三疣梭子蟹核受体基因HR38的克隆及其在蜕皮中的表达分析

Cloning and expression analysis in molting cycle of nuclear receptor HR38 gene in Portunus trituberculatus

采用RACE技术,克隆出三疣梭子蟹(Portunus trituberculatus)核受体基因HR38的c DNA全长,命名为PTHR38。该基因c DNA序列全长为2950 bp,5′和3′非编码区(UTR)长分别为101 bp和551 bp,开放阅读框(ORF)长2298 bp。推测编码765个氨基酸,预测分子量为84.2 k D,理论等电点为6.3。生物学分析预测,PTHR38基因编码的蛋白属于不稳定蛋白,不具有跨膜结构域。同源性分析表明,PTHR38基因编码的氨基酸序列与大型溞(Daphnia magna)的同源性最高。实时荧光定量分析表明,PTHR38基因在蜕皮周期的各个组织中均有差异表达,在眼柄中表达差异最大。去除单侧眼柄后,PTHR38的表达量在整体上呈现不同程度的上升趋势。

The nuclear receptor superfamily contains a large number of transcription factors that mediate many physiological process, such as cell differentiation, growth and development. Family members have a conserved functional domain that includes a DNA-binding domain and a ligand-binding domain. HR38, a member of the nuclear receptor superfamily, is an orphan receptor for which no ligand is known. HR38 is involved in an unusual ecdysteroid signaling pathway in Drosophila. Portunus trituberculatus is a major Chinese aquaculture species and is also an important Chinese export. The crustacean molting cycle is a physiologically important growth process. To investigate its function in the molting cycle of Portunus trituberculatus, c DNA encoding nuclear receptor HR38 of Portunus trituberculatus was cloned by rapid amplification of c DNA ends（RACE） and named PHR38. The full-length PTHR38 c DNA is 2950 bp, including a 101-bp 5′ untranslated region（UTR） and a 551-bp 3′ UTR. The 2298-bp open reading frame（ORF） encodes a 765-amino acid polypeptide. Bioinformatic analysis revealed that PTHR38 is anunstable protein and has no transmembrane domains. Homology analysis showed that PTHR38 of Portunus trituberculatus has the highest homology to the HR38 gene of Daphnia magna. Six crabs ateach main molting stage（inter-, pre- and post-molting） were selected by morphological feature observation. Eyestalks were excised, the crabs were depigmented, their exoskeletons were removed, and different tissues were sampled before being stored in Trizol Reagent for RNA extraction. Quantitative real-time PCR showed different expression patterns of PTHR38 at each stage of the molting cycle, which suggested that PTHR38 functions in the molting cycle of Portunus trituberculatus. It also showed a similar expression tendency to ecdysone in hemocytes and thus might be associated with ecdysone. In muscle and the hepatopancreas, PTHR38 was upregulated in the post-molting stage. Previous studies showed that HR38 participates in the metabolism of carbohydrates, which suggested that PTHR38 might function in energy metabolism processes. After eyestalk ablation, the expression of PTHR38 was upregulated, which also suggested a connection between PTHR38 and ecdysone.