Inducible EGFP expression under the control of the nitrate reductase gene promoter in transgenic Dunaliella salina
- 489 Downloads
The upstream region of the nitrate reductase (NR) gene is able to provide an ideal promoter for inducible expression of the heterologous genes because NR transcripts are induced by nitrate and repressed by ammonium. To improve the expression of heterologous proteins in transgenic Dunaliella salina, we developed a novel vector with the ability to switch on or off the expression of the heterologous genes using the upstream region of the NR gene. In the present study, a 1.2-kb fragment containing the promoter (Pnr) and a 0.9-kb fragment containing the terminator (Tnr) of the NR gene were cloned from the genome of D. salina. The results of mobility shift assays indicated that a GATA element in the isolated Pnr was involved in the nitrogen-dependent regulation. Additionally, the transcription start site of the D. salina NR gene was located at 133 bp upstream of the start codon ATG. Gene splicing by overlap extension (SOEing) was employed to construct a recombinant vector, p7NET, containing a Pnr-EGFP-Tnr expression cassette. On day 2 after the p7NET was introduced into the D. salina cells, EGFP-expressing transformants of D. salina appeared, in which expression of EGFP was induced by nitrate but repressed by ammonium. RT-PCR and PCR-Southern blots showed that the EGFP gene was correctly transcribed in transgenic D. salina. The findings of the present study suggest that the promoter of the NR gene from D. salina may be used to drive the inducible expression of heterologous proteins in transgenic D. salina.
KeywordsDunaliella salina Chlorophyceae Enhanced green fluorescent protein (EGFP) Heterologous expression Nitrate reductase Promoter
This study was supported by the National Natural Science Foundation of China (No.30270031).
- Geng DG, Wang YQ, Li WB, Sun YR (2002) Transient expression of gus gene in Dunaliella salina. High Tech Lett 2:35–40Google Scholar
- Jia YL, Hou WH, Li J, Liu HT, Chen HY, Wang JM, Xue LX (2005) Isolation and identification of nitrate reductase deficient mutants of Dunaliella salina. J Zhengzhou Uni 40:245–247Google Scholar
- Jimenez M, Ramazanov Z, Garcia-Reina G (1993) Dark induction of nitrate reductase in the halophilic alga Dunaliella salina. Planta 192:40–45Google Scholar
- Li ZX, SunY, Liu ZX, Song RT, Xu ZK (2005) Transient expression of foreign ble gene in Duanliella salina. Acta Biol Exp Sin 38:411–416Google Scholar
- Li J, Qu DJ, Liu LL, Feng SY, Xue LX (2007) Comparison of stable expressions of foreign genes driven by different promoters in transgenic Dunaliella salina. China Biotech 27:47–53Google Scholar
- Perdomo G, Navarro FJ, Medina B, Machin F, Tejera P, Siverio JM (2002) Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation. Plant Mol Biol 50:405–413PubMedCrossRefGoogle Scholar
- Xie H, Xu PR, Jia YL, Li J, Lu YM, Xue LX (2007) Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina. J Appl Phycol doi: 10.1007/s10811-007-9162-y
- Xu ZQ, Gong LG, Huang X (2004) Transgenic maize plants with low copy number of foreign genes were produced with maize Ubi-1 promoter. Chin J Biotech 20:120–125Google Scholar