留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码
王影, 范金凤, 张晴, 陈瑛. 浮萍棘尾虫Adss基因克隆及表达载体构建[J]. 水生生物学报, 2022, 46(5): 754-760. DOI: 10.7541/2022.2021.0204
引用本文: 王影, 范金凤, 张晴, 陈瑛. 浮萍棘尾虫Adss基因克隆及表达载体构建[J]. 水生生物学报, 2022, 46(5): 754-760. DOI: 10.7541/2022.2021.0204
WANG Ying, FAN Jin-Feng, ZHANG Qing, CHEN Ying. CLONING AND SEQUENCE ANALYSIS OF STYLONYCHIA LEMNAE ADSS GENE AND CONSTRUCTION OF EXPRESSION VECTOR[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(5): 754-760. DOI: 10.7541/2022.2021.0204
Citation: WANG Ying, FAN Jin-Feng, ZHANG Qing, CHEN Ying. CLONING AND SEQUENCE ANALYSIS OF STYLONYCHIA LEMNAE ADSS GENE AND CONSTRUCTION OF EXPRESSION VECTOR[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(5): 754-760. DOI: 10.7541/2022.2021.0204

浮萍棘尾虫Adss基因克隆及表达载体构建

CLONING AND SEQUENCE ANALYSIS OF STYLONYCHIA LEMNAE ADSS GENE AND CONSTRUCTION OF EXPRESSION VECTOR

  • 摘要: 为研究腺苷酸基琥珀酸合成酶(Adenylosuccinate synthase, 简称Adss)在原生动物中的作用, 对浮萍棘尾虫(Stylonychia lemnae)腺苷酸基琥珀酸合成酶Adss基因进行PCR克隆, 获得全长为1396 bp左右的序列, 其编码458个氨基酸, 保守结构域含有一个P-loop NTPase结构域, 蛋白结构预测Adss蛋白是由α-螺旋、β-折叠和无规卷曲组成的复杂结构。多重序列比对和系统进化分析显示浮萍棘尾虫Adss蛋白与第四双小核草履虫、多子小瓜虫和嗜热四膜虫等纤毛虫同源性较高。通过克隆浮萍棘尾虫Adss基因的启动子序列, 并采用增强型绿色荧光蛋白EGFP作为报告基因, 构建了启动子序列调控的表达载体pEGFP-N1-Adss, 通过转染细胞确定浮萍棘尾虫Adss蛋白定位于细胞质中。文章报道了原生动物纤毛虫Adss基因, 证实浮萍棘尾虫Adss蛋白属于典型的真核生物Adss, 为进一步揭示Adss在单细胞真核生物中的作用机制提供了新的参考。

     

    Abstract: Adenylosuccinate synthase (Adss) is an essential enzyme in the purine biosynthesis process. Since it was found in Escherichia coli in 1956, Adss has been reported about 30000 different gene sequences in NCBI. It was found in various species from bacteria to mammals, reflects a high level of genetic diversity. This gene plays an important role in the metabolism of nucleotide cycles. Therefore, it has been used as a target molecule for the researches of various drugs. In the study of the mechanism of hEGF action on asexual reproduction of Stylonychia lemnae, an unicellular animal model, Adss gene was found to be possibly involved in the regulatory process of cell division and cell cycle. In order to further develop the functional and molecular mechanism of Adss gene in protozoa, we cloned the Adss gene of Stylonychia lemnae by PCR. The sequence structure, basic physical and chemical properties and conserved domain of the protein were analyzed. The ML phylogenetic tree based on Adss amino acid sequences of different organisms was illustrated. Furthermore, the expression and localization vector of Adss gene was constructed by double enzyme digestion of Adss cDNA and pEGFP-N1 vector to confirm the subcellular localization of Adss protein with fluorescence staining. The results showed that the total length of the S. lemnae Adss gene is about 1396 bp, encoding 458 amino acids. Its conserved domain containes a P-loop NTPase. Tertiary structure prediction suggested that Adss protein is composed of α-helix, β-sheet and random coil. The Adss gene of S. lemnae has high homology and close genetic relationship with Paramecium tetraurelia, Ichthyophthirius multifiliis and Tetrahymena thermophila according to multiple sequence alignment and phylogenetic analysis. With a promoter sequence-regulated expression vector pEGFP-N1-Adss, the S. lemnae Adss protein mainly distribute in the cytoplasm. These results provide many valuable data and basics for the research on the function and molecular mechanism of protozoan Adss gene.

     

/

返回文章
返回