Cloning and initial characterization of 5”flanking region and 3” flanking region of the NR gene from Dunaliella Salina
To improve expression of heterologous proteins in transgenic Dunaliella salina(D.salina),highly efficient homologous promoter and terminator are required.In addition,continuous expression of the selectable marker gene can be problematic after stable transformants are screened.Inducible nitrate reductase(NR)promoter may provide a novel method to construct a transformation system of marker-free for D.salina with an ability to switch on or off the expression of the selectable gene.In the present study,a 5”franking region(Pnr) of 1.2kb and a 3”flanking region(Tnr) of 0.9kb were cloned from genome of D.salina by genomic walking LA-PCR and standard PCR,respectively,and then the fragments were ligated to the pMD 18-T vector and characterized.The result of sequencing revealed that the 5”-franking region contained conserved motifs such as CAAT-box,GAGA-box,etc,which are related to regulation of transcription,and the putative binding sites of transcriptional factors such as EBP,EFⅡ,NF-E1,LV,etc.The 3”flanking region harbored the 3UTR and two putative poly(A) signals.After a fusion gene Pnr-EGFP was produced by gene splicing by overlap extension(SOEing),the Pnr-EGFP and Tnr were inserted into plasimid pEGM-7zf orderly to generate a recombinant plasmid p7NET with HindⅢ sites at the two ends of Pnr-EGFP-Tnr expression cassette.After being transformed into D.salina with the plasmid p7NET,EGFP-expressing transformants of D.salina appeared,in which expression of EGFP was induced by nitrate but repressed by ammonium.Besides,PCR and PCR-southern bolts analysis showed that the EGFP gene was integrated into the genome of D.salina.The findings suggest that the Pnr-Tnr of the NR gene can be exploited as an inducible promoter-terminator cassette for the expression of a variety of heterologous genes in transgenic D.salina.
Dunaliella salina nitrate reductase 5”-flanking region 3”-flanking region function
LI Jie XUE Le-xun
Laboratory for Cell Biology, The First Affiliated Hospital,Zhengzhou University, Zhengzhou 450052, China
国内会议
郑州
英文
152-173
2006-06-01（万方平台首次上网日期，不代表论文的发表时间）