Cloning and molecular characterization of delta12-fatty acid desaturase gene from Mortierella isabellina.
OBJECTIVE
To clone delta(12)-fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.


METHODS
Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of delta(12)-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli (E.coli) strain BL21 using CaCl(2) method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1.


RESULTS
Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of delta(12)-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active delta(12)-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.


CONCLUSIONS
Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.
Li
Ming-Chun
MC
Department of Microbiology, The College of Life Science, Nankai University, Tianjin, China.
Li
Hang
H
Wei
Dong-Sheng
DS
Xing
Lai-Jun
LJ
D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors; D020094:Mortierella / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013696:Temperature
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase