四膜虫rDNA表达载体的改造及用于异源基因的高效表达  

Modification of Tetrahymena rDNA Expression Vector Used for Efficient Expression of Exogenous Gene

作　　者：袁冬霞[1] 冯立芳[2] 熊杰[1] 缪炜[1] 

机构地区：[1]中国科学院水生生物研究所,水生生物多样性与保护重点实验室,武汉430072 [2]浙江工商大学食品与生物工程学院,杭州310012

出　　处：《基因组学与应用生物学》2013年第4期548-556,共9页Genomics and Applied Biology

基　　金：中国科学院知识创新工程重要方向项目(KSCX2-EW-G-6-4);湖北省自然科学基金杰出青年项目(ZRZ0251)共同资助

摘　　要：基于四膜虫小核rDNA改造得到的pD5H8载体，在转染进接合生殖时期的四膜虫后能发育成9000～10000拷贝的大核rDNA，因此pD5H8载体是四膜虫异源基因表达的研究热点；但其载体上匮乏合适的克隆位点和无启动子结构阻碍了该载体的进一步发展。本研究在四膜虫启动子HSP70—2和终止子HSP70-I之间，通过稀有酶切位点I-SceI导入细菌绿色荧光蛋白NGFP）这一筛选标记，由此组成的DNA片段（HSP70-25’UTR．I-SceI—HGFP．I—SeeI—HSP70-13’UTR）整合进pD5H8，将之改造成pD5H8-HGFP表达载体。来自真核生物的绿色荧光蛋白（GFP）藉由l-SceI酶切位点一步导入pD5H8一HGFP表达载体后，在四膜虫中大量表达且具有生物学活性。因此，改造后的pD5H8一HGFP表达载体拥有一步法引入异源基因，几乎无酶切位点的限制，有效的筛选标记，高效的异源基因表达能力，以及环境友好、便捷可控等诸多优点，这对于深入开展四膜虫基因功能的过表达研究和开发异源基因在四膜虫这一真核表达系统中的应用奠定了技术基础。Vector ofpD5H8, modified from rDNA of Tetrahymena small nuclear, can develop into rDNA of large nuclear with 9 000-10 000 copies after introducing into conjugation cells, which make it to be an ideal expression vector of exogenous genes. However, both insufficient appropriate clone sites and lacking promoter region hinder the further application of pD5H8. In this study, a bacterium green fluorescence protein gene （HGPF） was inserted between the 5＇ UTR （untranslated regions） of liSP70-2 gene and the 3＇ UTR of liSP70-1 gene, and these three DNA fragment were connected by the rare restriction enzyme site ofI-Sce I. The constructed DNA fragment （HSP70-2 5＇ UTR-I-Sce I -HGFP-I-Sce I -HSP70-1 3＇ UTR） was then integrated into the pD5H8 vector, which became an expression vector ofpD5H8-HGFP. The HGFP fragment ofpD5H8-HGFP vector could be one-step replaced by an eukaryote green fluorescence protein （GFP） gene via restriction enzyme site of I-Sce I, and the recombinant vector was able to express a large number of products with biological activity in Tetrahymena. Therefore, the modified vec- tor ofpD5H8-HGFP had many advantages, such as one-step introduction of exogenous gene, unlimited restriction enzyme sites, effective selection marker, efficient expression level of exogenous gene, environment-friend and con- venient controllable, which will facilitate the research of gene over-expression, and pave the way for wildly applica- tion of exogenous genes expression in Tetrahymena.

关 键 词：四膜虫 RDNA pD5H8 pD5H8-HGFP 异源基因表达 

分 类 号：Q78[生物学—分子生物学]