关键词: 果蝇, 自噬, Ras家族蛋白, Rag GTPase, 基因敲除

Abstract: 【Aim】 Rag GTPases are highly conserved Ras family proteins in eukaryotes, which play important roles in regulating mechanic target of rapamycin complex 1 (mTORC1) activity and autophagy. In order to study the physiological function of Rag GTPases, the RagA-deleted mutant of Drosophila melanogaster was constructed and its phenotype was analyzed. 【Methods】 A plasmid expressing gRNAs targeted RagA gene was introduced into D. melanogaster to express Cas9 protein. The RagA mutants of D. melanogaster with deletion of the coding region of RagA were screened by PCR. The reproduction and survival of RagA mutants of D. melanogaster were analyzed by genetic hybridization. The FLP-FRT system was used to induce cell clones in the fat bodies and female ovaries to analyze the cell growth and autophagy level in RagA mutant cells, respectively. 【Results】 RagA gene was successfully knocked out using CRISPR-cas9 combined with microinjection technology. Mutation of RagA led to embryonic death in D. melanogaster. At the cellular level, knockout of RagA resulted in significantly slowed cell growth and the accumulation of autolysosome marked by LAMP1 and Rab7. 【Conclusion】 This study verifies the function of RagA in regulating cell metabolism, and provides a foundation for further analysis of RagA gene function and related mechanism in development using RagA mutant Drosophila.

Key words: Drosophila, autophagy, Ras family protein, Rag GTPases, gene knockout