绿色荧光蛋白基因的重组构建及在金鱼体内的整合与表达
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摘要
绿色荧光蛋白(Green fluorescent protein，GFP)是来源于多管水母(Aequorea Victoria)等海洋无脊椎动物的一种蛋白质，该蛋白质在体外经适当波长的光激发便发出绿色荧光，并且这种荧光的发射不需要任何底物和辅助因子的诱导。绿色荧光蛋白这种独特的生物学特性，使其作为报道基因在现代生命科学研究领域中，如细胞内基因产物的动态过程，蛋白质在细胞内的定位，药物的筛选，以及转基因个体的初步鉴定等等有着广泛的应用。本研究利用绿色荧光蛋白的这种独特生物学特性，作为转基因个体筛选的标志，来提高转基因个体检测效率，并为进一步研究外源基因在高等动物内整合与表达调控机理提供参考。
实验通过分子重组技术，采用定向克隆法将绿色荧光蛋白基因亚克隆到pUC118A上鲤鱼β-肌动蛋白基因启动子下游，构建成能在真核生物体内表达的表达载体pAGFP，经双酶酶切法序列鉴定后，回收带启动子和目的基因片段。采用显微注射法将这种重组基因转化1细胞期的金鱼受精卵，实验结果如下：
(1)显微注射后，根据胚胎发育分期，胚胎在显微注射后36小时开始能在紫外灯下观察到荧光，原肠期发荧光的胚胎比例为25％，后期发育荧光率逐渐下降，肌肉效应期后又相对稳定。
(2)分别以10~5、10~6、10~7和10~8拷贝数转化金鱼受精卵，结果显示在这四个不同级别当中以10~7转化率最高，说明导入的拷贝数与表达率之间不存在简单的线性相关关系。
(3)随机提取12尾转化个体肌肉总DNA进行PCR和Southern杂交检测。PCR检测结果显示，有5尾转化个体的总DNA能扩增出一条478bp的特异性分子带，说明转化个体整合有GFP基因，整合率为42％。Southern杂交进一步证实，其中有2尾GFP基因完全整合在转化个体的基因组上，另外3尾可能是GFP基因未完全整合到宿主基因组上。对于基因组上整合有GFP基因的2尾转化个体，GFP基因在基因组上的整合位点和拷贝数并不相同，说明GFP基因作为外源基因在转基因个体上的整合位点和拷贝数是非确定性的。
Green fluorescent protein (GFP) was extracted from jellyfish (Aequerea Victoria) of ocean invertebrate. GFP can emit green fluorescent when it is illuminated by light of suitable wavelength. The emissions of fluorescence need not any additional factors, such as substrate, supplementary factor. Green fluorescent protein is widely applied in researches of modern life science, such as gene product moved-process in vivo, protein localization, drug screening and preliminary selection of transgenic individual as a molecular marker. In this study, the distinctive character of green fluorescent protein was using as a screening method to improve determination efficiency of transgenic individuals, and simultaneously to provide reference concerning foreign gene integration and regulation.
In the research of transgenic fish, green fluorescent protein gene was sub-cloned to downstream of carp P -actin gene promoter that was cloned in pUCUSA by molecular recombination technology. Thus pAGFP plasmid was constructed successfully. The recombination was determined by digestion of restriction enzyme and sequencing. Recombination gene was cut-down and introduced into the nuclei of oocytes or the cytoplasm of goldfish at one-cell stage via microinjection. The results as follows:
(1) Fluorescence was observed from embryo under suitable UV light after microinjection 36 hours. The fluorescent ratio of Gastrula embryo period was up to 25%. During developing backward, fluorescent ratio was gradually descending. It was relatively stable after muscle effect period.
(2) Goldfish zygote were transformed by different copies 10\ 10\ 10 and 10" respectively, results showed that foreign copies was not existed simple linear relativity.
(3) Genomic DNA was extracted from 12 preliminary screening fish. The
transgenic goldfish were detected and screened by PCR, Southern blot. The results showed that PCR amplify a 478bp specific molecular band in 5 individuals, Southern blot results showed a strong signal in transgenic fish. It is concluded that ' green fluorescent protein gene has been integrated and expressed in at least 2 transgenic fish, it is also showed that foreign gene integrating positions and copy numbers are differ in different individuals.
实验通过分子重组技术，采用定向克隆法将绿色荧光蛋白基因亚克隆到pUC118A上鲤鱼β-肌动蛋白基因启动子下游，构建成能在真核生物体内表达的表达载体pAGFP，经双酶酶切法序列鉴定后，回收带启动子和目的基因片段。采用显微注射法将这种重组基因转化1细胞期的金鱼受精卵，实验结果如下：
(1)显微注射后，根据胚胎发育分期，胚胎在显微注射后36小时开始能在紫外灯下观察到荧光，原肠期发荧光的胚胎比例为25％，后期发育荧光率逐渐下降，肌肉效应期后又相对稳定。
(2)分别以10~5、10~6、10~7和10~8拷贝数转化金鱼受精卵，结果显示在这四个不同级别当中以10~7转化率最高，说明导入的拷贝数与表达率之间不存在简单的线性相关关系。
(3)随机提取12尾转化个体肌肉总DNA进行PCR和Southern杂交检测。PCR检测结果显示，有5尾转化个体的总DNA能扩增出一条478bp的特异性分子带，说明转化个体整合有GFP基因，整合率为42％。Southern杂交进一步证实，其中有2尾GFP基因完全整合在转化个体的基因组上，另外3尾可能是GFP基因未完全整合到宿主基因组上。对于基因组上整合有GFP基因的2尾转化个体，GFP基因在基因组上的整合位点和拷贝数并不相同，说明GFP基因作为外源基因在转基因个体上的整合位点和拷贝数是非确定性的。
Green fluorescent protein (GFP) was extracted from jellyfish (Aequerea Victoria) of ocean invertebrate. GFP can emit green fluorescent when it is illuminated by light of suitable wavelength. The emissions of fluorescence need not any additional factors, such as substrate, supplementary factor. Green fluorescent protein is widely applied in researches of modern life science, such as gene product moved-process in vivo, protein localization, drug screening and preliminary selection of transgenic individual as a molecular marker. In this study, the distinctive character of green fluorescent protein was using as a screening method to improve determination efficiency of transgenic individuals, and simultaneously to provide reference concerning foreign gene integration and regulation.
In the research of transgenic fish, green fluorescent protein gene was sub-cloned to downstream of carp P -actin gene promoter that was cloned in pUCUSA by molecular recombination technology. Thus pAGFP plasmid was constructed successfully. The recombination was determined by digestion of restriction enzyme and sequencing. Recombination gene was cut-down and introduced into the nuclei of oocytes or the cytoplasm of goldfish at one-cell stage via microinjection. The results as follows:
(1) Fluorescence was observed from embryo under suitable UV light after microinjection 36 hours. The fluorescent ratio of Gastrula embryo period was up to 25%. During developing backward, fluorescent ratio was gradually descending. It was relatively stable after muscle effect period.
(2) Goldfish zygote were transformed by different copies 10\ 10\ 10 and 10" respectively, results showed that foreign copies was not existed simple linear relativity.
(3) Genomic DNA was extracted from 12 preliminary screening fish. The
transgenic goldfish were detected and screened by PCR, Southern blot. The results showed that PCR amplify a 478bp specific molecular band in 5 individuals, Southern blot results showed a strong signal in transgenic fish. It is concluded that ' green fluorescent protein gene has been integrated and expressed in at least 2 transgenic fish, it is also showed that foreign gene integrating positions and copy numbers are differ in different individuals.
引文
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