世界上包括中國和巴基斯坦在內(nèi)的50多個國家都有棉花種植。由于其具有重要的經(jīng)濟價值,棉花被喻為“白金”。但是,棉花的生長過程中會面臨許多問題,如蟲害,雜草和CLCuD,特別是在巴基斯坦,這些情況尤為嚴(yán)重。蟲害可以被殺蟲劑防治,而由CLCuD引起的曲葉病卻仍然無法防控,常常造成巨大的經(jīng)濟損失。生物技術(shù)在解決棉花蟲害和病害上展現(xiàn)出了極大的潛力。Bt棉花即是棉花生物技術(shù)產(chǎn)生的一個奇跡。然而,自從種植Bt棉花以來,也出現(xiàn)了對于鱗翅目昆蟲產(chǎn)生抗性的報道。因此,培育具有高Bt毒素水平且對CLCuD具有強耐受性的多抗棉花種質(zhì),對棉花生產(chǎn)的發(fā)展具有重要意義�；诖�,本研究構(gòu)建了攜帶不同啟動子組合(即CaMV35S,NOS和PS-1)的Cry1Ac或Cry2Ab4的多基因表達載體,并在煙草中進行了表達;同時,進行了 CLCuD致病的的生化基礎(chǔ)研究,以為棉花育種提供參考。研究結(jié)果表明:Cry1Ac或Cry2Ab4在轉(zhuǎn)基因煙草中得到成功表達。對轉(zhuǎn)基因煙草進行RT-PCR和qRT-PCR的結(jié)果證明,與包含兩個表達盒的載體相比,含三個表達盒的表達載體,其Cry1Ac和Cry2Ab4基因的表達水平更高,其中Cry1...

【文章頁數(shù)】：70 頁

【學(xué)位級別】：博士

【文章目錄】：
摘要
Abstract
Chapter 1: Introduction
    1.1 Transgenic Bt cotton development
    1.2 Types of insecticidal crystal (Cry) proteins
    1.3 Expression of Bt gene
    1.4 Strategies to develop the elite Bt cotton genotypes
    1.5 Use of different promoter combinations with Bt genes
    1.6 Introduction of elite Bt germplasm and CLCuD
    1.7 Approaches to develop the CLCuD tolerance
    1.8 Objectives
Chapter 2: Materials and Methods
    2.1 Experiment No.1
        2.1.1 Construct development and evaluation for high Bt toxin level
        2.1.2 Construction of gene cassettes
        2.1.3 Extraction of plasmid
        2.1.4 Restriction and purification of plasmid
        2.1.5 Ligation of plasmid
        2.1.6 Transformation and cloning of ligated plasmid in E. coli
        2.1.7 Selection of positive colonies and confirmation of ligated fragment
        2.1.8 Development of expression vector
        2.1.9 Stable transformation of tobacco transformation
        2.1.10 Characterization of transgenic tobacco plants
        2.1.11 Expression analysis
    2.2 Experiment No.2
        2.2.1 Plant material
        2.2.2 Development of breeding population and experimental design
        2.2.3 Field evaluation and disease rating
        2.2.4 Sample collection for biochemical analysis
        2.2.5 Leaf chlorophyll (Chl) and carotenoid contents determination
        2.2.6 Determination of total soluble proteins (TSP) contents
        2.2.7 Determination of total soluble sugar (TSS) contents
        2.2.8 Enzymatic antioxidants assay
        2.2.9 Statistical analysis and data representation
Chapter 3: Results
    3.1 Experiment No.1
        3.1.1 Construction of gene cassette and expression vectors
        3.1.2 Tobacco Transformation and immunostrip analysis
        3.1.3 PCR based expression analysis
    3.2 Experiment No.2
        3.2.1 Field disease rating
        3.2.2 Total soluble protein (TSP) contents
        3.2.3 Superoxide dismutase activity (SOD)
        3.2.4 Polyphenol oxidase activity (PPO)
        3.2.5 Peroxidase activity (POD)
        3.2.6 Catalase activity[CAT]
        3.2.7 Phenylalanine ammonia-lyase activity (PAL)
        3.2.8 Chl a, b and total chlorophyll (a+b) contents (mg/g of FW)
        3.2.9 Carotenoid contents (mg/g of FW)
        3.2.10 Total soluble sugar contents (mg/g of FW)
        3.2.11 Correlation Co-efficient estimates
Chapter 4: Discussion
    4.1 Development and expression of multiple gene cassettes vector
    4.2 Biochemical basis of CLuCD
Conclusions
References
Acknowledgments
Curriculum Vitae



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