| Assimilatory nitrate reductase (NR) is a soluble, multiple center redox enzyme that catalyses the two-electron reduction of nitrate to nitrite using pyridine nucleotide as a electron donor. Transformation systems based on complementing nitrate reductase-deficient mutants have been developed in a number of filamentous fungi and algae. The nitrate assimilation system offers several inherent advantages for transformation, such as high frequencies, ease of recipient isolation through resistance to chlorate, and low background growth after transformation.Dunaliella salinaiD. salina)belongs to Chlorophyta, Chlorophyceae, Volvocales. PPT and antibiotics resistances have been used as selectable markers for nuclear transformation of D. salina. However, low efficiency of transformation limits their application.Here, we reported cloning of cDNA sequence of the NR gene from D. salina and construction of eukaryotic and prokaryotic expression vectors, and expression of the D. salina NR gene in E.coli. The study may lay a solid foundation for the construction of transformation systems based on complementing nitrate reductase-deficient mutants in D. salina.Methods and Results1. Cloning and characterization of the NR cDNA fragment of D. salina 1.1 Cloning and analysis of the NR cDNA fragment of D. salina by PCR with degenerate primersA pair of degenerate primers was designed according to the conserved motifs of homologous amino acid sequences and used to amplify a NR cDNA fragment from total RNA of D.salina by RT PCR. The resulting PCR product was inserted into a T vectorwhich would be transformed into E.coli JM109. The recombinant plasmid detected by amplicillin resistance and blue-white screen was sequenced.Homologous analysis showed that the deduced amino acid sequences shared high homo logy with assimilatory NR of other microalgae.1.2 Amplification of 5' and 3' cDNA fragments of the D. salina NR gene by using 5' and 3' RACE5'RACE and 3' RACE were used to amplify 5' and 3' ends of cDNA fragments of the D. salina NR gene. PCR products each was cloned into the T vector, and then sequenced and blasted against GenBank. The results also showed that each fragment shared high homo logy with the assimilatory NR gene.1.3 Cloning of full-length cDNA sequence of the D. salina NR geneTwo pairs of primers were designed according to the sequence obtained above and used to amplify the long NR 5'cDNA fragment that overlaps the PCR product of 3'RACE by 610bp. The full-length cDNA sequence was achieved by restriction enzyme cleavage and ligation of the two overlapping products.The obtained cDNA was 3696bp long and had an open reading frame encoding 900 amino acids residues. The putative amino acid sequence of conserved domains was identical to the NR gene of other microalgae. Examination of synonymous-codon bias in the NR gene showed that codons with high G+C content were used much more frequently than those with high A+U content. 2. Construction of expression vectors and the prokaryotic expression in E.coli2.1 Construction of eukaryotic expression vector for the NR geneAfter the cloning vector containing full-length NR cDNA sequence was digested with KpnI, the NR cDNA sequence was recovered and then blunted by T4 DNA polymerase. At the same time, a expression vector containing promoter of D. salina carbonic anhydrase, a bar gene and Nos-polyA was digested with SmaI to remove the bar gene from the the vector. Subsequently the NR cDNA fragment was fused into the recovered fragment of the expression vector to form the D. salina eukaryotic expression vector that could be introduced into NR deficient mutants. The correct construction was confirmed by restriction endonuclease analysis.2.2 Construction of prokaryotic expression vector for the NR gene and its expression in E.coliThe full-length NR cDNA sequence purified from recombinant cloning plasmid was inserted into the prokaryotic expression vector pMAL-c2X. SDS-PAGE analysis revealed that the molecular weight of the expressed BMP-NR fusion protein was about 140...
|
PDF Full Text Request