δ15脱氢酶基因克隆、表达与其活性分析

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1、AbstractPolyunsaturatefattyacids(PUFAs)arevitaltothehealthofhumanbyplayingimportantrolesinpreventionandtreatmentofseveraldiseases，suchas，cardiovasculardisease．Alpha-linolenicacid(ALA)astheoneofthe心3PUFAsalsoplaysimportantroleinthehealthofhuman,themoreimportantroleisthatALAistheprecur

2、sorofthelongchainPUFAs,suchas，eicosapentaenoieacid(EPA)anddocosahexenoicacid(DHA)．TwoofthemicroalgaesChlamydomonasreinhardtiiandMonodussubterraneusarefullofPUFAs，especiallyco-3PUFAs．Delta-15fattyaciddesaturaseisthekeyenzymeofthebiosynthesisofALA．whichin订oducesadoublebendbetweentheexi

3、stingdoublebendandcarboxylterminusattheA-15positionoffattyacylchainoflinoleicacid．Inthisstudy,thewholelengthgeneofthe△一15fattyaciddesaturasefromCreinhardtiiandArabidopsisthalianawereclonedwiththemethodofRT-PCR,andthebiologicalfunctionofthegeneswereverifiedviathetransformantsofPichiap

4、astorisandtobacco．Atthesametime，thetranscriptomeofM．subterraneuswassequencedandanalyzedwithbioinformaticssoftwaretofindthenewgenesofthebiosynthesisofPUFAs．TIlemainresultsareasfollows：TotalRNAoftheexponentialphasescellofCreinhardtiiandthefreshleafofA．thalianawereextracetedandpurified,

5、thegeneswereclonedwiththemethodofRT-PCR,andsubclonedinthepMDl9-Tvector,thenwassequenced．ThenucleotidesequenceswereanalyzedwithBlastintheNCBI．TIlefulllengthofthegeneofA-15fattyaciddesaturasefromCreinhardtiiwas1,362bp，encoding419aminoacidreciduesjandthatfromA．thalianawas1,525bp，encodin

6、g447aminoacidrecidues．Bothofthe△·15fattyaciddesaturasegeneswereinsertedintoPichiapastorisexpressionvectorpPIC3．5Ktogeneraterespectivelytherecombinantplasmidp3．5K·Ldl5andp3．5K-Ndl5，whichweresubsequentlytransformedintoPichiapastorisstrainGS15forheterologousexpressionbyelectropomtionmet

7、hod．AfterPCRdetection,totalfattyacidsofPichiapastoriswereextractedfromtheinducedcellsandmethylated．11他resultantfattyacidsmethylesterswereanalyzedwithgaschromatography(GC)，theresultsshowedthatA一15fattyaciddesatumsegenewereexpressedinthePichiapastor妇．ComparedwiththetransformantofpPIC3．

8、5K。theconten