Act-1核心启动子转录的EGFP基因在秀丽隐杆线虫体内的表达

Act-1 core promoter region introduces high-performance transcription of EGFP gene expressed in Caenorhabditis elegans

摘要:

克隆获得的秀丽隐杆线虫(Caenorhabditis elegans)Act-1基因的核心启动子, 经Bgl Ⅱ和Hind Ⅲ限制性内切酶消化后, 与用相同酶消化的pEGFP-4.1载体连接(由pEGFP-N1去掉CMV启动子形成), 构建重组表达载体Pact-EGFP。通过脂质体介导转染Vero细胞, 结果发现EGFP在Vero细胞中有表达, 但表达量很低。通过显微注射将Pact-EGFP与pRF4共注射到C. elegans性腺, 结果发现EGFP能够在C. elegans的皮层、副皮层以及咽部表达, 根据表达部位不同, 获得了2种转基因线虫株。研究结果显示: EGFP在C. elegans体内的表达水平明显高于在Vero细胞内的表达, 表明C. elegans Act-1基因的核心启动子区域可能存在与转录水平密切相关的独特的转录调节元件。该研究为进一步实现寄生性线虫基因在C. elegans表达提供了参考。

Abstract:

To construct the recombinant vector Pact-EGFP, the Act-1 core promoter region was amplified from the pUCm-T/Act-1 and subcloned into pEGFP-4.1 vector (derived from pEGFP-N1 with the removal of human cytomegalovirus immediate early promoter), by restriction enzymes Bgl II and Hind III. Transfection of Pact-EGFP vector into Vero cell by liposome indicated that Act-1 core promoter regulated the expression of EGFP gene in lower level in Vero cells. After Pact-EGFP microinjection into the gonad of Caenorhabditis elegans with pRF4 as a gene marker, green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. According to the locations, two different transgene lines were separated. The expression level of EGFP expressed in C. elegans was more than that in Vero cell. Some unique motifs might exist in Act-1 core promoter region of C. elegans, which was closely related to the expression level of EGFP. These results lay the foundation for the further research on gene function of parasitic nematodes using C. elegans.