水稻叶色基因OsBML和OsPL的基因定位和功能分析

发布时间：2021-03-03 03:34

　　水稻是世界上重要的粮食作物之一,也是中国的主要粮食作物。稻米养活了世界一半以上的人口,中国近65%的人口是以稻米为食,培育出产量更高更稳的水稻新品种有利于保证国家的粮食安全。近年来,水稻基因组学的发展给水稻优良品种的选育带来了新的机遇和挑战。正常的叶色有利于叶片进行光合作用,而一些特殊的叶色也可作为鉴定水稻材料的有效标志性状。叶色的变化与植株衰老有着直接或间接的关系,对水稻的产量和品质影响很大。目前,在水稻中已经发现了一些与叶色有关的基因,但是对于调控叶色与叶片衰老的遗传机理和分子机制仍缺乏充分的了解和认识。深入研究这些叶色基因的生物学功能和代谢途径有助于叶色标记性状在水稻遗传育种中的应用,在水稻抗衰老育种中也有重要的理论和实践意义。本研究采用甲基磺酸乙酯（EMS）诱变技术从籼稻品种浙农34的M2群体中分离到两个不同的叶色突变体,分别命名为水稻叶片主脉褐色突变体bml和紫叶色突变体pl。并采用表型观察、图位克隆和基因表达分析等方法分析了突变基因的功能。结果表明,两个叶色基因控制的表型能够稳定遗传,在叶片衰老过程中均起着重要作用,其中OsBML和OsPL分别参与了早期和后期的叶片衰老调控... 

【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校

【文章页数】：107 页

【学位级别】：博士

【文章目录】：
Acknowledgements
List of Abbreviations
Abstract
摘要
1 Introduction
2 Literature review
    2.1 Research progress of color mutants
    2.2 Source of leaf color mutants
    2.3 Types and physiological changes of leaf color mutants
        2.3.1 Types of leaf color mutants
        2.3.2 Physiological changes in leaf color mutant
    2.4 Factors affecting rice leaf coloring
        2.4.1 Environmental factors affecting leaf senescence in rice
        2.4.2 Cultivation conditions affecting leaf senescence in rice
        2.4.3 Plant factors affecting the leaf coloring of rice leaves
    2.5 Molecular mechanism of rice leaf color
        2.5.1 Chloroplast development related genes
        2.5.2 Chlorophyll metabolism related genes
        2.5.3 Gene mutations in the heme metabolism pathway
        2.5.4 Gene mutations encoding other chloroplast proteins
        2.5.5 Gene mutation not directly related to the photosynthetic system
    2.6 Leaf senescence and its regulation of coloration in rice
        2.6.1 Genetic mechanism of rice leaf senescence
        2.6.2 Chloroplast development and chlorophyll metabolism related genes
        2.6.3 Transcription factor gene
            2.6.3.1 Classification of MYB transcription factor genes
            2.6.3.2 Metabolic path way for MYB transcription factor
            2.6.3.3 Advances in the regulation of anthocyanin synthesis
        2.6.4 Hormone metabolism related genes
        2.6.5 Protein modification related genes
            2.6.5.1 Analysis and comparison of RLCK and RLK characteristics
            2.6.5.2 Functional analysis of RLCK
        2.6.6 Other aging related genes
    2.7 The genetic analysis for the leaf color and leaf senescence
    2.8 Purpose and significance of present study
3 Materials and methods
    3.1 Rice materials and growth conditions
    3.2 Agronomic traits measurement
    3.3 Gene mapping program
        3.3.1 DNA extraction method
        3.3.2 SSR and InDel molecular marker analysis method
    3.4 Gene mapping
    3.5 Phylogenic analysis
    3.6 Different physiological parameters analysis
        3.6.1 Estimation of the chlorophyll content, photosynthetic and chlorophyllfluorescence parameters
        3.6.2 Anthocyanin content measurement
        3.6.3 Microscopic structure analysis
        3.6.4 Transmission electron microscopy （TEM）
        3.6.5 Histochemistry assay
        3.6.6 Measurement of antioxidant and enzyme activity
        3.6.7 Measurement of endogenous phytohormones
        3.6.8 Dark-induced treatment
        3.6.9 Inoculation with bacterial blight pathogen
        3.6.10 Temperature treatment on pl
    3.7 Total RNA extraction
    3.8 Quantitative qRT-PCR analysis
    3.9 Construction of artificial RNA interference vector of OsBML
    3.10 Genetically modified tissue culture procedures in rice
        3.10.1 Callus induction and subculture
        3.10.2 Infection of rice callus
        3.10.3 Co-cultivation
        3.10.4 Selection of resistant callus
        3.10.5 Pre-distribution
        3.10.6 Differentiation
        3.10.7 Rooting
    3.11 Statistical Analysis
4 Results and analysis
    4.1 Phenotypic analysis of mutants
        4.1.1 The analysis for the bml mutants
            4.1.1.1 Phenotypic trait performance of the bml mutants
            4.1.1.2 Agronomic and yield related traits characterization of the bml mutant394.1.1.3 Chlorophyll content, photosynthesis and chlorophyll fluorescence ofthe bml mutant
            4.1.1.4 Ultrastructure changes in mesophyll cells and leaf chloroplasts of bmlmutant
            4.1.1.5 Activities of antioxidant enzymes and reactive oxygen species in thebml mutant
            4.1.1.6 Measurements of phytohonnones in the bml mutant
        4.1.2 The analysis for the pl mutant
            4.1.2.1 Phenotypic trait characterization of the pl mutant
            4.1.2.2 Chlorophyll contents, chlorophyll fluorescence in pl mutant
            4.1.2.3 Anthocyanin contents analysis
            4.1.2.4 Ultra structure alterations in leaf mesophyll cells in the pl mutant
            4.1.2.5 Antioxidant enzymes, MDA and TSS contents in the pl mutant
            4.1.2.6 Phytohormones measurement in the pl mutant
    4.2 Genetic analysis and gene mapping of color leaf mutants
        4.2.1 Genetic analysis, map-based cloning and confirmation of the mutation of theOsBML gene
            4.2.1.1 Genetic analysis and map-based cloning of the OsBML gene
            4.2.1.2 Confirmation of the mutation of the OsBML gene
        4.2.2 Genetic analysis and map-based cloning of OsPL gene
    4.3 Phylogenetic analysis and domain location
        4.3.1 Phylogenetic analysis and domain location of OsBML gene
        4.3.2 Phytogenic tree analysis of OsPL gene
    4.4 Expression pattern analysis
        4.4.1 Expression pattern analysis of OsBML gene
        4.4.2 Leaf senescence-associated gene expression in the bml mutant
        4.4.3 Disease response and upregulation of pathogenesis-related marker genes
        4.4.4 Expression pattern analysis of OsPL gene
        4.4.5 Photosynthesis and anthocyanin biosynthesis associated gene expression inthe pl mutant
        4.4.6 Effect of high temperature on pl mutant
5 Discussions
    5.1 Change of normal leaf color leads directly or indirectly to leaf senescence
    5.2 BML gene involved in the induction of defense responses against disease
    5.3 OsPL gene involved in anthocyanin synthesis, leaf senescence and heat stressresponse
6 References
7 List of publications


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