利用基因工程途径强化小麦叶酸和花青素含量的研究
发布时间：2021-01-03 20:21
世界上很多人正在遭受营养不良的困扰,而谷类和块茎类作物无法提供所有类型的必需的营养物质。为了解决这一问题,人们尝试了多种方法,比如营养添加剂、食品营养强化和粮食作物生物强化。小麦作为“谷物之王”,是谷物中非常重要的粮食作物,并被认为是全球粮食安全的关键作物。因此,小麦具有很大的生物强化潜力。叶酸是维持细胞功能的基本要素,包括植物在内的许多生物可以自己合成叶酸。人类与其他脊椎动物一样,靠食物来摄入叶酸,并常因叶酸摄入不足而患上许多疾病。花青素是高等植物合成的次生代谢产物,负责花和果实的着色,也用作常规和分子育种中的可见标记。植物源的花青素也很重要,花青素具有抗炎、抗癌活性,所以摄入花青素可以预防冠心病。MYB和bHLH转录因子参与到花青素生物合成途径中,并与WD40蛋白形成一个三元复合物调控花青素合成通路中的结构基因。在本研究中,我们评估了中国不同的小麦种质,探索不同小麦种质中叶酸含量的自然变化。此外,通过代谢工程对小麦籽粒进行生物强化以获得高叶酸含量。首先,将参与叶酸前体蝶呤和对氨基苯甲酸合成的大豆基因GmGCHI（GTP cyclohydrolase I）和GmADCS（aminod...
【文章来源】：中国农业科学院北京市
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviation
Chapter 1:Introduction
1.1 Malnutrition in the world
1.1.1 Economic impact of malnutrition
1.1.2 Causes of malnutrition/micronutrients deficiencies
1.1.3 Strategies to combat the malnutrition
1.2 Wheat as a potential crop for biofortification
1.3 Folates biosynthesis and biofortification
1.3.1 Folate biosynthesis
1.3.2 Problems caused by folate deficiency
1.3.3 Strategies to fight against folate deficiency
1.4 Anthocyanin biosynthesis and biofortification
1.4.1 Anthocyanins and their nutraceuticals properties
1.4.2 Anthocyanin biosynthesis
1.4.3 Biofortification for anthocyanins
1.5 Objectives of the present study
Chapter 2:Folate content analysis of wheat cultivars developed in the North China Plain
2.1 Introduction
2.2 Materials and methods
2.2.1 Plant materials
2.2.2 Chemicals and reagents
2.2.3 Folate extraction and deglutamylation
2.2.4 Folate determination by HPLC–MS/MS
2.2.5 Statistical analysis
2.3 Results
2.3.1 Overall variation of the total folate levels in wheat samples
2.3.2 Screening of wheat genotypes with high folate content in different regions
2.3.3 Distribution of folate forms in Chinese wheat genotypes
2.3.4 Association of wheat folate contents with environment
2.4 Discussion
2.5 Summary
Chapter 3:Folate fortification of wheat by genetic engineering approach
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Construction and transformation of overexpression vectors
3.2.3 Identification of transgenic wheat plants through Quickstix method
3.2.4 Identification of transgenic plants through PCR amplification of transgenes
3.2.5 Determination of expression levels of transgenes in the grains of transgenic plants
3.2.6 Obtaining of homozygous wheat transgenic plants through double haploids
3.2.7 Chromosome preparation and fluorescent in situ hybridization
3.2.8 Determination of levels of folate and its precursors contained in transgenic plants
3.2.9 Investigation the effect of transgenes GmGCHI and GmADCS on agronomic traits of transgenic wheat plants
3.2.10 Statistical analysis
3.3 Results
3.3.1 Co-expression of GmGCHI and GmADCS in wheat transgenic plants
3.3.2 Co-expression of codon-optimized soybean GmGCHI and tomato LeADCS in wheat
3.4 Discussion
3.5 Summary
Chapter 4:Anthocyanin accumulation in wheat through expression of maize transcriptional factors
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant material and growth conditions
4.2.2 Construction of expression vectors containing transcriptional factors involved in anthocyanin biosynthesis
4.2.3 Agrobacterium-mediated transformation using wheat immature embryos
4.2.4 Detection of bar protein through Quickstix strip method
4.2.5 DNA extraction and PCR amplification
4.2.6 Southern blot analysis
4.2.7 Chromosome preparation and fluorescent in situ hybridization
4.2.8 Obtaining of homozygous wheat transgenic plants through double haploids
4.2.9 RNA extraction and quantitative real-time PCR assay
4.2.10 Determination of pigment contents in the transgenic wheat plants
4.3 Results
4.3.1 Purple phenotype of expressed R2R3-MYB and bHLH type TFs in wheat immature embryos and derived tissues after transformation
4.3.2 Agrobacterium-mediated transformation efficiency and obtaining of wheat transgenic plants
4.3.3 Quickstix detection of bar protein in wheat transgenic wheat plants
4.3.4 PCR detection of transgenic wheat plants
4.3.5 Southern blot analysis
4.3.6 Production of doubling haploid wheat plants through gynogenesis
4.3.7 Production of doubling haploid plants through colchicine application
4.3.8 Identification of stable transgenic wheat plants through fluorescent in situ hybridization analysis（FISH）
4.3.9 Phenotype of three types of stable transgenic lines expressing ZmC1 and/or ZmR genes
4.3.10 Expression profiling of the two target genes ZmC1 and ZmR as well as their wheat homologous genes in the three types of transgenic lines
