牙鲆碱性磷酸酶基因调控序列的功能分析及甲状腺激素对其调节作用  

Function analysis of regulation sequences of alkaline phosphatase gene in Japanese flounder(Paralichthys olivaceus) and regulating effect of thyroid hormone

作　　者：施志仪[1] 胡燕林[1] 张俊玲[1] 靳雨丽[1] 

机构地区：[1]上海海洋大学,农业部水产种质资源与养殖生态重点开放实验室,上海201306

出　　处：《海洋渔业》2010年第3期244-250,共7页Marine Fisheries

基　　金：国家自然科学基金项目(30571420);国家教育部博士项目基金(20040264001);上海市重点学科水生生物学建设项目(S3070)

摘　　要：为分析牙鲆碱性磷酸酶基因5'调控区和第一内含子调控序列的功能,本研究运用DNA重组技术将PCR扩增得到的牙鲆碱性磷酸酶基因的5′调控区序列、第一内含子序列及5′调控区和第一内含子串联序列分别插入启动子缺失的增强型绿色荧光蛋白表达载体pAcGFP1-1中,得到pAcGFP1-5′ALP、pAcGFP1-Intron1和pAcGFP1-AI报告基因表达载体。通过脂质体介导重组质粒转染中国仓鼠卵巢细胞(CHO)并检测荧光信号。转染后细胞状态良好,24 h后发现,在倒置荧光显微镜下均可看到不同强度的绿色荧光信号,且荧光信号随时间的延长而增强,其中转染pAcGFP1-AI后的荧光信号强度最强。以上结果表明,牙鲆碱性磷酸酶基因5′调控区和第一个内含子序列均具有启动子活性,且第一内含子与5′调控区存在协同效应,两者协同能增强基因的表达。为了解甲状腺激素T3对调控序列启动子活性的调节作用,用不同浓度的T3处理转染的CHO细胞,24 h后发现加入50、75和100 nM的T3都增强了绿色荧光蛋白的表达,而且信号强度随T3浓度的增加而增强。这表明甲状腺激素T3对牙鲆碱性磷酸酶基因调控序列的启动子活性有一定的增强作用。本研究为深入阐明牙鲆碱性磷酸酶基因转录表达的分子机制提供了实验依据。In order to analyze the function of regulation sequences of 5′ regulation region and the first intron of alkaline phosphatase gene（ALP） in Japanese flounder,the two fragments amplified by PCR were inserted into promoterless pAcGFP1-1 and enhanced green fluorescence protein vector to construct combined expression vector pAcGFP1-5′ALP,pAcGFP1-Intron1 and pAcGFP1-AI by using DNA combination.By liposome-mediated plasmid transfection of Chinese hamster ovary cells（CHO）,the fluorescence signals of three combined expression vectors were detected.Results showed that cells were in good condition and different green fluorescence intensities of three combined expression vectors could be detected at 24h after transfection,and the intensity was enhanced with time lasting.Therein transfected pAcGFP1-AI was the strongest signal between these combined expression vectors.So as to understand thyroid hormone T3 regulation of promoter activity of regulation sequences,transfected CHO were treated with different concentrations of T3.Results showed that the fluorescence intensity was enhanced with 50,75 and 100 nM T3 treament.Moreover,it was enhanced with increasing concentration.Results indicated that both of the 5′ regulation region and the first intron of ALP had promoter activity,and 5′ regulatory region and first intron had synergistic effect of enhanced gene expression.In addition,the thyroid hormone T3 also enhanced the promoter activity of regulation sequences of 5′ regulation region and the first intron of ALP in Japanese Flounder.The research provided an experimental basis for further study on molecular mechanism of the expression and regulation of ALP.

关 键 词：牙鲆 碱性磷酸酶 5′调控区 第一内含子 调控序列 甲状腺激素T3 

分 类 号：S917[农业科学—水产科学] Q786[生物学—分子生物学]