30684.擬南芥micrornas序列特征分析及人工合成amirnagfp載體構(gòu)建

1、石河子大學(xué)碩士學(xué)位論文擬南芥micrNAs序列特征分析及人工合成amiRNAGFP載體構(gòu)建姓名：劉沖申請學(xué)位級別：碩士專業(yè)：作物遺傳育種指導(dǎo)教師：孫杰王磊20090601AbstractRNAinterference(RNAi)isallefficientgenesilencingtechnologyItisanaturalprocessbywhichdoublestrandedRNAdirectssequencespecificpos

2、ttranscriptionalsilencingofhomologousgenesRNAicallbetriggeredbysmallRNAmoleculessuch鷦siRNAandmiRNAmiRNAsilencesgeneexpressioninasequence—specificmannerandthroughtheactionsofsmallpiecesofsinglestrandedRNAsmicroRNAs(miRNAs

3、)areendogenouslyexpressedsmallssRNAsequencesof～22nucleotideinlength，whichnaturallydirectgenesilencingthroughcomponentssharedwiththeRNAipathwayThematuremiRNAscanregulategeneexpressionbymRNAcleavageortranslationalrepressio

4、nComparedwithsiRNAmiRNAalsoplaysamoree仃icientrepressionrolebecauseoftheflexibilityofmiRNAinbindingwithpartiallycomplementarymI酬AtargetsRecentlyalargenumberofstudiesshowthatamiRNAwithmicroRNAexpressionframecanbeintroduced

5、intocellsandinhibittheexpressionoftargetgenesWeexaminedthesequencecharacteristicsofa11knownArabidopsismiRNAsandthebasepairingrulesbetweenthemiRNAsandtheirtargetsTheamiRNAGFPexpressionplasmidswhichtargetedGUSGFPgenesitesw

6、asconstructedWhetheramiRNAGFPcouldmediatedRNAinterferenceandthereforeblockreplicationofGUS—GFPmRNAorproteingeneexpressionweredetectedThemainresultswereasfollows：1W|eexaminedthesequencecharacteristicsofallknownArabidopsis

7、miRNAsandfoundthat：themiRNAsequencesdisplayed5’instabilitywithhigherU。Acontentatthe5’end，andhigherUcontentatposition1andhigherCcontentatthe3’endaroundposition192ThemiRNAwerefurtherscreenedbasedonthestatisticanalysisofbas

8、emismatchesbetweenmicroRNAsandtheirtargets：(1)onlyfivemiRNAcouldmatchthetargetgenecompletelyinthe254sequence，about18％：42withonebasepairmismatch，about5％；68withtwobasemismatch，about24％；92withthreebasemismatch，about37％；41wi

9、thfoDrbasemismatch，aboutl4％；(2)mismatchesoccurfrequentlyatpositionl，2or21，respectively583％and43％butalmostneveroccuratposition3616—173RTPCRandGUSactivitywereperformedtodetermineGUS—GFPRNAandproteinlevel，theresultsshowthat

10、theamiRNAGFPplasmidhadobviouseffect72hafterposttransfectioncomparedwiththecontr01theGUS—GFPmRNAWasinhibitedby50％andtheGUS—GFPproteinreducedby556％4AmiRNAsequencewereintroducedtothemiRNAl69precursorbythemethodofdigestionOu