番茄Gr基因的表达及作用机制研究

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1、中国农业大学博士学位论文番茄Gr基因的表达及作用机制研究姓名：谢远红申请学位级别：博士专业：食品科学指导教师：罗云波;朱本忠20070501AbstractEthyleneisallendogenousplanthormonethatinfluencesmanyaspectsofplantgrowthanddevelopment．ForthecfimaOeficfruit,asthematurehormone,ethyleneisveryimportantforthematureandsenes

2、eenc：eoffruit．Theethyleneresponseisconductedbyethylenesignaltransductionpathwayintheplant．Inthispaper,toperfectthetheoryofethylenesignaltransduction,thetomatomutantGreen-ripe(Gf)wasusedtoinvestigatethefunctionandpattern0fGrgene．Someworkshavebeendonea

3、sfollows：(1)CloningofGrgeneandexpressinginprokaryotic：RT-PCRwasusedtoamplifytheGfgenefromgreenripetomatofruitThePCRproductwagsubclonedintoprokaryoticproteinexpressionvectorspET-30atogeneraterecombinantplasmidsinE．coilBL21．Andtheproteinwasinducedby0．1

4、mM／LIPTGforthreehours．(2)PurificationofGfproteinandpreparationofpolyclonalantFoodies：theGrproteinwaspurifiedbyNi-NTAagarosecolumn．Whentheimidazoleofwashingbufferwas40mM／L,theGfproteinwaspurifiedperfectly．Andthepurificationlevelwasabout96％．Theanti-cas

5、erumwasobtainedbyimmu丑izingrabbits,andthetik口,oftheanti-case／umwasabovel0000．PurifiedbytheDEAE-52ion-column,thehighpurificationlevelofanti-capolyclonalantibodywasobtained．IdentifiedbyWestemblotting,theanti-Capolyclonalantibodyhadspeci丘cimmunizationfu

6、nction．(3)TheexpressionpatternandthelocalizationofCa：tostudytheexpressionpatternofGr缸tomato，andtheinfectionoftheethylene．RT-CPRwasusedintheRNAleveltodemonstratethattheexpressionofGrwagspecialized缸∞lnccultures．ExpressionlevelofGfwashigherinseed,flower

7、andgreenripefruitthanothers,andtheexpressionlevelwerereducedbyethyleneintheflowerandgreenripefrailTheWesternblottingwasusedtoinvestigatetheexpressionpatternofGrinproteinlevel．TheresultsdemonstratedthattheexpressionlevelofGfinproteinlevelwascompiledwi

8、ththeexpressioninRNAlevel．(4)The咖dyofthepattembetweentheGfproteinandethylenereceptorproteinsby’YeastTwo-Hybridassay：bythemethodofYeastTwo-Hybridassay,theOrandtheethylenereceptorgenesweresubclonedintoYeastTwo-HybridvectorpGAD-TTandpGBK-T7respectivelya