Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China.
Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China; College of Life Science, Shanxi University, Taiyuan, Shanxi 030006, China.
Insect Biochem Mol Biol. 2018 Jan;92:1-11. doi: 10.1016/j.ibmb.2017.11.001. Epub 2017 Nov 4.
During growth and development of insects, the steroid hormone 20-Hydroxyecdysone (20E) regulates the molting process through activation of a series of genes including E74, E75 and HR3 by the 20E receptor EcR. Here, we analyzed the function of LmHR3 in the migratory locust Locusta migratoria. By sequence comparison, we first identified and characterized the putative nuclear receptor protein (LmHR3) based on L. migratoria transcriptome data. The full length cDNA is 2272 bp long encoding a protein of 455 amino acids that contains a DNA binding domain (zinc finger) and a ligand binding domain. Phylogenetic analyses showed that LmHR3 has a high homology with the ortholog from Blattaria. RT-qPCR results revealed that LmHR3 has a low level expression in the early days of 5th instar nymphs, and then increases and peaks at day 6, followed by a decrease to low levels before ecdysis. The LmHR3, hence, coincides with the profile of circulating 20E levels. Indeed, we show that transcription of LmHR3 is induced by 20E in vivo, and significantly suppressed by successfully knocking down expression of LmEcR. After injection of dsRNA for LmHR3 (dsLmHR3) at day 2 of earlier instar nymphs (3rd and 4th instar) and final instar nymphs (5th instar), none of the nymphs were able to molt normally, and eventually died. Chitin staining and ultra-structural analysis showed that both the synthesis of the new cuticle and the degradation of the old cuticle were blocked in the dsLmHR3 treated nymphs. Especially, chitin synthesis genes (LmUAP1 and LmCHS1) and chitinase genes (LmCHT5 and LmCHT10) were significantly down-regulated in the dsLmHR3 treatment group. Together, our results suggest that LmHR3 is involved in the control of chitin synthesis and degradation during L. migratoria molting.
在昆虫的生长发育过程中，类固醇激素 20-羟基蜕皮酮（20E）通过 20E 受体 EcR 激活一系列基因，包括 E74、E75 和 HR3，从而调节蜕皮过程。在这里，我们分析了 LmHR3 在飞蝗 Locusta migratoria 中的功能。通过序列比较，我们首先根据 L. migratoria 转录组数据鉴定并表征了假定的核受体蛋白（LmHR3）。全长 cDNA 长 2272bp，编码 455 个氨基酸的蛋白质，包含一个 DNA 结合域（锌指）和一个配体结合域。系统发育分析表明，LmHR3 与 Blattaria 的同源物具有很高的同源性。RT-qPCR 结果显示，LmHR3 在 5 龄若虫早期表达水平较低，然后增加并在第 6 天达到峰值，然后在蜕皮前降至低水平。LmHR3 与循环 20E 水平的变化模式一致。事实上，我们表明 LmHR3 的转录在体内被 20E 诱导，并且在 LmEcR 表达被成功敲低后显著受到抑制。在第 2 龄若虫（第 3 龄和第 4 龄）和末龄若虫（第 5 龄）早期注射 LmHR3 的 dsRNA（dsLmHR3）后，没有一只若虫能够正常蜕皮，最终死亡。几丁质染色和超微结构分析表明，dsLmHR3 处理的若虫中，新表皮的合成和旧表皮的降解都被阻断。特别是，dsLmHR3 处理组中几丁质合成基因（LmUAP1 和 LmCHS1）和几丁质酶基因（LmCHT5 和 LmCHT10）显著下调。综上所述，我们的结果表明 LmHR3 参与了飞蝗蜕皮过程中几丁质合成和降解的调控。
