Gal80^(ts)与Gal4组合灵敏控制果蝇中UAS转基因的表达水平

Combination of Gal80^(ts) and Gal4 flexibly manipulates the expression levels of UAS transgenes in Drosophila

【目的】Gal80^(ts)与Gal4组合驱动UAS转基因表达是黑腹果蝇Drosophila melanogaster研究中常用的转基因过表达遗传学工具,通过温度控制实现对UAS转基因表达的灵活开关。Gal80^(ts)是一种温度敏感型蛋白,低温下(18℃)与Gal4蛋白结合并抑制其转录活力,高温下(29℃)解除对Gal4的抑制,从而允许Gal4结合UAS位点,启动UAS转基因的表达。但是从18~29℃的开关只能强烈过表达UAS转基因,而不能灵活调控转基因的表达水平。本实验系统研究一系列温度下转基因的表达水平,从而实现该体系对转基因的表达水平的灵活控制。【方法】以果蝇翅芽这一常用器官组织为研究模型,以2种Gal4品系(dpp-Gal4和en-Gal4,分别由decapentaplgic和engrailed基因的启动子驱动)分别与tub-Gal80^(ts)(微管蛋白基因tubulin启动子驱动)基因重组后,再分别与UAS-wg(wingless)转基因品系杂交;在一系列温度(18,25,27.5,28,28.5和30℃)下进行子代幼虫培养,通过免疫组化染色揭示并量化分析转基因wg在3龄幼虫翅芽上的表达水平。【结果】18~25℃培养条件下,Gal80^(ts)与Gal4组合系统中的UAS转基因不能表达;30℃时培养,转基因强烈地过表达;在25~30℃区间内,随着温度升高,转基因表达水平逐渐上升。【结论】在25~30℃之间的温度调控可以实现对Gal80^(ts)与Gal4组合系统中的UAS转基因表达水平的调控。本研究结果对调控转基因表达程度有重要价值。

【Aim】The combination of Gal80^（ts）and Gal4 is commonly used to regulate the UAS transgene overexpression in Drosophila,and flexibly turns on or off the transgene expression in a temperaturedependent manner. Gal80^（ts）,a temperature-sensitive protein,binds Gal4 and inhibits its transcription activity at low temperature（ 18℃）,but relieves the inhibition of Gal4 protein and allows its binding to the UAS sites to drive the expression of UAS transgenes at high temperature（ 29℃）. However,the switch from 18 to 29℃ could only achieve intensive overexpression of UAS transgenes,but not flexible manipulation. Thus in this study we systematically studied the flexible control of UAS transgene expression levels by this system at a series of temperatures. 【Methods】Two Gal4 lines（ dpp-Gal4 and en-Gal4 driven by decapentaplgic and engrailed promoter,respectively,in Drosophila wing disc）,after recombination with tub-Gal80^（ts）（ driven by microtubulin gene tubulin promoter）,were hybridized with UAS-wg（ wingless） lines. The progeny larvae were cultured at a series of temperatures（ 18,25,27. 5,28,28. 5 and 30℃）,and immunohistochemistry was used to detect wg transgene expression levels,which were further quantified and analyzed in the 3rd instar larvae. 【Results】The wg transgene was not expressed at 18- 30℃,but overexpressed significantly at 30℃. In the range of 25- 30℃,with the increasing temperature,the wg transgene expression levels（ fluorescent intensity） in en-Gal4 and dppGal4 regions commonly exhibited a gradient uptrend. 【Conclusion 】 The system of Gal4 and Gal80^（ts） combination can flexibly manipulate UAS transgene expression levels between 25- 30℃. This result has an important value in the regulation of transgene expression level.