陆地棉抗除草剂基因的遗传转化及其抗镉胁迫的遗传效应探讨

论文摘要

陆地棉抗除草剂基因的遗传转化及其抗镉胁迫的遗传效应探讨棉花是一种重要的经济作物。棉花遗传改良可以通过传统的育种技术和现代生物技术实现。现代生物技术通过DNA重组技术将外源性状转入到寄主基因组，可实验基因在不同生物界之间的自由交换和流动，对于增加作物产量、提高产品质量、减少生产成本和环境胁迫均具有重要的作用。本研究通过比较二种非基因型限制的外源基因遗传转化方法，将抗除草剂基因（Bar和Cyp81A6）导入到陆地棉，获得转基因植株，并育成抗除草剂的转基因陆地棉种质系（BR001）。同时研究了抗除草剂转基因种质系的生产性能、纤维品质、抗性遗传等，并以抗虫转基因抗虫转基因推广品种（GK30）和它的遗传背景亲本为对照，研究了转基因抗除草剂种质系（BR001）在镉胁迫下的生长发育和组织生理反应。主要结果如下：1．本研究探讨了两种非基因型依赖性的遗传转化方法，并研究了Bar基因在转基因后代的分离和遗传特点，以及抗除草剂棉花种质系（BR001）的生产性能和纤维品质。PCR和Southern杂交分析表明，外源基因已成功地插入到两种方法获得的转基因植株的基因组中。尽管项尖农杆菌介导方法的转化效率高于花粉管通道法，但获得的转基因植株数量无明显差异。田间试验结果表明，抗除草剂转基因棉花种质系（BR001）的农艺性状略差于亲本，但纤维品质略优于对照。本试验结果表明两种方法均可用于棉花外源基因的转化。2．本研究用珂字棉312和珂字棉201胚性愈伤组织为外植体，通过农杆菌介导方法，将来源于水稻抗苯拉松除草剂基因（Cyp81A6）导入棉花基因组。将以VaMV35启动子、Cyp81A6基因、NPⅡ基因构建于质粒并导入到农杆菌菌系LBA444，与胚性愈伤组织共培养48h后脱菌，接到含100mg／L卡那霉素和卡那霉素+苯拉松的筛选培养基上筛选3次，并在非筛选培养基上继代2～3次后获得大量的体细胞胚状体，并获得再生小植株。卡那霉素和苯拉松鉴定，以及PCR分析结果均表明，外源抗草剂基因已成功地导入两个基因型的基因组中。目前，转基因植株已移至温室。3．本文研究了不同浓度的鎘对抗除草剂转基因陆地棉种质系（BR001）及其抗虫转基因推广品种（GK30）和它的亲本Coker 312在形态和结构上的影响。结果表明低浓度（10μM、100μM）的鎘对两个转基因栽培品种的发芽起到了促进作用，但是高浓度的鎘（1000μM）对发芽起到的是抑制作用。在Coker312的发芽实验中，鎘的毒害作用是很强烈的，无论是低浓度的还是高浓度的对Coker312的发芽起到的都是副作用。本文还探讨了不同浓度的鎘对两个转基因品种的全植株和根的生理和形态产生的不同影响。幼苗根鎘的积累率比茎高。在本实验中GK30的鎘积累率是最高的，随后是BR001，最少的是珂字312。两个转基因品种和珂字312的根尖细胞结构的变化主要取决于鎘的浓度。随着鎘浓度的增加它们的根的微细胞结构会随之发生变化。两个转基因品种表现出了原生质体分离，液泡和细胞核增大的现象。但是珂字312除了液泡增大、线粒体增多外，根尖的亚显微结构也发生了变化。在参试的三个材料的液泡和细胞壁上可以看到鎘颗粒的堆积。这些结果显示鎘对棉花幼苗期毒害作用是比较大的。BR001对鎘敏感，GK30和珂字312对鎘具有一定的抗性。4．鎘对抗除草剂转基因陆地棉种质系（BR001）及其抗虫转基因推广品种（GK30）和它的亲本Coker 312植株在形态和生理上影响的研究结果表明，在0，10，100，1000μM鎘浓度下，根、叶片、茎、叶片的厚度和它们的鲜重、干重都呈直线下降趋势。鎘对根、茎、叶的吸水性有一定的刺激作用，其中对叶片的作用最高，其次是根和茎。在本实验中鎘在BR001的富集率是最高，其次是GK30珂字312。根和茎对鎘的吸收是相互关联的，它们的鎘的吸收量影响了根、茎、叶的吸水能力。随着鎘的浓度的提高叶肉细胞的形态结构发生了改变，BR001表现最明，显其次是GK30和珂字312。鎘对光合器官也存在一定影响，在本实验中叶绿体和线粒体的大小和数量都发生了变化。三个参试材料的液泡和细胞壁中可以清楚的看到鎘粒子。液泡膜没有发生变化。这表明这些鎘的浓度没有影响到水分的吸收，还说明了这些材料对鎘有不同的耐性。5．本文研究了增加镉浓度对转基因抗除草剂种质系和转基因抗虫棉品种及其野生型棉花镉诱导氧化代谢的作用。研究结果表明，不同浓度的镉处理显著地影响了抗氧化酶活性和脂质过氧化反应。镉胁迫对转基因抗虫棉品种和转基因抗除草剂种质系及其野生型之间的酶活水平没有显著的差异。与其它器官相比，镉更能诱导叶片的酶活水平。此外，参试材料的抗氧酶活与耐镉性之间没有直接关系。本实验的材料中这些酶的不同表型表明它们在细胞和分子水平上可能对不同的耐性机制，并需要进行验证。

论文目录

• Acknowledgement
• Summary
• 中文摘要
• SECTION-A
• CHAPTER 1.GENERAL INTRODUCTION
• 1.1 Cotton-an important cash crop
• 1.1.1 Cotton plant and its classification
• 1.1.2 Genetic improvement approaches
• 1.2 Herbicides menace in plants
• 1.2.1 Development of herbicide resistance
• 1.2.1.1 Natural resistance
• 1.2.1.2 Introduced resistance
• 1.2.2 Crop herbicide selectivity
• 1.2.3 Genetic engineering for herbicide tolerance
• 1.3 Agrobacterium-a small genetic engineer
• 1.3.1 T-DNA region of Agrobacterium
• 1.3.2 Steps in Agrobacterium-mediated gene transformation
• 1.4 Cadmium toxicity syndrome in plants
• 1.5 Aims,strategies and overview of the present study
• 1.5.1 Aims of the study
• 1.5.2 Stepwise approaches/strategies to accomplish the intended objectives
• 1.5.3 An overview of the whole study
• CHAPTER 2.LITERATURE REVIEW
