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《農(nóng)桿菌介導(dǎo)小麥成熟胚愈傷組織的遺傳轉(zhuǎn)化研究.pdf》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫(kù)。
1、麥類作物學(xué)報(bào)2007,27(2):188-192JournalofTriticeaeCrops農(nóng)桿菌介導(dǎo)小麥成熟胚愈傷組織的遺傳轉(zhuǎn)化研究111,211陳立國(guó),后猛,王玉海,崔發(fā),王洪剛(1.山東農(nóng)業(yè)大學(xué)農(nóng)學(xué)院/國(guó)家小麥改良中心泰安分中心,山東泰安271018;2.嶧城區(qū)農(nóng)業(yè)局,山東嶧城277300)摘要:為建立和優(yōu)化小麥成熟胚愈傷組織遺傳轉(zhuǎn)化體系,以篩選出的適合小麥成熟胚組織培養(yǎng)再生的普通小麥山農(nóng)2618為材料,采用農(nóng)桿菌介導(dǎo)法對(duì)其成熟胚愈傷組織進(jìn)行轉(zhuǎn)化,對(duì)影響轉(zhuǎn)化效率的因素如侵染菌液的濃度、侵染時(shí)間、超聲波處理、真空處理等進(jìn)行了研究。結(jié)果表明,這些因素對(duì)小麥成
2、熟胚遺傳轉(zhuǎn)化效率有很大影響。菌液侵染濃度OD600為0.8、侵染時(shí)間為60min時(shí)轉(zhuǎn)化效率最高。在此基礎(chǔ)上輔以超聲波處理、真空處理可明顯提高轉(zhuǎn)化效率,最高轉(zhuǎn)化率可達(dá)8.1%。對(duì)轉(zhuǎn)化愈傷組織和轉(zhuǎn)化植株分別進(jìn)行GUS染色和PCR檢測(cè),結(jié)果表明外源基因已整合到小麥基因組中。關(guān)鍵詞:小麥;農(nóng)桿菌;成熟胚;愈傷組織;遺傳轉(zhuǎn)化中圖分類號(hào):S512.1;S336文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):100921041(2007)0220188205GeneticTransformationthroughMatureSeed2derivedCallusofCommonWheatbyAgrob
3、acteriumTumefaciens111,211CHENLi2guo,KOUMeng,WANGYu2hai,CUIFa,WANGHong2gang(1.AgronomyCollege,ShandongAgriculturalUniversity/TaianSubcenterofNationalWheatImprovementCenter,Taian,Shandong271018,China;2.YichengBureauofAgriculture,Yicheng,Shandong277300,China)Abstract:Agrobacterium2medi
4、atedtransformationofcommonwheatthroughcallusinitiatedfromim2matureembryoshasprovedtobeaneffectiveway.But,obtainingalargenumberofimmatureembry2osoverashortperiodoftimeisalaboriouswork.Toovercomthislimitation,analternativewayofu2singcallusinitiatedfrommatureseedshastobeestablished.Alth
5、oughtherewasalsoaproblemoflowtransformationrate.InthisessaythecommonwheatSN2616wasused,theregenerationofwhichhasbeenestablishedbefore.Celldensity,inoculationtimeandtreatmentsofsonicationandvacuuminfiltrationonthetransformationefficicencybeenassayed.Agrobacterium2mediatedtransformatio
6、nassistedbyacombinationof7.5minsonicationwith7.5minvacuuminfiltrationresultedinthehigh2esttransformationfrequency.TheblueblotshadimprovedtheGUSexpressedinthecallusthroughhistochemicalGUSdetection.AndtheexistenceandintegrationofBG2geneintransgenicplantswereconfirmedbyPCR.Keywords:Whea
7、t;Agrobacterium;Matureembryos;Callus;Genetictransformation近十幾年來(lái),小麥遺傳轉(zhuǎn)化研究取得了長(zhǎng)足抗除草劑基因bar導(dǎo)入小麥,首次獲得了小麥轉(zhuǎn)[1]進(jìn)展。1991年,Vasil等利用基因槍法轉(zhuǎn)化小基因植株。1993年,Vasil等又用基因槍法直接麥懸浮細(xì)胞獲得了轉(zhuǎn)化的愈傷組織,次年,利用同轟擊幼胚,獲得了可育轉(zhuǎn)基因小麥株系,使轉(zhuǎn)化效[2][3]樣的方法轉(zhuǎn)化長(zhǎng)期培養(yǎng)的胚性愈傷組織,將編碼率提高到了1%左右。1994年Becker等、[4]β2葡萄糖苷酸酶(β2glucuronidase)的gus基因和Nehr
8、a等分別利用基因槍法轟擊
