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《白樺抗蟲基因遺傳轉(zhuǎn)化研究及轉(zhuǎn)基因植株的抗蟲性》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、摘要應(yīng)用農(nóng)桿菌介導(dǎo)法向白樺(BetulaplatyphyllaSuk)基因組中導(dǎo)入抗蟲基因(蜘蛛殺蟲肽與Bt基因C肽序列連接在一起表達的一種融合蛋白基因)，并首次獲得抗蟲轉(zhuǎn)基田植株。為白樺的遺傳改良與高新技術(shù)育種開辟了一條新途徑，也為其他林木樹種的遺傳改良提供了理論依據(jù)。利用雙元載體的根癌農(nóng)桿菌LBA4404菌株(Agrobacteriummmefaciens)，食質(zhì)粒pYHY(目的基因及nptll、gus基困)，對白樺試管苗莖段，葉柄，葉片三種外植體進行侵染，結(jié)果表明：大葉片生氏勢強，為轉(zhuǎn)基因的最優(yōu)外植體，轉(zhuǎn)化率能夠達到22％。轉(zhuǎn)基因}鄖霉素抗性植株
2、中，GUS表達陽性率為43％，隨機抽樣的方法進行殺蟲肽和nptll基岡的PCR檢測陽性率均為100％。f經(jīng)過分子生物學(xué)檢測表明，Southern斑點雜交765％植株表現(xiàn)陽性。轉(zhuǎn)基因植目的基因(蜘蛛殺蟲肽+Bt基因的C肽)的PCRSouthern雜交陽性率為87．5％。經(jīng)過Southern印跡雜交47．2％的GUS陽性苗雜交呈陽性，說明外源基因已穩(wěn)定整合到白樺基闋組中，多次繼代后，仍很穩(wěn)定。Northern斑點雜交表明，外源基因在轉(zhuǎn)基因植株中正常表達／o轉(zhuǎn)基因植株的殺蟲實驗結(jié)果表明，轉(zhuǎn)基因植株的殺蟲率能達到30％以上。舞毒蛾幼蟲發(fā)育推遲，15犬時處理為4
3、齡，但對照仍為2齡，發(fā)育推遲了2個齡期；舞毒蛾生長受到抑制，對照的體重為處理的9．7倍與6．4倍，外源基因嚴重抑制幼蟲的生長，說明外源基岡在轉(zhuǎn)基因白樺中能夠正常表達，轉(zhuǎn)基因植株表現(xiàn)一定的抗蟲性。關(guān)鍵詞自樺遺傳轉(zhuǎn)化分子雜交抗蟲性StudiesonInsectResistantGeneTransformationofBetulaPlatyphyllaandtheInsectResistanceofTransgenicPlantsAbstractThepaperfirstreportedthattransgenicplantsofBetullaPlatyph
4、yllawereobtainedbyAgrobacteriummediatedmethodinChina．ItreplenishedtheblanknessofBirchgenetictransformation，andprovidedanewpathforgeneticimprovementinthisspecies．ThematerialsasexplantintransformationcomefromBirchlear,stemsegmentandleafstalk,andtheSpidertoxingenewasusedasforeignge
5、neforthistransformationexperiment．ItshowedthatthebestexplantwasthebigIeaf,onwhichthetransformationfrequencywas22％．ByGUSdetection．therewere43percentoftheplantswithkanamycinresistance，and100percentofpositiveresult，byPCRamplification，wasgottenfromrandomsampling．Itindicatedthat76．5p
6、ercentofpositivefrequencyofalltheplantswasachievedbyusingSoutherndothybridization，87．5percentoftheplantswithGUSpositiveactivitywasgottenbyPCRsouthernblottingand42percentoftheplantswithGUSpositiveactivitywasgottenbysouthernblotting．Itmeansthatinsectresistantgenewastransferredandi
7、ntegratedintogenomeoftheplants．Bynortherndotblotting，italsoshowedthattheforeigngenecouldexpressinthetransgenicplants．TheinsectfeedingtestindicatedthatthedevelopmentofLymantriadisparwasclearlyputoff,inwhichtheinsectsfeededtransgenicplantsWere2ages，whentheinsectsasthecontrolwere4a
8、gesaftertreating15days．Besides，theinsectgrowthw
