Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga Dunaliella salina.
Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.
Wang
Tian-yun
TY
Department of Biochemistry and Molecular Biology, Xinxiang, Medical University, Jinsui Road, Xinxiang 453003, Henan, China. wty@xxmu.edu.cn
Jing
Chang-Qin
CQ
Dong
Wei-Hua
WH
Zhang
Jun-He
JH
Zhang
Yu
Y
D048948:14-3-3 Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D000460:Chlorophyta / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D020869:Gene Expression Profiling; D009711:Nucleotides / Q000032:analysis; D011817:Rabbits; D055049:Salt Tolerance / Q000235:genetics; D017422:Sequence Analysis, DNA
D048948:14-3-3 Proteins; D009711:Nucleotides