gata 5相互作用蛋白的筛选和mir 142 3p在造血过程中的功能研究-screening of gata 5 interacting protein and functional study of mir 1423p in hematopoietic process.docx

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2018-06-04 发布于上海
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gata 5相互作用蛋白的筛选和mir 142 3p在造血过程中的功能研究-screening of gata 5 interacting protein and functional study of mir 1423p in hematopoietic process.docx

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gata 5相互作用蛋白的筛选和mir 142 3p在造血过程中的功能研究-screening of gata 5 interacting protein and functional study of mir 1423p in hematopoietic process

AbstractObjective:WescreentheproteinsinteractingwithGATA-5throughYeastTwo Hybridization,andlayfoundationsforthefurtherresearchofthefunctionandmechanismofGATA-5.Methods:WeextractedthetotalRNAfromthezebrafisheggs (day1andday2)usetheTrizolreagent,andusedthetotalRNAasreversetranscriptivetemplate,CDSIIIandSMARTIIIasprimertosynthesizethefirststrandofcDNA, TheproductswegotwhichpossessedthesamehomologousarmsthroughthelongdistancePCR（LD-PCR）.Atthebaseofthisproperty,wehaveaccomplishedthehomologousrecombinationthroughtransformingtheSMARTcDNA,Sma1linearizedPGADT7-RecplasmidintotheyeaststrainofY187.Meanwhilewetransformed PGBKT7-Rec-GATA-5recombinantplasmidintoyeaststrainofAH109fortestthetranscriptionalactivationandtoxicity,Thenpickedtheyeastclonieswhichgrewon platesviathesetwokindsofyeaststrainmating,Wemadethepreliminaryresearchof genesthanmaybeinteractingwithGATA-5byaseriesofexperimentalmethods（Colony-lift Filter Assay, yeast PCR , Basic Local Alignment Search Tool ）Results:TheSMARTcDNAfromthezebrafisheggshasbeenbuildfinished,afteragaroseelectrophoresis,thecoverageofcDNAsegmentswerefrom250bpto3000bp. Now,wehavescreenedoutfivegenesthatmaybeinteractwithGATA-5.Conclusion: ThediversityandcapacityofthecDNAlibraryfitthescreeningrequirementsand qualifiedenoughforthereseachoftheotherheartdevelopmentrelatedgenes,butthegenesthathavealreadybeenscreenedoutremainingneedtobefurtheridentifiedMicroRNAs(miRNAs)invirusesandeucaryotesareendogenoussmallnon-proteincodingandsinglestrandRNAwhichhave21-23nucleotides．TheycandegradetargetedmRNAsorinhibittheirtranslationbybindingto3‘UTRregionsoftheirtargetedmRNAsaccordingtotheprincipleofcomplementarybasepairing.SofarhundredsofmiRNAgeneshavebeenidentifiedineucaryotesandmanymiRNAsareevolutionarilyconserved.ItissupposedthatmiRNAsregulateupto30%ofthegenesinhumangenome.andplayanimportantroleingrowth,developmentandoccurrenceofdisease．Inrecentyears,modelorganismsextensivelyusedpromotethefootsofscientificresearch.Inthispaper,weusedthezebrafishasatoolforresearcht