• 2.1 Genetic engineering in upland cotton via Agrobacterium-mediated technique
• 2.2 Genetic engineering for herbicide tolerance in cotton
• 2.2.1 Glyphosate-tolerant cotton
• 2.2.2 Bromoxynil-resistant cotton
• 2.2.3 Gluphosinate-resistant cotton
• 2.2.4 Imidazolinone-resistant cotton
• 2.3 Cadmium toxicity syndrome in plants
• 2.3.1 Cellular mechanisms in response to cadmium stress
• 2.3.1.1 Phytochelatins
• 2.3.1.2 Vacuolar compartmentalization
• 2.3.1.3 Metallothioneins
• 2.3.1.4 Stress Proteins
• 2.3.2 Cadmium uptake and translocation
• 2.3.3 Generation and detoxification of ROS in plants
• SECTION-B
• CHAPTER 3.Studies on genetic transformation of the Bar gene and its inheritance and segregation in the resultant transgenic cotton germplasm
• Abstract
• 3.1 Introduction
• 3.2 Materials and methods
• 3.2.1 Plant materials
• 3.2.2 Agrobacterium strain and plant species
• 3.2.3 Transformation methods
• 3.2.3.1 Pollen Tube Pathway via Ovarian Injection method （Method-Ⅰ）
• 3.2.3.2 Shoot Apex Culture method （Method-Ⅱ）
• 3.2.3.2.1 Preparation of shoot apex explants
• 3.2.3.2.2 Agrobacterium co-cultivation
• 3.2.3.2.3 Selection of transgenic plants
• 3.2.3.2.3 Putative transgenic plants
• 3.2.4 Selection and screening of putative transgenic seedlings
• 3.2.5 Polymerase chain reaction （PCR） analysis
• 3.2.6 Southern blot hybridization analysis
• 3.2.7 Progeny tests
• 3.2.8 Identification of herbicide resistance
• 3.2.9 Inheritance of the herbicide resistant trait in transgenic cotton germplasm （BR001）
• 3.2.10 Agronomic characters and fiber quality of the resultant transgenic germplasm
• 3.2.11 Data analysis
• 3.3 Results and analysis
• 3.3.1 Kanamycin test for the presence of nptⅡ gene
• 3.3.2 Bioassay test for basta（?） and the comparative analysis of the two transformation methods
• 3.3 3 PCR analysis
• 3.3.5 Analysis of T1 plants
• 3.3.6 Inheritance and segregation of exogenous Bar gene in transgenic cotton germplasm （BR001）
• 3.3.7 Identification of herbicide resistance
• 3.3.8 Agronomic characters and fiber quality of BR001
• 3.4 Discussion
• 3.5 Conclusions
• CHAPTER 4.Agrobacterium-mediated genetic transformation of Cyp81A6 gene in upland cotton using embryogenic callus as explant
• Abstract
• 4.1 Introduction
• 4.2 Materials and methods
• 4.2.1 Plant materials and embryogenic callus induction
• 4.2.2 Transformation and plant regeneration process
• 4.2.2.1 Inoculation,co-culture and selection processes
• 4.2.3 Agrobacterium tumefaciens strain and plasmid vector
• 4.2.4 Kanamycin and bentazon assays
• 4.2.5 PCR analysis for putative transformants
• 4.3 Results and analysis
• 4.3.1 Embryogenic calli growth and proliferation on selective medium （MSB3）
• 4.3.2 Confirmation of transformation event by kanamycin and bentazon assay
• 4.3.3 Molecular analysis of the putative transgenic embryogenic calli
• 4.3.4 Somatic embryos production,their germination,maturation and conversion to plantlets
