AIM： To clone △^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 use...AIM： To clone △^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 △^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 CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 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 IN- VSc1.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 △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^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.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30200167 Tianjin Natural Science Foundation, No. 013802511
文摘AIM： To clone △^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 △^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 CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 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 IN- VSc1.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 △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^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.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.
