ABSTRACT
Aim:
Clone and characterize of the 5′- flanking region of the
nitrate reductase (NR)
gene derived from Dunaliella salina(D. salina).
Methods:
The genomic
DNA from D. salina was respectively digested with BamHI, EcoRI, HindIII, Pst I, Sal I and Xba I. A genomic
walking cassette was ligated to the ends of the digested
DNA fragments, and then genomic
walking libraries comprising BL, EL, HL, PL, SL and XL were constsucted. The 5′- flanking region of the NR
gene from genomic
walking libraries of D. salina was amplified by LA-
PCR. The
DNA sequences were analyzed with the
software - Promoter Predictions. Isolated 5′-flanking regions fused to the GUS
gene were tested for
transient expression in the alga.
Results:
A single specific
PCR product of about 1200bp in length from the HL
library was generated. Also, several conserved motifs, such as CAAT-box, GAGA-box were found, which are related to
regulation of transcription, and the putative
binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the
DNA sequences shared high homology with 5′-
upstream region of the NR
gene from Dunaliella viridis. The isolated 5′-flanking regions were able to strongly
drive GUS
reporter gene expression, suggesting that it contains the promoter
elements necessary for the transcription of the NR
gene. The expression pattern of the GUS
gene and the NR
gene were
similar, both ware induced by
nitrate and repressed by
ammonium.
Conclusion:
The cloned 5′- flanking sequences of NR
gene derived from D. salina might be a specific promoter with the
ability to“switch on or off” an expression of the heterologous
gene in transgenic D. salina.
