摘要
利用双式荧光报告基因检测团头鲂β-actin启动子在家蚕BmN细胞和在家蚕体内的活性。通过脂质体介导法将转基因载体pigA3GFPAβdsRed和辅助质粒(1∶3)混合均匀后转染BmN细胞,48h后,可以检测到同时发绿色荧光和红色荧光的家蚕细胞,表明来自团头鲂的β-actin启动子在家蚕BmN细胞具有活性,能驱动DsRed基因在家蚕BmN细胞中表达。以gfp、dsred的特异引物,可从pigA3GFPAβdsRed转化家蚕BmN细胞基因组内扩增出gfp、dsred相应大小的片段,提示外源基因已经插入到细胞基因组中。随着转化细胞的传代,绿色荧光稳定存在,而红色荧光会逐渐减弱直至检测不到,暗示β-actin启动子在家蚕细胞中容易受到家蚕BmN细胞因子的影响而活性减弱。将转基因载体pigA3GFPAβdsRed和辅助质粒(1∶3)利用精子介导法导入家蚕受精卵,7天后,在部分受精卵中可观察到绿色荧光和红色荧光,进一步证明了β-actin启动子在家蚕体内具有活性。结果显示,含双式荧光报告基因的转基因载体为鉴定β-actin启动子等外源启动子在家蚕中的活性提供了快捷准确的鉴定方法。
In order to explore the β-actin promoter activity of the bluntnose black bream （ Megalobrama amblycephala） in the silkworm BmN cell line and in the silkworm fertilized eggs, the transgenic vector pigA3GFPAβdsRed and the helper vector pigA3 （1：3 ） were mixed to transfect the silkworm BmN cells based on lipofectin transformation technique, 48 hours later, both the green fluorescence and the red fluorescence could be observed in the BmN cells. The result illustrated that the β-actin promoter of bluntnose black bream functioned in the BmN cells. What＇ s more, the gfp, β-actin and dsRed fragments could be amplified from the genome of the transfected BmN cells with the specified primers, which indicated that the exogenous genes have been inserted into the genome of the BmN cells. With the passage of the transformed BmN cells, the red fluorescence would be reduced gradually until undetectable but the green fluorescence maintained stably, suggesting that the activity of β-actin promoter from the bluntnose black bream was weakened because of the effects of cytokine in the BmN cells. In addition, the transgenic vector pigA3GFPAβdsRed and the helper vector pigA3 （ 1：3 ） were transferred into the silkworm fertilized eggs by the sperm-mediated transferring method, 7 days later, both the green and red fluorescence were observed in the silkworm fertilized eggs. The result further proved that the β-actin promoter of the bluntnose black bream was active in the silkworm fertilized eggs.
出处
《江苏蚕业》
2010年第2期1-5,共5页
Jiangsu Sericul Ture
