同，用液体法对3种革兰氏阳性菌和2种革兰氏阴性菌进行检测。已经有报道称，黑腹金黄色葡萄球菌不是革兰氏阴性细菌，而是革兰氏阳性细菌。因此，雄激素的抗菌特异性在不同物种之间没有区别；对革兰氏阳性细菌有效，而对革兰氏阴性细菌不起作用。虽然氨基酸序列有很大的差异，但人们认为在已被研究过的物种中，雄激素保持其抗菌活性，在枯草芽孢杆菌中，铁氏杆菌雄激素的抗菌活性比黑腹杆菌的低。较少

In the previous study, we showed that Andropin has a rapid evolutionary pattern and amino acid sequences are not conserved between closely-related species. If a some kind of positive selection operates the Andropin evolution, it is considered that its function might slightly differ from each species. In this study, we tried to have a series of functional analysis ofAndropin in order to compare the gene expression pattern and protein activity between species.First, we determined the sequence of 51 upstream regulatory region of Andropin between melanogaster closely-related species. It is not clear which transcription factor binding motif is necessary for tissue and stage-specific expression of Andropin. However, predicted transcription factor binding motifs are well conserved between the examined species. Interestingly, in these region, we found two TCTAA motifs, which may have a regulatory role of the tissue-specific expression pattern in the Drosophila male ejaculatory duct, referring … More the case of Glucose dehydrogenase (Gld) gene. This motif is also conserved between species. It may be considered that the expression pattern of Andropin is not different between species and that the diversification of Andropin from ancestral gene is accelerated by acquirement of such a motif in the regulatory region.Second, we examined the expression patterns of Andropin between species by RT-PCR analysis. It is dear that Andropin gene expressions are shown among all of the melanogaster closely-related species, restricted in the adult male reproductive organ. In adult female, Andropin expression was not observed.Next, in order to examine whether Andropin conserved its function among species, an antibacterial activity assay was performed. Andropin peptides were artificially synthesized for melanogaster and teissieri D. teissieri Andropin amino acid sequence shows the lowest amino acid identity with D. melanogaster Andropin and the predicted secondary structure was different from each other Assay was performed with the liquid method using three kinds of gram-positive bacteria and two of gram-negative bacteria. It has been already reported that D. melanogaster Andropin Mils not gram-negative bacteria, but gram-positive bacteria. As a result, antibacterial specificity of Andropin is not different between species ; active for gram-positive bacteria and not active for gram-negative bacteria. Although amino acid sequences vary considerably, it is considered that Andropin keeps its antibacterial activity between species in which have been examined Notably, hi the case of Bacillus subtilis, D. teissieri Andropin exerted its antibacterial activity in lower concentration than D. melanogaster Andropin. Less