[Aim] The combination of Gal80tsand 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.Gal80ts,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-Gal80ts (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 Gal80ts combination can flexibly manipulate UAS transgene expression levels between 25-30℃.This result has an important value in the regulation of transgene expression level.%[目的]Gal80ts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.Gal80ts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-Gal80ts(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,Gal80ts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 ～ 30C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Gal80ts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.
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