水杨酸和谷胱甘肽调控铬胁迫下油菜不同耐性品种生理生化和基因组变化的作用机理

摘要

土壤中重金属的毒害是制约油菜产量的主要因素之一。筛选对重金属抗性高的基因型是最佳解决方法之一，使用不同的植物生长调节剂提高非生物胁迫下长势也有显著效果。本研究包括四部分试验内容，取得结果如下:
　　(1)试验以四个不同甘蓝型油菜品种(浙双758、浙大619、浙油50、浙大622)的6天幼苗为试验材料，研究铬(Cr)的毒害作用。结果表明，随着铬浓度的升高，所有品种的幼苗生长均受到抑制，光合色素含量和抗氧化酶活性显著降低，丙二醛(MDA)和活性氧(ROS)的含量上升。相对于其他品种，浙大622的各个部位的铭含量均较高。电镜结果表明，400μM铬处理下，叶肉和根尖细胞超微结构被完全破坏，浙大622的破坏效果最显著。因此，浙大622相对于其他3个油菜品种表现更敏感。
　　(2)选取了水培生长下的四个不同甘蓝型油菜品种来研究铬的毒性。结果表明，随着铬浓度的升高，油菜的生物量有所降低。而浙大622下降幅度最为显著，其光合作用降低，而浙双758在铬的毒害下相对表现较好。不同浓度的铬诱导了活性氧和丙二醛的积累。然而，为了清除活性氧和丙二醛的积累，相关的酶活性在叶片和根中均升高。电镜结果表明，400μM铬处理下浙大622的叶肉和根尖细胞超微结构损伤更严重，可在叶片超微结构中发现破裂的细胞壁、不成熟的细胞核、损坏的细胞膜，线粒体数量增加，以及破裂的类囊体膜和膨大的液泡和淀粉粒。在损伤的根细胞中还能发现高尔基体的破裂和扩散的细胞壁。据此，我们推断在铬的毒害下浙大622最敏感，其次是浙油50、浙大619和浙双758。
　　(3)有研究表明水杨酸(SA)能诱导植物增强对许多生物和非生物胁迫的抗逆性。本试验研究了水杨酸如何调节铬胁迫下甘蓝型油菜（浙大622、浙双758）的防御机制。结果表明，外源添加水杨酸能够促进植物的生长，积累干物质量，加强活性氧的清除，减轻铬对细胞器的毒害。浙大622（黄籽）叶和根中铬和水杨酸的含量显著高于浙双758（黑籽）。为了清除随着铬浓度的增长而积累的活性氧，植物防御体系被激活，水杨酸进一步增强植物的防御能力。基因表达分析表明，铬和水杨酸的复配处理上调了抗氧化剂相关基因的表达。以上结果证实了水杨酸能够诱导铬毒害下叶和根中抗氧化酶的活性与相关基因的表达，以及次生代谢和转录水平上相关蛋白的表达。
　　(4)除了生理参数，我们对两个不同铬抗性品种（浙双758、浙大622）进行转录组分析，希望能够找到其抗性机制。结果表明，在铬胁迫处理下，两个品种中DEG均显著降低。此外，GO分析发现涉及了铬毒性胁迫及其正向响应调控机制，包括生物调节、逆境和外界刺激产生的响应。KEGG途径分析阐述了铬毒害表现为抑制玉米素、氮素吸收和光合作用天线蛋白通路被阻断。此外，数据表明，铬胁迫抑制了色氨酸表达的相关途径，但另一方面增强了核糖体的代谢途径。本研究还阐述了外源还原型谷胱甘肽(GSH)恢复铬胁迫下上述的有关指标。而且研究发现，外源谷胱甘肽能诱导铬处理下转导蛋白的活性，这类蛋白与细胞应对外来逆境胁迫直接有关。

目录

声明

ACKNOWCEDGEMENTS

CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABBREVIATIONS

Abstract

摘要

CHAPTER 1 GENERAL INTRODUCTION

1．1．What is oilseed rape?

