桃多聚半乳糖醛酸酶(PG)基因克隆及反義表達(dá)載體的構(gòu)建.pdf

1、甘肅農(nóng)業(yè)大學(xué)碩士學(xué)位論文桃多聚半乳糖醛酸酶（PG）基因克隆及反義表達(dá)載體的構(gòu)建姓名：陳鑫申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：作物遺傳育種指導(dǎo)教師：李唯20090601甘肅農(nóng)業(yè)大學(xué)2009屆碩士學(xué)位論文SummaryPeachfruit，aclimactericfruit，isverydifficulttobestoragedandtransportedbecauseitisverytobesoftenafterharvested。Thetraddi

2、tionalstorage，whichnotonlyneedhighchargebutalsohavealimitedfreshperiod，CannotsolutethesoftingproblemofpeachfruitultimatelyIthashopetosolvetheproblembytranditionalbreeding，butpeachisperennialplantSObreedingtimeisverylonga

3、ndgermresourcehaslimitationSoitishardtogetbreakthroughinshorttimeThesofteningandmatureofpeachfruitrelatedtOdegradationofalargenumberofpectin，whichmainlyduetotheroleofPolygalacturonase(PG)TosilencetheexpressionofPGwhichle

4、adpeachfruitsoftbyantisensegenetechnologymayincreasethestoragingfunctionofpeachfruitandobtainnewvarietiesofpeachThistechnologyhasbeensucceedintomato，Itlaiddownasolidfoundationandprovidedbeneficialinformationforsolvingthe

5、difficultsoilingproblemofpeachfruitusingantisensegenetechnologyTheobjectiveofthestudywastoobtainthecompletesequenceofPGgeneandconstructtheplantexpressionvectorThestudylaiddownasolidexperimentfoundationforfurtherexplanttr

6、ansformationofpeachvarietieswi也thePGgenetoimprovethequalityofpeachfruitTheresultswereasfallows：(1)TotalRNAwasextractedfrommaturefruitsofBaifenpeachbyTrizol，improvedTrizolmethodTheimprovedTrizolmethodisthebettermethodinth

7、eRNAextractByusingthespecificprimersdesignedfromthePGgeneinGeneBank，afragmentabout1188bpwithanopenreadingframeof393aminoacidswasproducedbyreversetranscriptionandpolymerasechainreactionTheamplifiedfragmentWasclonedintopGE

8、MTvectoranditWasidentifiedbysequenceanalysisafterPCRandrestrictionenzymeTheeDNAnucleotidesequenceanddeducedaminoacidsequencewerehi曲lyhomologoustothatofthereportedpeachPGeDNA，theresultsshowedthatthisfragmentWasPGgenefragm

9、entsofPeach(2)PlasmidpGEMPGandpBI121weredoubledigested謝threstrictionendonucleasesBamHIandSacItoretrievethePGgeneandthelinearvectorwithoutgusgeneThusthelinearpBI121andthePGhadreverselymatchingendsAfterligatingthetwofragme

10、ntsDNA，transformedintoEcobcellsIndentificationofrestrictionmapbyBamHI／SacIdigestionindicatedthat12kbfragmentWasobtainedThus，theintermedialplantexpressionvectorpBIaPGcontainingreversePGgeneWassuccessfullyconstructedItlaid