Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.
Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.
复制标题
性别二态性鹿角甲虫金属脂质中性别测定基因的鉴定和功能分析。
DOI：
10.1186/s12864-016-2522-8
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发表时间：
2016-03-22
期刊：
影响因子：
4.4
通讯作者：
Lavine LC
中科院分区：
文献类型：
--
作者：
Gotoh H;Zinna RA;Warren I;DeNieu M;Niimi T;Dworkin I;Emlen DJ;Miura T;Lavine LC
性别决定途径中的基因是性二态动物性状的重要调节因子，包括精心设计和夸张的雄性装饰物和性选择武器。在这项研究中，我们确定和功能分析的性别决定基因家族的成员在金色的金属雄鹿甲虫Cyclommatus metallifer，表现出极大的差异，男性和女性之间的下颌骨大小。我们构建了一个C。来自幼虫和预蛹发育阶段以及雄性和雌性的组织的Metallifer转录组学数据库。使用Roche 454焦磷酸测序，我们从总共1，223，516个原始读段生成了从头组装的数据库，这导致10，794个同源群（推定的鉴定基因）中包含14，565个同源群（推定的转录物同种型）。我们在这个数据库中查询了C。metallifer保守的性别决定基因，并确定了14个候选性别决定途径基因。然后，我们的特点是在这个物种的极端性二型性状的发展中的几个这些基因的作用。我们用RT-PCR和RNAi技术对三个C.金属虫候选基因-性致死（CmSxl）、Transformer-2（Cmtra 2）和间性（Cmix）。这三个基因的表达模式在性别之间没有差异。在RNAi基因敲除实验中，我们发现只有Cmix对性二态形态有任何影响，并且这些模拟了Cmdsx敲除在雌性中的影响。敲低CmSxl对鹿角甲虫表型没有可测量的影响，而敲低Cmtra 2导致在预蛹期完全致死。这些结果表明，与果蝇相比，CmSxl和Cmtra 2在性别决定级联中的作用可能在鹿角甲虫中有所不同。我们的研究结果还表明，Cmix在这一途径中具有保守的作用。除了这三个基因，我们还对C. metallifer dsx基因（Cmdsx），以确定使用外显子特异性RNAi更充分地调节二型性的同种型。我们确定了总共16个Cmdsx基因的选择性剪接变体，其编码多达14个单独的外显子。尽管Cmdsx基因的RNA剪接产物的变化，只有四个蛋白质亚型的预测。我们的外显子特异性RNAi的结果表明，必需的CmDsx异构体的胚后雄性分化是CmDsxB，而胚后雌性特异性分化主要是由CmDsxD调节。总之，我们的研究结果强调了研究许多昆虫物种中高度保守的性别决定途径功能的重要性，特别是那些具有戏剧性和夸张的性二型性的昆虫，因为蛋白质结构的保守并不总是转化为下游功能的保守。本文的在线版本（doi：10.1186/s12864-016-2522-8）包含补充材料，可供授权用户使用。
Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females. We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes – Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD. Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function. The online version of this article (doi:10.1186/s12864-016-2522-8) contains supplementary material, which is available to authorized users.