甜瓜果实八氢番茄红素合成酶基因的克隆、表达分析与遗传转化

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1、甜瓜果实八氢番茄红素合成酶基因的克隆、表达分析及遗传转化导法将正、反义表达载体分别对甜瓜进行遗传转化，已成功获得了Kan抗性的转化苗。关键词：甜瓜；八氢番茄红素合成酶；克隆；HPLC；原核表达；遗传转化2山东农业大学硕士学位论文AbstractMelon(CucumismeloL.)isoneofthemostimportanthorticulturalcropsintheworld,itcontainsalotofcarotenoids,especiallyβ-carotene.β-caroteneistransformedintothevitaminA

2、afterdigestedandabsorbedinthebody,sotheβ-carotenealsoknownastheoriginalvitaminA.Medicalstudieshaveshownthatcarotenoidshaveimportantrolesinenhancinghumanimmunity,preventingcardiovasculardiseaseandcancer.Phytoenesynthase(PSY)isthekeyenzymeinthemeloncarotenoidbiosynthesispathway,andp

3、laysakeyroleinthesynthesisofcarotenoidsinmelon.Toimprovethequalityofmelon,ithasimportantsignificancetostudymelonphytoenesynthaseactivityandits’regulatorymechanismbytheuseofmodernmolecularbiologymethods.Inthisstudy,wemeasuredβ-carotenecontentatdifferentdevelopmentalstagesofmelonfru

4、itbyHPLC.WeclonedthefulllengthofmelonPSYcDNAbyRT-PCR.WealsoanalyzedthePsyexpressioncharacteristicanditsgenetictransformationtomelon.Themainresultsareasfollows:1.β-carotenecontentindifferentgenotypes’muskmelonfruitatdifferentdevelopmentstagesweremeasuredbyHPLC.Theresultsshowedthati

5、nthefruitdevelopmentofthreegenotypes(M01-3,HomokenandJ-01),thereareverylittleβ-carotenecontentinearlymelonfruitdevelopmentandβ-carotenecontentrapidlyincreasedinnearlymaturefruitandfollowedbydeclinedaftermature.OrangeflashHomokenhadthehighestβ-carotenecontentinnearlymaturefruitands

6、ignificantlyhigherthanwhiteflashM01-3andlightgreenflashJ-01.1.PrimersweredesignedaccordingtoPsygeneconservativesequenceinGenBank,A1443bpcDNAwasclonedfromM01-3melonfruitbyRT-PCR,itencoded422aminoacidsandtheregistrationnumberinGenBankisGU361622.Real-timeRT-PCRwasusedtoanalyzePsyexpr

7、essioncharacteristic.TheresultsshowedthatPsyexpressionlevelweredifferentamongroot,stem,leaf,flowerandfruittissues.ThePsyexpressionlevelwasverylowintheearlydevelopmentstageoffruitandreachedthemaximumatnearlymaturefalloweddecreasedaftermature.TheexpressiontrendofPsywasconsistwiththe

8、trendofβ-carotenecontentinthedeve