45477.athsc701在擬南芥葉綠體磷酸丙糖磷酸轉(zhuǎn)運(yùn)蛋白tpt膜定位中的作用

1、Y938323分類號：密級：單位代碼學(xué)號氣嚼巖≮武等碩士學(xué)位論文10019S030599AtHsc701在擬南芥葉綠體磷酸丙糖／磷酸轉(zhuǎn)運(yùn)蛋白(TPT)膜定位中的作用TheFunctionofAtHsc701During7FriosePhosphateTranslocator(TPT)TargetingtotheInnerMembraneofChloroplastinArabidopsis研究生：塞蘊(yùn)墓指導(dǎo)教師：睦塑熬攫申請學(xué)位fJ類級別

2、：堡堂亟專業(yè)名研究方稱：生毖絲堂：生筮壬生塑堂向：墮量自墨值墨技昱所在學(xué)院：生物堂暄二零零六年六月AbstractProteinimportintochloroplastsisahi曲lyregulatedprocessMostproteinsarecodedbynucleus，andpreproteinsaresynthesizedinthecytos01Ahighlyimport—competentguidancecomplexis

3、formedbytheinteractionofprecursorproteinswithchaperonfactorinthecytosolforthepreciseimportofchloroplastsproteinsintotheorganelleTherefore，adetailedinvestigationontherecognitionandinteractionofatransitpeptideandachaperonf

4、actorissignificantfortheillustrationofmoleculemechanismunderingthepreciselocalizationofprecursorproteins，aswellasforthecomprehensiveunderstandingofthecommunicationandrelationshipsbetweennucleus—cytosol——chloroplastduring

5、translocationprocessofeukaryoticmembraneproteinsTriosephosphmetranslocator(TPT)consistsofhomo—dimerproteinintheinnermembraneofchloroplastAsoneofthemostefficienttranslocatorintheinnermembraneofchloroplast，TPTcatalyzesthee

6、xportoftriosephosphatesandphosphme，aformoffixedcarbon，andimportofphosphatescytosol，functioningasabridgebetweencarbonmetabolismofphotosynthesisandothercytosolicmetabolismBecauseofthismaintransportroleTPTiskeyfactortoregul

7、atethepartitionofassimilatesbetweensot／iceandsinkHowerverbynowthereisneportontheinteractionofpreTPTtransltpeptide(preTPT)andcytosolicchaperonfactor，Inthisstudy，weinvestigatedtheinteractionofHsc701andpreTPTtpinAmbidopsisT

8、hefull—lengthCDSencodingHse70andpreTPTtpwereisolatedfromArabidopsisTheArabidopsisTPTeDNAencodesaprecursorproteinof410aminoacidresiduesAbioinfornmticspredictionshowedthatthelengthoftransitpepfidesis83aminoacidsandanalysis

9、ofhydrophobicityrevealedthat唧has8transmembraneregionsTheprokaryoticexpressionVeCtOrSofpGEX—Hsc70andpGEXtpwereconstructedThefusionproteinsofGSTHsc70andGSTtpwereinducedbyIPTGandpurifiedbyaffinitybindingofGSTSepharosebeads，

10、thenusedtoprepareantiserumagainstrabbit，thetitleofantiserumreachedto1：104and1：105respectivelyFollowingdigestionofGSTHsc70withproteasethrombinandcentrifution，Hsc70waspurified，incubatedinvitrowithGST—SepharosebeadsboundGST

11、tpIntheGSTpulldownassay，a70kDproteinbandwasdetectedbywesternblottingusingantibodyagainstHsc70，indicatinganinteractionbetweenHscT0andpreTPTtpTovalidatethisinteractionofthecandidateproteinHsc70andpreTPTtpinvivo，theyeasttwo

12、hybridformembraneproteinDualmembraneWasperformed，ThebaitvectorofpTFBl一tpandthepreyvectorofpDL2NH70wereconstructedandcotransformedintoDSY1(yeastreporterstrain)PositivecolonieswereselectedonSD／Trp—LeuHismedium，anddetecdedb

13、yIjgalactosidaseactivitytestResultdemonstratedthatDSY一1COtransformedwithpTFBltpandpDL2NHsc70grewonSD／TrpLeuHismedium，andturnedblueinfiveminutes；whereas，Butthenegativecontroldisplayednogrowth，providingevidencethatHsc70int