Δ-15脱氢酶基因克隆、表达及其活性分析的开题报告.docxVIP

Δ-15脱氢酶基因克隆、表达及其活性分析的开题报告

Introduction:

Delta-15desaturase(D15D)isakeyenzymeinthebiosynthesisofhighlyunsaturatedfattyacids(HUFA)biosynthesis,whichplayssignificantphysiologicalrolesinthegrowthanddevelopmentofaquaticorganisms,especillaymarineinvertebrates.Thus,cloning,expressionandactivityanalysisoftheD15DgeneisimportantonbetterunderstandingsthemetabolismpathwayofHUFAinorganisms.

Objectives:

Theobjectivesofthisstudyistoclone,express,andanalyzetheactivityoftheD15Dgenefrommarineinvertebrates,inordertoexploreitspotentialrolesinHUFAsynthesis.

Methods:

1.CloningofD15Dgene:

TotalRNAwillbeisolatedfromthetissuesamplesofmarineinvertebratesusingTrizolreagent.ThecomplementaryDNA(cDNA)willbesynthesizedbyreversetranscriptionofRNAusingMMLVreversetranscriptase.TheD15DgenewillbeobtainedbyPCRamplificationusinggene-specificprimers.ThePCRproductswillbepurifiedandthenclonedintoexpressionvectors,suchaspET-28aorpcDNA3.1(+),toconstructrecombinantplasmids.

2.ExpressionofD15Dgene:

Therecombinantplasmidswillbetransformedintoexpressionhosts,suchasE.coliorCHOcells,andtheninducedtoexpresstheD15Dprotein.Theexpressedproteinwillbepurifiedusingaffinitychromatography.

3.ActivityanalysisofD15Dprotein:

TheactivityofD15DwillbedeterminedbymeasuringtheconversionofprecursorfattyacidstoHUFAusinggaschromatographyorliquidchromatography-massspectrometry(LC-MS)analysis.Theoptimumenzymeconditions,suchastemperatureandpH,willbedeterminedtomaximizeD15Dactivity.TheeffectsofdifferentsubstratesandinhibitorsontheactivityoftheD15Dwillbeinvestigated.

Expectedresults:

Weexpecttosuccessfullyclone,expressandpurifytheD15Dproteinfrommarineinvertebrates.Theoptimizedconditionsforenzymeactivitywillbeestablished,andthekineticsoftheenzymewillbeexplored,wh