• 4.4 Discussion
• 4.5 Conclusions
• CHAPTER 5.Cadmium-induced functional and ultrastructural alterations in roots of herbicide resistant transgenic cotton germplasm （BR001）
• Abstract
• 5.1 Introduction
• 5.2 Materials and methods
• 5.2.1 Seed surface sterilization and treatment process
• 5.2.2 Seed germination assay and measurement of hypocotyl and radical lengths
• 5.2.3 Plant growth parameters and tolerance index
• 5.2.4 Determination of Cd content
• 5.2.5 Transmission electron microscopy
• 5.2.6 Statistical analysis
• 5.3 Results
• 5.3.1 Germination assay and radical and hypocotyl lengths
• 5.3.2 Biomass production,tolerance indices and root tip percentage
• 5.3.3 Root morphological traits
• 5.3.4 Bioaccumulation of cadmium in roots and shoots of plants
• 5.3.5 Ultrastructural observations and features
• 5.4 Discussion
• 5.4.1 Effect of Cd treatments on plant's qualitative and quantitative parameters
• 5.4.2 Effect on root morphological parameters
• 5.4.3 Effect on Cd accumulation in roots and shoots
• 5.4.4 Effect on root ultrastructure
• 5.5 Conclusions
• CHAPTER 6.Cadmium-induced ultramorphological and physiological changes in leaves of herbicide resistant transgenic cotton germplasm （BR001）
• Abstract
• 6.1 Introduction
• 6.2 Materials and Methods
• 6.2.1 Seed surface sterilization and treatment process
• 6.2.2 Plant growth parameters and Water Absorption Capacity （WAC）
• 6.2.3 Transmission electron microscopy
• 6.2.4 Statistical analysis
• 6.3 Results
• 6.3.1 Plant growth responses
• 6.3.2 Biomass production
• 6.3.3 Water Absorption Capacity （WAC）
• 6.3.4 Correlation among qualitative and quantitative traits of the seedlings
• 6.3.5 Ultrastructural changes under Cd treatments
• 6.4 Discussion
• 6.4.1 Plant growth responses
• 6.4.2 Biomass production
• 6.4.3 Water Absorption Capacity （WAC）
• 6.4.5 Correlation among qualitative and quantitative traits of the seedlings
• 6.4.6 Ultrastructural changes under Cd stress
• 6.5 Conclusions
• CHAPTER 7.Cadmium-induced oxidative metabolism in herbicide resistant transgenic cotton germplasm （BR001）
• Abstract
• 7.1 Introduction
• 7.2 Materials and methods
• 7.2.1 Plant materials and growth conditions
• 7.2.2 Determination of antioxidant enzymes activities and Lipid peroxidation
• 7.2.3 Statistical analysis
• 7.3 Results
• 7.3.1 Effect of Cd on APOX activity
• 7.3.2 Effect of Cd on POX activity
• 7.3.3 Effect of Cd on SOD activity
• 7.3.4 Effect of Cd on CAT activity
• 7.3.5 Effect of Cd on MDA activity
• 7.4 Discussion
• 7.4.1 APOX activity under Cd stress
• 7.4.2 POX activity under Cd stress
• 7.4.3 SOD activity under Cd stress
• 7.4.4 CAT activity under Cd stress
• 7.4.5 MDA activity under Cd stress
• 7.5 Conclusions
• CHAPTER 8.Major findings and future recommendations
• 8.1 Major Findings
• 8.2 Future Recommendations
• References
• Curriculum Vitae