Identification and functional characterization of doublesex gene in the testis of Spodoptera litura
Identification and functional characterization of doublesex gene in the testis of Spodoptera litura
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斜纹夜蛾睾丸双性基因的鉴定及功能表征
DOI：
10.1111/1744-7917.12608
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发表时间：
2019-12-01
期刊：
影响因子：
4
通讯作者：
Liu, Lin
中科院分区：
文献类型：
--
作者：
Du, Qian;Wen, Liang;Liu, Lin
睾丸融合现象在大多数鳞翅目昆虫中都存在，包括斜纹夜蛾这种重要的多食性害虫。斜纹夜蛾的睾丸融合对雄性繁殖有利，但其融合的分子机制尚不清楚。双性基因影响果蝇和家蚕的生殖器形成、求偶行为以及性别二态性特征，对性别分化至关重要。然而，它在斜纹夜蛾睾丸中的作用仍不清楚。斜纹夜蛾的双性基因（Sldsx）具有雄性特异性的Sldsx(M)和雌性特异性的Sldsx(F)异构体，且在雄性睾丸中表达水平较高。在睾丸融合阶段（6龄第6天，L6D6），与融合前和融合后时期相比，Sldsx表达量最高。此外，Sldsx在睾丸的腹膜鞘中的表达高于卵泡中的生殖细胞。利用CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR相关蛋白9）技术对斜纹夜蛾进行研究以确定Sldsx的作用。在产卵后2小时内将单向导RNA信使RNA和Cas9蛋白的混合物（每种300纳克/微升）注入卵中。CRISPR/Cas9在G₀代成功诱导了Sldsx的基因组突变。突变的雄性个体睾丸较小，周围的气管也较少。此外，突变的雄性个体外生殖器异常，无法与野生型雌性完成交配。另外，几乎所有突变的雄性个体睾丸都发生了融合。结果表明，Sldsx与睾丸融合无关，但对斜纹夜蛾的睾丸发育以及外生殖器的形成和功能是必需的。双性基因在雄性中的主要作用与其他昆虫相似。
Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit‐fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male‐specific SldsxM and female‐specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre‐fusion and post‐fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild‐type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.