SOEing法构建EGFP真核表达载体及其在杜氏盐藻中的表达  

CONSTRUCTION OF AN EGFP EUKARYOTIC EXPRESSION VECTOR BY SOEING AND ITS TRANSFORMATION TO DUNALIELLA SALINA

作　　者：李杰[1] 闫红霞[1] 曲东京[1] 刘红涛[1] 鲁照明[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室郑州大学第一附属医院,郑州450052

出　　处：《水生生物学报》2007年第4期546-551,共6页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金资助项目(30270031)资助

摘　　要：通过重叠区扩增基因拼接法(Gene splicing by overlap extension,SOEing)构建含有杜氏盐藻(Dunaliella salina)硝酸盐还原酶(NR)基因5′-上游序列(Pnr)and 3′-端序列(Tnr)的EGFP真核表达载体,并将其转化杜氏盐藻。利用改进的SOEing法,将杜氏盐藻NR基因Pnr与报告基因EGFP cDNA融合,并与pEGM-7zf克隆载体连接,顺序将盐藻NR基因Tnr序列与融合片段相连,构建含Pnr-EGFP-Tnr表达盒的盐藻真核表达载体p7NET。电击法转化杜氏盐藻,在盐藻转化株中观察到了EGFP的瞬时表达。此研究为转基因杜氏盐藻研究和成功建立杜氏盐藻生物反应器奠定了实验基础。Previous studies, including some from our own laboratory, have demonstrated that Dunaliella salina （ D. salina ） was one of the most extremely halotolerant eukaryotes and able to live in a variety of salt concentrations ranging from 0.05 to 5M solution of sodium chloride. The simple and cheap culture of D. salina makes it have a great potential in bioengineering for producing valuable polypeptides and proteins. However, lack of efficient expression systems has been one of major limitations in the genetic manipulation of this microalga. In order to produce high levels of heterologous proteins in transgenic D. salina, it is necessary to obtain a high-efficiently endogenous promoter for expressions of the heterologous genes under controlled conditions. We investigated whether 5＇-flanking region （Pnr） and 3＇-flanking region （Tnr） of D. salina nitrate reductase （NR） would control expression of the heterologous genes. Gene splicing by overlap extension （SOEing） was modified according to following steps： （1） synthesis of two individual DNA fragments of interest with 36 bp overlap by PCR with high-fidelity Pyrobest DNA polymerase; （2） six cycles of pre-extension without flanking primers after mixing the two fragments above, which ensures the overlap extension between the two individual templates; （3） 25 cycles of post-extension with flanking primers after cooling the product of preextension. An enhanced green fluorescence protein（EGFP） eukaryotic expression vector harboring Pnr-Tnr cassette was constructed based on the method of SOEing and transformed into D. salina using electroporation. Using the modified SOEing PCR, a special fusion fragment Pnr-EGFP was obtained with the equal molar Pnr and EGFP cDNA from the first stage PCRs being templates, and a recombinant expression vector p7NET containing Pnr-EGFP-Tnr expression cassette was constructed by inserting Pnr-EGFP and Tnr, which was obtained from the genome of D. salina by PCR, into vector pEGM-7zf. The resulting recombinant

关 键 词：杜氏盐藻 重叠区扩增基因拼接法(SOEing) 增强型绿色荧光蛋白(EGFP) 真核表达载体 

分 类 号：Q754[生物学—分子生物学]