Sequence Analysising And Effect On Arachidonic Acid Production Of △5-, △6- Desaturase Gene In M.Isabellina

Arachidonic acid is an essential dietary component for human beings, and has various physiological functions and plays an important role in infant nutrition..It is a new way on preparation arachidonic acid by microbial fermentation. Which can reduce production costs and increase productivity. Arachidonic acid synthesis is based on dehydrogenation and carbon-chain extension of fatty acid.â–³5 -,â–³6 -desaturase is the key enzyme of arachidonic acid synthetic pathway. Evaluating the effect of desaturases on arachidonic acid synthesis and studying the relation of ARA production with activity of desaturase and relation of gene expressed quantity can further establish its basis on high-yield strains of arachidonic acid by metabolic engineering.Full DNA of Mortierlla isabellina AS3.3410,W35s2-153,Z80s2-109,Y-69,YZ-124 5 strainâ–³5-,â–³6 -desaturase gene was obtained by PCR and gene clone based on the original strain of Mortierlla isabellina and the mutant strain of high-yield ARA and its sequence was analyzed. The result showed that the full-length DNA sequence of 5 strain Mortierlla isabellinaâ–³6-desaturase gene was 1374bp and 457 amino acids were coded.â–³5-desaturase gene was 1341bp and 446 amino acids were coded.With biological information software, DNA and amino acid sequence by homology comparison can be found that theâ–³5-,â–³6 -desaturase gene sequence of Mortierlla isabellina AS3.3410 original strain had higher homology with Genebank published sequence, while nucleotide sequences of the mutant strain had low variation. Animo acid sequences of desaturase gene of the mutant strain had lower homology with original strain sequence, which can show the protein of mutant strain probable had a certain variation. The activity of 5 strain Mortierlla isabellina at the same culture time and high-yield ARA strain YZ-124 at the different culture time was determined by TTC-desaturase activity determining method and the experimental condition was established by using fluorescence quantitative PCR method to determine the mRNA expression levels of Mortierlla isabellinaâ–³5 -,â–³6-desaturase gene. The results showed that the desaturase activity of different strain and different cultured time was different and the desaturase activity of original strain AS3.3410 was lowest, the mutant strain YZ-124 being highest. The desaturase activity of 3-5d cultures increased obviously and which 5 d later was stable basicly.The result of fluorescence quantitative PCR testing showed the mRNA expression level of different strainâ–³5 -,â–³6 -desaturase gene was different.The desaturase gene mRNA expression quantity of Mortierlla isabellina YZ-124 at different cultured time was different,which theâ–³5,â–³6-desaturase mRNA expression quantity of 3-8d cultures gradually increased with bacterium age increasing, the expression quantity reaching the highest at 8d.The activity and expression levels of desaturase and ARA quantity in cultured oil presented a positive correlation with ARA acquisition.