4.3.11 Expression profiling of wheat native anthocyanin biosynthesis related genes in the three types of transgenic lines
4.3.12 Pigment contents in the seeds of transgenic wheat plants
4.4 Discussion
4.5 Summary
Conclusion
References
ACKNOWLEDGEMENTS
RESUME
附件
本文编号：2955428
【文章来源】：中国农业科学院北京市
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Abbreviation
Chapter 1:Introduction
1.1 Malnutrition in the world
1.1.1 Economic impact of malnutrition
1.1.2 Causes of malnutrition/micronutrients deficiencies
1.1.3 Strategies to combat the malnutrition
1.2 Wheat as a potential crop for biofortification
1.3 Folates biosynthesis and biofortification
1.3.1 Folate biosynthesis
1.3.2 Problems caused by folate deficiency
1.3.3 Strategies to fight against folate deficiency
1.4 Anthocyanin biosynthesis and biofortification
1.4.1 Anthocyanins and their nutraceuticals properties
1.4.2 Anthocyanin biosynthesis
1.4.3 Biofortification for anthocyanins
1.5 Objectives of the present study
Chapter 2:Folate content analysis of wheat cultivars developed in the North China Plain
2.1 Introduction
2.2 Materials and methods
2.2.1 Plant materials
2.2.2 Chemicals and reagents
2.2.3 Folate extraction and deglutamylation
2.2.4 Folate determination by HPLC–MS/MS
2.2.5 Statistical analysis
2.3 Results
2.3.1 Overall variation of the total folate levels in wheat samples
2.3.2 Screening of wheat genotypes with high folate content in different regions
2.3.3 Distribution of folate forms in Chinese wheat genotypes
2.3.4 Association of wheat folate contents with environment
2.4 Discussion
2.5 Summary
Chapter 3:Folate fortification of wheat by genetic engineering approach
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Construction and transformation of overexpression vectors
3.2.3 Identification of transgenic wheat plants through Quickstix method
3.2.4 Identification of transgenic plants through PCR amplification of transgenes
3.2.5 Determination of expression levels of transgenes in the grains of transgenic plants
3.2.6 Obtaining of homozygous wheat transgenic plants through double haploids
3.2.7 Chromosome preparation and fluorescent in situ hybridization
3.2.8 Determination of levels of folate and its precursors contained in transgenic plants
3.2.9 Investigation the effect of transgenes GmGCHI and GmADCS on agronomic traits of transgenic wheat plants
3.2.10 Statistical analysis
3.3 Results
3.3.1 Co-expression of GmGCHI and GmADCS in wheat transgenic plants
3.3.2 Co-expression of codon-optimized soybean GmGCHI and tomato LeADCS in wheat
3.4 Discussion
3.5 Summary
Chapter 4:Anthocyanin accumulation in wheat through expression of maize transcriptional factors
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant material and growth conditions
4.2.2 Construction of expression vectors containing transcriptional factors involved in anthocyanin biosynthesis
4.2.3 Agrobacterium-mediated transformation using wheat immature embryos
4.2.4 Detection of bar protein through Quickstix strip method
4.2.5 DNA extraction and PCR amplification
4.2.6 Southern blot analysis
4.2.7 Chromosome preparation and fluorescent in situ hybridization
4.2.8 Obtaining of homozygous wheat transgenic plants through double haploids
4.2.9 RNA extraction and quantitative real-time PCR assay
4.2.10 Determination of pigment contents in the transgenic wheat plants
4.3 Results
4.3.1 Purple phenotype of expressed R2R3-MYB and bHLH type TFs in wheat immature embryos and derived tissues after transformation
4.3.2 Agrobacterium-mediated transformation efficiency and obtaining of wheat transgenic plants
4.3.3 Quickstix detection of bar protein in wheat transgenic wheat plants
4.3.4 PCR detection of transgenic wheat plants
4.3.5 Southern blot analysis
4.3.6 Production of doubling haploid wheat plants through gynogenesis
4.3.7 Production of doubling haploid plants through colchicine application
4.3.8 Identification of stable transgenic wheat plants through fluorescent in situ hybridization analysis（FISH）
4.3.9 Phenotype of three types of stable transgenic lines expressing ZmC1 and/or ZmR genes
4.3.10 Expression profiling of the two target genes ZmC1 and ZmR as well as their wheat homologous genes in the three types of transgenic lines
4.3.11 Expression profiling of wheat native anthocyanin biosynthesis related genes in the three types of transgenic lines
4.3.12 Pigment contents in the seeds of transgenic wheat plants
4.4 Discussion
4.5 Summary
Conclusion
References
ACKNOWLEDGEMENTS
RESUME
附件
本文编号：2955428
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