植物脂氧合酶基因是一個多基因家族,它參與植物體內(nèi)茉莉酸等生長調(diào)節(jié)劑的生物合成。植物對逆境脅迫的響應(yīng)及其組織器官的膨大均可誘導(dǎo)多種脂氧合酶基因的表達(dá)。前人研究表明,馬鈴薯脂氧合酶基因POTLX-1參與對馬鈴薯塊莖生長和發(fā)育的調(diào)節(jié),但該基因是否參與馬鈴薯塊莖發(fā)芽和塊莖休眠過程仍不清楚。因此,研究POTLX-1基因在馬鈴薯生長發(fā)育階段尤其是塊莖發(fā)芽過程中的功能對闡明POTLX-1基因功能的多樣性和復(fù)雜性具有重要的意義。為了研究脂氧合酶在馬鈴薯發(fā)芽過程中的功能,本研究構(gòu)建了POTLX-1基因RNA干擾載體,并進(jìn)行了農(nóng)桿菌介導(dǎo)的馬鈴薯莖段遺傳轉(zhuǎn)化實(shí)驗(yàn),同時,使用萘普生（脂氧合酶的抑制劑）和茉莉酸（脂氧合酶途徑的中間產(chǎn)物）,預(yù)測脂氧合酶在馬鈴薯塊莖發(fā)芽和薯塊形成中的功能。獲得的主要實(shí)驗(yàn)結(jié)果如下:1.通過TOPO克隆,將POTLX-1基因干擾片段連入pENTR,成功構(gòu)建入門載體pENTRPOTLX-1。2.根據(jù)GATEWAY CLONING TECHNOLOGY進(jìn)行LR重組,將入門載體pENTR-POTLX-1連入pHELLSGATE12,成功構(gòu)建干擾表達(dá)載體pPOTLX-1-RNAi。3.采用冷融... 

【文章來源】：蘭州理工大學(xué)甘肅省

【文章頁數(shù)】：80 頁

【學(xué)位級別】：碩士

【文章目錄】：
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER1.INTRODUCTION
    1.The function of lipoxygenase gene family in solanaceae plants development
        1.1.Studies on lipoxygenase gene function in solanaceae plants
        1.2.Solanaceae lipoxygenase gene identity and the analysis of motifs organization of lipoxygenase gene system in solanaceae
        1.3.Lipoxygenase pathway and products
        1.4.Lipoxygenase gene family is involved in maturation and development of solanaceae plants
        1.5.Lipoxygenase function in defense and environmental stress of solanaceae plants
        1.6.Previous studies on lipoxygenase(POTLX-1 Gene)in potato sprouting
    2.RNAi technology
        2.1.RNAi mechanism
        2.2.Methods for constructing RNAi vectors
            2.2.1.Constructions of RNAi vector by traditional restriction enzyme ligation
            2.2.2.Gateway cloning methods to construct RNAi vectors
    3.Plant genetic transformation technology
        3.1.Binary agrobacterium tumefaciens vectors
        3.2.Hormone regulates tissue regeneration during potato genetic transformation
        3.3.Antibiotic concentration affects transgenic plants regeneration and agrobacterium elimination.
    4.Naproxen(lipoxygenase inhibitor),JA and methyl jasmonate inhibits sprouting of potato tuber disc and affect tuberization
    5.The objective and significance of this study
    6.Research experimental procedure
CHAPTER2.MATERIALS AND METHODS
    2.1.Experimental methods
        2.1.1 Potato tissue culture
        2.1.2 Extraction of total RNA from the potato tissue
        2.1.3 cDNA was obtained by reverse transcript kit
        2.1.4 Amplification of the interference cloning fragment and RNA template
    2.2.Construction of RNA interference expression vectors for POTLX-1 gene
        2.2.1.Downregulation of POTLX-1 with RNAi
        2.2.2.LR Cloning or LR Reaction(Entry clone(pENTR-POTLX-1) +Destination Vector(pHELLSGATE12) –Expression clone(pPOTLX-1-RNAi)
        2.2.3.E.coli transformation
    2.3.Potato genetic transformation
        2.3.1.Transformation of potato tuber stem with antibacterial genes containing our silenced gene of interest
        2.3.2.Preparation of freeze/thaw-competent agrobacterium cells
        2.3.3.Transformation of agrobacterium through freeze/thaw method
        2.3.4.Freeze-thaw method:p POTLX-1-RNAi vector was transferred to agrobacterium tumefaciens GV
        2.3.5.Studying the transgenic plants
    2.4.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
        2.4.1.Stock solutions preparation
        2.4.2.The tuber disc eyes treatment with naproxen(LOX inhibitor),methyl jasmonate,gibberellic acid
        2.4.3.Atlantic potato stems cultured in MS medium supplemented with jasmonic acid,methyl Jasmonates and naproxen
CHAPTER3.RESULTS AND ANALYSIS
    3.1.Construction of pPOTLX-1-RNAi vector
        3.1.1.Extraction of total RNA from plants
        3.1.2.RNAi interference fragment amplification
        3.1.3.pENTR-POTLX-1 entry Clone vector construction
        3.1.4.Ligation of digested pENTR-POTLX-1 and pHELLSGATE12 carrier vector
        3.1.5.PCR detection of recombinants
        3.1.6.Restriction enzyme analysis of the expression vector
    3.2.Potato transformation
        3.2.1.Detection of pPOTLX-1-RNAi in agrobacterium tumefaciens
        3.2.2 Callus regeneration from the potato stems
    3.3.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
    3.4.Jasmonic acid,methyl jasmonate and naproxen affect tuberization of favorita potato stolons cultured in vitro
CHAPTER4.DISCUSSION AND CONCLUSION
    4.1.DISCUSSION
        4.1.1.Construction of pPOTLX-1-RNAi vector
        4.1.2.Potato transformation
        4.1.3.Preliminary studies about POTLX-1 and what they indicated
        4.1.4.Naproxen(Lipoxygenase Inhibitor)and methyl jasmonate inhibits sprouting of potato tuber disc
        4.1.5.LOX activity as well as JA expression affect tuber initiation in vitro and the tuber skin color
    4.2.CONCLUSION
CHAPTER5.SUMMARY AND FUTURE DIRECTIONS
    5.1.Summary of the results
    5.2.Future directions
REFERENCES
DEDICATION
ACKNOWLEDGEMENTS



本文編號：3351003