Small Rna Based Genetic Engineering for Virus-resistant Pota

发布时间：2021-08-03 06:25

　　从人们消费的角度来看,目前马铃薯是仅次于小麦和水稻的第三大作物。中国是世界上最大的马铃薯生产国,其产量9000万吨,几乎占全球马铃薯产量的四分之一,并且其种植面积约占总耕地面积的28%。然而,由于马铃薯是营养繁殖作物,病毒可在植株中世代积累并通过引起块茎内部和表面坏死而影响品质。单个病毒就可使马铃薯潜在总产量损失50%以上,而混合病毒则可损失80%以上。因此,病毒是马铃薯生产中最重要的经济性生物限制因素之一。近年来,大量研究表明全世界感染马铃薯的病毒种类约40种,我国仅发现11种。在这11种病毒中,有6种病毒被认为是马铃薯的主要病毒:PLRV、PVA、PVM、PVS、PVX和PVY。然而,病毒已经发展出各种机制来克服植物的抗性,使得对几乎所有病毒都缺少相应的抗性种质。因此,在本研究中,我们利用双链RNA来启动RNA干扰,以靶向马铃薯病毒六大基因组中的不同保守区域,即PLRV、PVA、PVM、PVX、PVY、PVS。我们以微管和节间作为转化外植体来进行比较。与微管相比,十天龄的节间转化效率和转化频率都较高。因此,利用节间可以降低碳源成本和微管培养成本。并且利用节间进行微繁殖,可使转化保持... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：60 页

【学位级别】：硕士

【文章目录】：
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


【参考文献】：
期刊论文
[1]不同处理方法对马铃薯茎尖成苗率的影响[J]. 于仙萍,陈炜,卞春松,金黎平.  中国马铃薯. 2010(04)
[2]马铃薯主产区病毒病发生情况调查[J]. 张威,白艳菊,高艳玲,申宇,范国权,耿宏伟,孟宪欣.  黑龙江农业科学. 2010(04)
[3]我国马铃薯生产存在的主要问题及对策研究[J]. 李勤志,冯中朝.  长江蔬菜. 2009(18)
[4]应用免疫电镜对几种马铃薯病毒毒源的鉴定研究[J]. 朱光新,郭福寰,肖志敏,李芝芳,周淑琴.  中国马铃薯. 1992(02)



本文编号：3319121