Orphan genes are genes with no sequence homologues in other species. Here, we identified two orphan genes, namely, Tssor-3 and Tssor-4, in Plutella xylostella. Both genes contained a signal peptide sequence, suggesting their functions as secreted proteins. Expression pattern analysis based on real-time quantitative PCR (qPCR) showed that both orphan genes were specifically expressed in all male gonads except the testes. The expression of both the orphan genes peaked at the male adult stage. Immunofluorescence assays suggested that the two proteins were seminal proteins, indicating their potential roles in male reproductive regulation. To further explain their functions, we knocked down the expression of these two genes by RNA interference (RNAi). The results showed that the expression of Tssor-3 and Tssor-4 was significantly downregulated at 24 h after injection compared to that of the controls. Biological assays showed that the number of laid eggs and the hatching rate of offspring eggs were significantly reduced when the expression of Tssor-3 and Tssor-4 was reduced, suggesting that the two orphan genes played a role in male fertility in P. xylostella. Our results provide evidence that orphan genes are involved in male reproductive regulation, which is important for male fitness during evolution.

孤儿基因是在其他物种中没有序列同源物的基因。在这里，我们在小菜蛾中鉴定了两个孤儿基因，即Tssor-3和Tssor-4 。这两个基因都含有信号肽序列，表明它们具有分泌蛋白的功能。基于实时定量 PCR (qPCR) 的表达模式分析表明，这两种孤儿基因在除睾丸外的所有雄性性腺中均特异性表达。这两种孤儿基因的表达在成年雄性阶段达到峰值。免疫荧光分析表明这两种蛋白质是精液蛋白质，表明它们在男性生殖调节中的潜在作用。为了进一步解释它们的功能，我们通过 RNA 干扰 (RNAi) 敲低了这两个基因的表达。结果表明，Tssor-3和Tssor-4的表达与对照组相比，注射后 24 小时显着下调。生物学分析表明，Tssor-3和Tssor-4的表达降低时产卵数和后代卵孵化率显着降低，表明这两个孤儿基因在小菜蛾雄性生育中发挥了作用。我们的结果提供证据表明孤儿基因参与男性生殖调节，这对进化过程中的男性健康很重要。