Construction Of A Recombinant Vector For Mortierella Alpina By A Modified Overlap Extension PCR

Mortierella alpina is an oil fungus and saprophytic fungus, which has a unique ability tosynthesize a wide range of polyunsaturated fatty acids, and mainly accumulated ARA. So thisfungus is not only a good model to elucidate the mechanisms of lipid accumulation and PUFAbiosynthesis from fundamental viewpoints, but also used for industrial production of ARA.ω3desaturase could convert n-6PUFAs into n-3PUFAs effectively. In this study, weconstrust a expression vector, and apply it to knockout ω3gene by homologous recombination,making the M. alpina enriched more the ARA.Malic enzyme (ME) carries out decarboxylation of malate to pyruvate with the formationof NADPH from NADP+. It is then this NADPH which is vital for fatty acid biosynthesis. Noother NADPH-generating enzyme appears able to provide the requisite reducing power forfatty acid synthase to funtion. In M. alpina, ME has seven (A-G) distinct isomers and amongthem D and E isomer were found not only have relationship with lipid accumulation, but alsomaybe derived from the same gene. In this study, the cDNA we amplified has one more intronthan the previous studies, which proves that the D and E isomers from the same encodinggene. The sequences we obtained may encode the D-isomer.Overlap extension PCR is a powerful method, which is often used in kinds of experiments,including gene splicing which assembles different DNA fragments into a longer fragmentwith restriction endonuclease free, site-directed mutagenesis which introduce desiredsequences into targeted DNA fragment and PCR-based accurate systhesis of long DNAsequences with DNA template independence. This method has been applied in the research offunctions with promoter or coding gene. In this study, we modified the previous method into amore advanced method which lengthens the primer sequences corresponding to overlappingregion between two fragments such as the ω3aR(1), CBXBF and MECF. And uses successiveannealing in each single PCR cycles of the second PCR process. The annealing ofamplification for the HCT fragment is65℃(1s),60℃(1s),56℃(1s) and50℃(30s).This modified method has higher specificity and sensitivity, and is called successive annealoverlap extension PCR.The promoter and terminator are derived from plasmid pD4. Because of more DNA framents, we spliced part of the small framents into large framents first and used the splicingfragments to constrct vector. The fragments ω3(2), HCT and HMT were connected to thepEasy-Blunt simple cloning vector respectively, after digestion that fragments connected tothe carrier pB2. Owing to the polyT of ME gene, HMT frament fail to ligate to pB2O3CBvector. The vector pB2O3CB we obtained can be used to knockout ω3gene.