Cloning and prokaryotic expression of a salt-induced cDNA encoding a chloroplastic fructose-1,6-diphosphate aldolase in Dunaliella salina (Chlorophyta).
A salt-induced fructose-1,6-diphosphate (FDP) aldolase cDNA (DsALDP) in Dunaliella salina was cloned by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis of DsALDP revealed that the 1520 bp cDNA had an open reading frame (ORF) of 327 amino acid residues. BLAST Search showed that DsALDP shared an amino acid identity (73-66%) with AldP in other plants. Alignment with homologues in other plants indicated that all the conserved substrate-specific binding sites could also be found in DsALDP. Phylogenetic analysis further confirmed the deduced amino acid sequence of the D. salina DsALDP gene belonged to the same subfamily to AldP of other green algae. Southern blot analysis suggested possible presence of the D. salina DsALDP gene as a few copies and Northern blot analysis confirmed salt-induced expression pattern at the transcriptional level. A 62 kDa fusion protein generated by adding a Trx-His.tag at the N-terminal of DsALDP was induced by IPTG in Escherichia coli BL21. An improvement of salt tolerance in E. coli expressing DsALDP fusion protein was observed.
Zhang
Xiao-Ning
XN
Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan Research Centre of Gene Diversity and Designed Agriculture, Fudan University, 220 Handan Road, Shanghai 200433, People's Republic of China.
Wang
Hao
H
Qu
Zhi-Cai
ZC
Ye
Ming-Ming
MM
Shen
Da-Leng
DL
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012965:Sodium Chloride; D005634:Fructose-Bisphosphate Aldolase