RagA缺失果蝇的构建及表型分析  

Construction and phenotype analysis of RagA deletion in Drosophila

作　　者：傅媛媛 沈苏林 孟国强 刘倩倩 樊伟康 韦有恒 FU Yuan-Yuan;SHEN Su-Lin;MENG Guo-Qiang;LIU Qian-Qian;FAN Wei-Kang;WEI You-Heng(College of Biosciences and Biotechnology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学生物科学与技术学院,扬州225009

出　　处：《昆虫学报》2024年第9期1182-1189,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金面上项目(32370504)。

摘　　要：【目的】Rag GTPase是真核生物中高度保守的Ras家族蛋白,在调节雷帕霉素靶点复合体1(mechanic target of rapamycin complex 1, mTORC1)活性和自噬等方面发挥重要作用。为了研究Rag GTPase的生理功能,本研究构建了RagA基因编码区缺失的突变黑腹果蝇Drosophila melanogaster,对其表型进行分析。【方法】将靶向RagA基因的gRNA表达质粒导入表达Cas9蛋白的黑腹果蝇中,随后通过PCR方法筛选获得RagA编码区缺失的RagA突变黑腹果蝇;利用遗传杂交的方法对腹果蝇RagA突变体的生殖及存活情况进行分析;利用FLP-FRT系统分别在脂肪体和雌性卵巢中诱导产生RagA突变的细胞克隆,分别分析RagA突变细胞生长和自噬水平的改变。【结果】利用CRISPR-cas9结合显微注射技术成功敲除RagA基因;RagA突变导致黑腹果蝇在胚胎期死亡,在细胞水平上使细胞生长明显降低、细胞内LAMP1和Rab7标记的自噬溶酶体堆积。【结论】本研究验证了RagA基因调节细胞代谢的功能,为进一步利用RagA突变果蝇研究RagA基因在发育中的功能分析和机制解析奠定基础。【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.

关 键 词：果蝇 自噬 Ras家族蛋白 Rag GTPase 基因敲除 

分 类 号：Q963[生物学—昆虫学]