農(nóng)桿菌介導棉花胚性愈傷轉(zhuǎn)化體系的建立及轉(zhuǎn)AFP-SPCEMA基因棉花的獲得.pdf

1、西南農(nóng)業(yè)大學碩士學位論文農(nóng)桿菌介導棉花胚性愈傷轉(zhuǎn)化體系的建立及轉(zhuǎn)AFPSPCEMA基因棉花的獲得姓名：魯秀敏申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：裴炎2003.5.1transgeniccottonplantsInthepresentmethod，cottonembryogenicculturesareusedasanidealtargetmaterialforAgrobaaeriummediatedtransforma

2、tionMostoftheKmresistantandGUSpositiveembryogenicculturesregeneratedontheselectionmediumhavetheabilitytodevelopintotransgenicplants，whichincreasingthetransformationefficiencyThestepofinducingtransgenicembryogeniccallus，w

3、hichisapivotalstepintheregenerationoftransgenicplants，iselimitedandtherebytheperiodofregeneratingtransgenicplantsreducesatleast3qmonthsWeintroducedafpspCEMAgeneintothecottongenomewiththeimprovedtransformationsystemThemai

4、nresultsareasfollows：1DevelopmentofefficientAgrobacteriummediatedtransformationsystemofcottonembryogenicculturesThegus／ntrongenewasusedinexperimentstostudythefactorsinfluencingtransformationefficiencybytransientexpressio

5、nAnefficientAgrobacteriummediatedtransformationsystemofcottonembryoganiccultureswasdevelopedTheresearchindicatedthat100I_tmoi／Lacetosyringone(AS)suppliedintheinoculationandcocultivationmediumincreasedthetransientexpressi

6、onefficiencygreatlyforallthe3Agrobaeterinmtumefaeiensstrainsinvestigated(LBA4404，EHAl01andEHAl05)ThesupervimlentstrainEHAl01gavethehighesttransientefficiencyinthepresenceof100Irmol／LASornot，Themaximumtransientexpressionf

7、requencywasobtainedwhenembryogenicculturescocultivedwithAgrobacteriuratumefaciensstrainEHAl01for4daysThecotyledonembryowaseasilyinfectedbyAgrobacteriuratumefaciens2Stabletransformationofinterestgeneandregenerationoftrans

8、genicplantsAfteroptimizingthefactorsinfluencedtransformationfrequencyinterestgenewasintroducedintothecottongenomewithAgrobacteriumOtmefaciensstrainEHAl01inthepresenceof100mol／LASFollowingco—cultivation，theembryogeniccall

9、uswastransferredontoselectionmediumforselectingtransformedcellsAfter7to8weeks，kanamycinresistantculturesformedOiltheselectionmediumandwereverifiedtotransgenicceilsbyGUShistochemicalassayGUSpositivefrequencywerevariedgrea

10、tlyfrom20％90％withdifferentrepetitionAgreatdealofembryosproducedaftertheKmresistantandGUSpositiveembryogeniccallusculturedinliquidMSBfortwoweeksThedevelopedembryosweretransferredtoembryoelongatemediumforpromotingthemelong