從人們消費的角度來看,目前馬鈴薯是僅次于小麥和水稻的第三大作物。中國是世界上最大的馬鈴薯生產(chǎn)國,其產(chǎn)量9000萬噸,幾乎占全球馬鈴薯產(chǎn)量的四分之一,并且其種植面積約占總耕地面積的28%。然而,由于馬鈴薯是營養(yǎng)繁殖作物,病毒可在植株中世代積累并通過引起塊莖內(nèi)部和表面壞死而影響品質(zhì)。單個病毒就可使馬鈴薯潛在總產(chǎn)量損失50%以上,而混合病毒則可損失80%以上。因此,病毒是馬鈴薯生產(chǎn)中最重要的經(jīng)濟(jì)性生物限制因素之一。近年來,大量研究表明全世界感染馬鈴薯的病毒種類約40種,我國僅發(fā)現(xiàn)11種。在這11種病毒中,有6種病毒被認(rèn)為是馬鈴薯的主要病毒:PLRV、PVA、PVM、PVS、PVX和PVY。然而,病毒已經(jīng)發(fā)展出各種機(jī)制來克服植物的抗性,使得對幾乎所有病毒都缺少相應(yīng)的抗性種質(zhì)。因此,在本研究中,我們利用雙鏈RNA來啟動RNA干擾,以靶向馬鈴薯病毒六大基因組中的不同保守區(qū)域,即PLRV、PVA、PVM、PVX、PVY、PVS。我們以微管和節(jié)間作為轉(zhuǎn)化外植體來進(jìn)行比較。與微管相比,十天齡的節(jié)間轉(zhuǎn)化效率和轉(zhuǎn)化頻率都較高。因此,利用節(jié)間可以降低碳源成本和微管培養(yǎng)成本。并且利用節(jié)間進(jìn)行微繁殖,可使轉(zhuǎn)化保持... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：60 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
Abstract
VI.List of Abbreviations
1.Introduction
    1.1.The current situation of potato in food security
    1.2.The impact of virus diseases
    1.3.The six major potato viruses
    1.4.Traditional methods in use against potato viruses
        1.4.1.The cultural practices and The clean seed systems
        1.4.2.The host plant resistance
    1.5.Genetic transformation as a tool for developing virus-resistance
    1.6.The use of RNA interference(RNAi)as antivirus strategy
2.Materials and Methods
    2.1.Plant material
        2.1.1.Micropropagation
        2.1.2.Microtuberization
    2.2.Generation of transgenic plants based on Agrobacterium-mediated transformation
        2.2.1.Agrobacterium transfection
        2.2.2.Agrobacterium preparation
        2.2.3.Explant preparation
        2.2.4.Agrobacterium infection and co-cultivation
        2.2.5.Transformant selection and shoot initiation
    2.3.Molecular detection of putative transgene
        2.3.1.Extract genomic DNA using Shorty buffer method
        2.3.2.PCR detection
    2.4.Field growing transgenic plants assay
        2.4.1.Virus inoculation test
    2.5.Resistance analysis of the generated plants in the greenhouse
        2.5.1.Triple Antibody Sandwich (TAS) ELISA
3.Results and Analyses
    3.1.Transformation of Agrobacterium tumefaciens LB4404
    3.2.Production of the Explants
    3.3.Generation of Transformed AC142-01 plants
    3.4.Molecular screening and Resistance analysis of AC142-01 transgenic lines
        3.4.1.Analysis of AC142-01 transformants for PLRV virus
        3.4.2.Analysis of AC142-01 transformants for PVA virus
        3.4.3.Analysis of AC142-01 transformants for PVM virus
        3.4.4.Analysis of AC142-01 transformants for PVS virus
        3.4.5.Analysis of AC142-01 transformants for PVX virus
        3.4.6.Analysis of AC142-01 transformants for PVY virus
        3.4.7.Analysis of AC142-01 transformants for multiple viruses PLRV,PVA,PVX,PVY,PVM,PVS
4.Discussion
    RNA interference pathway between natural exist and enhancement
    Agrobacterium-mediated transformation
    Problems affecting transformation efficiency and safety issues
5.References
IX.Appendices Ⅰ Materials Reagents
IX.Appendices Ⅱ Vector Constructs
X.Acknowledgements


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期刊論文
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