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

在先前的研究中，我们发现雄激素具有快速的进化模式，并且氨基酸序列在近缘物种之间不保守。如果某种积极的选择操作雄激素的进化，它被认为是它的功能可能略有不同，从每个物种。本研究试图对Andropin进行一系列的功能分析，以比较不同物种间Andropin的基因表达模式和蛋白活性。首先，我们测定了黑腹果蝇近缘物种Andropin上游51个调控区的序列。目前还不清楚哪些转录因子结合基序是雄激素组织和阶段特异性表达所必需的。然而，预测的转录因子结合基序在所研究的物种之间是很保守的。有趣的是，在这些区域中，我们发现了两个TCTAA基序，它们可能对果蝇雄性射精管的组织特异性表达模式具有调节作用， ...更多信息 葡萄糖脱氢酶（Gld）基因的情况。这种模式在物种之间也是保守的。这可能是因为Andropin的表达模式在物种之间没有差异，而在调控区中获得这样的基序可以加速Andropin从祖先基因的多样化。其次，我们通过RT-PCR分析了Andropin在物种之间的表达模式。Andropin基因在所有与黑腹果蝇亲缘关系密切的物种中均有表达，仅限于成年雄性生殖器官。在成年雌性中，没有观察到Andropin的表达。接下来，为了检查Andropin在物种之间是否保守其功能，进行了抗菌活性测定。人工合成了黑腹果蝇（melanogaster）和泰氏果蝇（teissieri D. teissieri Andropin氨基酸序列与D.黑腹菌雄激素和预测的二级结构彼此不同。使用三种革兰氏阳性细菌和两种革兰氏阴性细菌用液体法进行测定。已有报道D.黑腹菌Andropin Mils不是革兰氏阴性菌，而是革兰氏阳性菌。因此，雄激素的抗菌特异性在物种之间没有差异;对革兰氏阳性菌有活性，对革兰氏阴性菌无活性。虽然氨基酸序列有很大的差异，但认为雄激素在已检测的物种之间保持其抗菌活性。teissieri Andropin在较低浓度下具有抑菌活性。黑腹菌雄激素少

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