Development of Bt Cotton Genotypes Using Pakistani Cultivars

发布时间：2023-04-09 20:41

　　世界上包括中国和巴基斯坦在内的50多个国家都有棉花种植。由于其具有重要的经济价值,棉花被喻为“白金”。但是,棉花的生长过程中会面临许多问题,如虫害,杂草和CLCuD,特别是在巴基斯坦,这些情况尤为严重。虫害可以被杀虫剂防治,而由CLCuD引起的曲叶病却仍然无法防控,常常造成巨大的经济损失。生物技术在解决棉花虫害和病害上展现出了极大的潜力。Bt棉花即是棉花生物技术产生的一个奇迹。然而,自从种植Bt棉花以来,也出现了对于鳞翅目昆虫产生抗性的报道。因此,培育具有高Bt毒素水平且对CLCuD具有强耐受性的多抗棉花种质,对棉花生产的发展具有重要意义。基于此,本研究构建了携带不同启动子组合(即CaMV35S,NOS和PS-1)的Cry1Ac或Cry2Ab4的多基因表达载体,并在烟草中进行了表达;同时,进行了 CLCuD致病的的生化基础研究,以为棉花育种提供参考。研究结果表明:Cry1Ac或Cry2Ab4在转基因烟草中得到成功表达。对转基因烟草进行RT-PCR和qRT-PCR的结果证明,与包含两个表达盒的载体相比,含三个表达盒的表达载体,其Cry1Ac和Cry2Ab4基因的表达水平更高,其中Cry1...

【文章页数】：70 页

【学位级别】：博士

【文章目录】：
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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