PROTEIN INTERACTION OF IAG IN CHERAX QUADRICARINATUS BASED ON NUCLEAR SYSTEM YEAST TWO-HYBRID TECHNOLOGY

Insulin-like androgenic hormone (IAG), secreted by androgenic glands (AG), plays a crucial role in the sex differentiation of crustaceans. In the case of red claw crayfish (Cherax quadricarinatus), IAG has been confirmed to have a close association with the formation of intersex individuals. However, the molecular regulatory pathways through which IAG exerts its effects have not been fully elucidated. In order to obtain candidate protein information that interacts with the IAG protein and explore the role of interacting proteins in the regulation of sex in C. quadricarinatus, we constructed a yeast library using the testes and spermaducts of C. quadricarinatus and employed the nuclear system yeast two-hybrid technique to screen for proteins that interact with IAG. The encoding gene of the key candidate proteins was cloned, and its expression was analysed. The results revealed that the primary library had a capacity of 1.12×10⁷, the secondary library had a capacity of 1.28×10⁷, 25 positive clones were successfully screened and 12 protein-encoding genes were annotated. Among these genes, the coding sequence (CDS) of the key candidate protein named muc5acl (Mucin-5AC-like) was cloned, and its full length was determined to be 474 bp. Semi-quantitative and fluorescence quantitative expression analysis demonstrated that muc5acl exhibits specific expression in the androgenic glands, testes, and spermaducts of C. quadricarinatus, with significantly higher levels of expression in the spermaducts of intersex individuals were significantly higher compared to males, and the expression levels in males androgenic gland were significantly higher than that of intersex individuals. This finding suggests that the muc5acl gene is involved in gonadal development in C. quadricarinatus and may be related to the secretion of male hormones and sperm transport processes. The results of this study provide important information for further elucidating the molecular mechanisms by which IAG regulates the formation of intersex individuals in C. quadricarinatus.