基于CRISPR/Cas9系统的Met-mcherry果蝇制备及其表达分析  

Construction and Expression Analysis of Met-mcherry Drosophila Based on CRISPR/Cas9 System

作　　者：王顺鑫 侯天兰 陈金霞 何倩毓 Wang Shunxin;Hou Tianlan;Chen Jinxia;He Qianyu(College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing163319)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2024年第6期93-100,共8页journal of heilongjiang bayi agricultural university

基　　金：国家自然科学基金项目(31970465);黑龙江省博士后科研启动基金(LBH-Q19165)。

摘　　要：保幼激素(juvenile hormone,JH)作为昆虫变态发育中的关键激素,其核内信号的传递主要依赖于胞内受体Methoprene-tolerant(Met)。JH生理功能的发挥取决于Met的组织定位。然而,受限于Met抗体的制备,目前有关Met的组织定位仍无法确定。实验采用CRISPR/Cas9系统,将红色荧光蛋白(mcherry)编码序列插入Met基因C末端,使果蝇表达Met-mcherry融合蛋白,构建获得原位表达Met-mcherry的果蝇品系。利用mcherry抗体从而可真实准确地反应Met的时空表达情况。免疫荧光染色结果发现游走早期,Met-mcherry主要定位于果蝇唾液腺、触角盘、前胸腺、脂肪体、神经索、腿盘等组织中。亚细胞定位结果表明,在果蝇游走期和产卵后96 h,Met-mcherry分别定位于细胞核和细胞质中,并且JH可促进Met-mcherry由细胞质转移至细胞核中。研究可为深入了解Met的功能提供基础。Juvenile hormone(JH)is a key hormone in insect metabolism,and its intracellular signaling is mainly dependent on intracellular receptor Methoprene-tolerant(Met).The physiological function of JH depends on the tissue localization of Met.However,due to the limitation of the preparation of Met antibodies,the tissue localization of Met is still uncertain.In the experiment,the CRISPR/Cas9 system was used to insert the coding sequence of red fluorescent protein(mcherry)into the C-terminal of Met gene to enable Drosophila melanogaster to express Met-mcherry fusion protein in situ.Mcherry antibody could be used to accurately reflect the spatio-temporal expression of Met.The results of immunofluorescence staining showed that Met-mcherry was mainly located in salivary gland,antennal disc,prothoracic gland,fat body,nerve cord and leg disc of Drosophila melanogaster at the early wandering stage.The results of subcellular localization showed that Met-mcherry was located in the nucleus and cytoplasm respectively during early wandering stage and 96 h after oviposition,and JH could promote the transfer of Met-mcherry from cytoplasm to nucleus.It could provide a basis for further understanding of the function of Met.

关 键 词：黑腹果蝇 MET CRISPR/Cas9系统 组织定位 

分 类 号：Q94-335[生物学—植物学]