Chen Ping, Xu Shan-Liang, Zhou Wei, Guo Xiao-Ge, Wang Chun-Lin, Wang Dan-Li, Zhao Yun-Long
School of Marine Sciences, Ningbo University, Ningbo 315211, China.
School of Marine Sciences, Ningbo University, Ningbo 315211, China.
Anim Reprod Sci. 2014 May;146(3-4):227-37. doi: 10.1016/j.anireprosci.2014.03.010. Epub 2014 Mar 29.
The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation.
利用RACE技术从枝角类大型溞中克隆出一个转化基因（Dptra）的全长cDNA。通过qPCR和全胚胎原位杂交技术评估了Dptra在不同生殖阶段的表达情况。Dptra cDNA全长1652bp，具有一个1158bp的开放阅读框，编码一个385个氨基酸的多肽，该多肽包含一个性别决定蛋白N端（SDP_N）超家族、八个推定的磷酸化位点以及N端富含精氨酸-丝氨酸（RS）的结构域。Dptra与大型溞、意大利蜜蜂、中华蜜蜂和熊蜂的直系同源基因的同一性分别为81%、53%、51%和45%。基于推导氨基酸序列的系统发育分析表明，Dptra聚集在膜翅目分支中，与大型溞和意大利蜜蜂的亲缘关系最为密切。qPCR结果显示，Dptra在不同生殖阶段的表达量显著增加（P<0.05），顺序如下：雄性、具卵鞍雌体、孤雌生殖雌体、休眠卵和幼龄雌体。Dptra在雄性和雌性之间的表达存在显著差异，在具卵鞍雌体和雄性中的表达量显著高于产夏季卵的孤雌生殖大型溞。全胚胎原位杂交显示，Dptra在雄性和雌性之间的表达水平不同。在雄性中，在第一触角、第二触角和胸肢中发现杂交信号，而在孤雌生殖雌体和具卵鞍雌体的相应部位表达水平相对较弱。这表明Dptra基因在生殖模式转换和性别分化中发挥着重要作用。
