Using lentiviral to investigate the effect of novel candidate gene MAEL identified by RNA-seq on sex differentiation in the Chinese Alligator (Alligator sinensis)

The sex differentiation of Chinese alligator displays temperature-dependent sex determination, which occurs in the temperature-sensitive period (TSP) of embryonic development. Despite studies identifying genes involved in sex determination in the Chinese alligator, the complex mechanisms underlying their role remain a scientific mystery awaiting further exploration. In this study, before and after sex differentiation, six sampling points, FPT (F1-F6) and MPT (M1-M6), were established to explore TSP, candidate genes for sex differentiation, and lentivirus validation. We discovered that F4-vs-F5 and M4-vs-M5 exhibited the highest number of differential genes, with distinctive morphological characteristics of oocytes and spermatotubules during the F5 and M5 stages, respectively. Consequently, we speculate that the TSPs are F4 and M4. Through RNA-seq analysis, we identified genes potentially, such as MAEL , influencing sex differentiation and constructed lentivirus to explore its mechanism. LV-MAEL-shRNA and LV-MAEL-OE successfully invaded the gonads of Chinese alligator in vitro and in vivo experiments. knockdown of MAEL can reduce the male sex determining gene Dmrt1 by about 70 %. These findings suggest that MAEL , acting as a transcription factor, may play a role in sex determination by regulating Dmrt1 . This study advances understanding of sex determination in Chinese alligator, establishing a foundation for future research. • F4 and M4 as the temperature-sensitive period for FPT and MPT, respectively. • Discovery of differential genes, such as MAEL , AMHR2 , MIF , HOXA11 , BMP6 . • LV-MAEL experiments suggest MAEL's role in sex determination via Dmrt1 regulation.