杜氏盐藻异养表达载体的构建及异养转化株的初步鉴定

陈涛; 刘红涛; 吕鹏举; 薛乐勋

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杜氏盐藻异养表达载体的构建及异养转化株的初步鉴定

首发时间：2008-12-19

陈涛 ¹ 刘红涛 ² 吕鹏举 ² 薛乐勋 ¹

1、郑州大学医学院肿瘤细胞生物学研究室
2、郑州大学医学院肿瘤细胞生物学研究室

摘要：目的：分别构建杜氏盐藻诱导型和组成型异养表达载体，初步筛选并鉴定异养转化藻株。方法：通过RT-PCR从人胎盘组织中克隆并鉴定人红细胞葡萄糖转运基因（Glut1），构建以诱导型双拷贝碳酸酐酶启动子（DCA）驱动Glut1表达的中间载体，和筛选标记bar盒连接形成盐藻诱导型异养表达载体pMDDGN-bar。此外，将pUΩGUS (简称G5)质粒的GUS基因去除，与Glut1基因连接，构建组成型启动子Ubiquitin驱动的异养组成型异养表达载体G5Glut1-bar。通过电击转化法转化盐藻，使Glut1得到表达,经草丁膦PPT筛选表达Glut1的盐藻转化株。提取转化株总RNA，RT-PCR检测目的基因的整合。结果：克隆获得了1479bp的Glut1序列，编码493个氨基酸。电泳检测各酶切结果，Glut1、DCA、NOS、bar盒已依次连接到相应的载体上构建表达载体。此外利用PPT筛选一周后，转化藻株生长良好，而对照野生藻株全部死亡。电泳检测RT-PCR产物表明两株转化株在相应位置（约250bp处）出现了较为特异的扩增条带，Blast同源性分析显示序列与人Glut1基因的同源性为100%。结论：诱导型和组成型启动子驱动的盐藻异养表达载体构建成功，Glut1基因确已整合到盐藻的基因组中，构建的表达载体可用于盐藻中Glut1基因的表达。

关键词：杜氏盐藻表达载体异养葡萄糖转运基因

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Construction of Dunaliella salina heterotrophic expression vector and preliminary identification of heterotrophically transformed algal strains

Chen Tao ¹ Liu Hongtao ¹ Lv Pengju ¹ Xue Lexun ¹

1、Laboratory for Tumor Cell Biology, Zhengzhou University Medical College

Abstract：Aim: To construct Dunaliella salina (D.salina) inducible and constitutive heterotrophic expression vectors and preliminarily identify heterotrophically transformed algal strains. Methods: The human glucose transporter gene (Glut1) was cloned from placenta tissues by RT-PCR technique and sequenced. A D.salina inducible heterotrophic expression vector pMDDGN-bar，including a duplicated carbonic anhydrase (DCA) promotor which is an inducible promotor and a bar selectable marker that can drive expression of the Glut1 gene in D.salina, was constructed. In addition, plasmid pUΩGUS (G5), from which the GUS gene was removed, was ligated with the Glut1 gene and then constructed the heterotrophic expression vector G5Glut1-bar, which contains a constructive Ubiquitin promotor. The two expression vectors were introduced into D.salina for expession of the Glut1 gene by electroporation method, and then screened the transformants with phosphinothricin (PPT). Total RNA of the transformants was extracted and the integration of the target gene (Glut1) was determined by RT-PCR. Results: The cloned Glut1 sequence was 1479 bp and encoded 493 aminno acids. Digestion with corresponding enzyme and agarose gel electrophoresis showed that the Glut1 gene, DCA, NOS and bar-box were ligated into corresponding vectors, respectively, to construct expression vectors. After screening by PPT for one week, the transfomants grew well while wild-type cells died completely. Agarose gel electrophoresis showed that the RT-PCR products of two transformants both have an about 250 bp specific band and the sequence homology was 100% compared with the human Glut1 sequence by BLAST analysis. Conclusions: Inducible and constitutive heterotrophic expression vectors of D.salina was constructed successfully and the Glut1 gene was integrated into the genome of D.salina. Expression vectors above can be used for the expression of the Glut1 gene in D.salina.

Keywords： Dunaliella salina expression vector heterotrophy glucose transporter1(Glut1)

基金：

1. 教育部高等学校博士学科点专项科研基金,国家自然科学基金（20050459007,30600006）

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陈涛，刘红涛，吕鹏举，等. 杜氏盐藻异养表达载体的构建及异养转化株的初步鉴定[EB/OL]. 北京：中国科技论文在线 [2008-12-19]. http://www.paper.edu.cn/releasepaper/content/200812-603.

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论文编号	200812-603
论文题目	杜氏盐藻异养表达载体的构建及异养转化株的初步鉴定
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