1．2．Economic importance

1．3．Promising heavy metals and their concentration in the soil of Zhejiang Province

1．4．Impacts of heavy metals on plants

1．5．Interaction of Cr and plants

1．6．Possible way outs

1．7．Aims of the study

1．8．Study outlines

CHAPTER 2 REVIEW OF LITERATURE

2．1．What is stress?

2．2．Cr as a toxic and a non-essential element

2．2．1．Occurrence

2．2．2．Cr species

2．3．Human health and Cr-toxicity

2．4．Hazardous effects of Cr on plants

2．5．Cr and Brassica napus interaction

2．6．SA as an elevator

2．7．Role of GSH to improve the stress impacts on plant

CHAPTER3 GENOTYPIC VARIATION OF THE RESPONSE TO CHROMIUM TOXICITY IN FOUR OILSEED RAPE CULTIVARS

3．1．Introduction

3．2．Materials and methods

3．2．1．Plant material and experimental conditions

3．2．2．Measurement of Plant physio-morphic attributes

3．2．3．Determination of MDA and singlet oxygen species

3．2．4．Assessment of enzyme based plant immune system

3．2．5．Ultrastructural observations

3．2．6．Statistical analysis

3．3．Results

3．3．1．Analysis of physiological parameters

3．3．2．Determination of Cr and chlorophyll contents

3．3．3．MDA and ROS contents estimation

3．3．4．Evaluation of defense related enzymes and TSP contents

3．3．S．TEM ultrastruetural observations of leaf

3．3．6．TEM ultrastructural observations of root

3．4．Discussion

3．5．Conclusions

CHAPTER 4 CHROMIUM-INDUCED PHYSIO-CHEMICAL AND ULTRASTRUCTURAL CHANGES IN FOUR CULTIVARS OF Brassica napus L.

4．1．Introduction

4．2．Materials and methods

4．2．1．Plant material and growth conditions

4．2．2．Morphological parameters

4．2．3．Analysis of lipid peroxidation and reactive oxygen species(ROS)

4．2．4．Biochemical analysis

4．2．5．Analysis of detoxification related antioxidants

4．2．6．Transmission electron microscopy

4．2．7．Statistical Analysis

4．3．Results

4．3．1．Morphological attributes

4．3．2 Photosynthetic gas exchange capacity

4．3．3．Estimation of MDA and ROS

4．3．4．Analysis of enzymatic anlioxidants and TSP

4．3．5．Analysis of detoxification related defense mechanism

4．3．6．TEM cell strueturai observations

4．4．Discussion

4．5．Conclusions

CHAPTER 5 EXOGENOUSLY APPLIED SALICYLIC ACID REGULATES THE PHYSIO-MORPHIC AND MOLECULAR CHANGES UNDER CHROMIUM TOXICITY IN BLACK AND YELLOW SEEDED Brassica napus

5．1．Introduction

5．2．Materials and methods

5．2．1．Plant material and growth conditions

5．2．2．Chlorophyll and Cr content determination

5．2．3．Extraction and estimation of total SA contents

5．2．4．Determination of MDA contents and ROS activity

5．2．5．Determination of antioxidant machinery

5．2．6．Total RNA extraction，cDNA synthesis，and quantitative real-time PCR(qRT-PCR)assays

5．2．8．Statistical analysis

5．3．Results

5．3．1．Morpho-physiological attributes

5．3．2．Lipid peroxidation and ROS determination

5．3．3．Antioxidants enzyme activities

5．3．4．Enzymatic anfioxidants transcript level

5．3．5．Cr and total salicylic acid contents

5．3．6．Detoxification and secondary metabolite related gene expression

5．3．7．Cell ultra-structural observations

5．4．Discussion

5．5．Conclusions

CHAPTER 6 REDUCED GLUTATHIONE MEDIATES CHROMIUM TOXICITY IN OILSEED RAPE CULTIVARS AS REVELAED BY COMPARATIVE FUNCTIONAL RNA SEQUENCE ANALYSES

6．1．Introduction

6．2．Materials and methods

6．2．1．Experimental material and growth conditions

6．2．2．Cr and GSH content determination

6．2．3．Total RNA extraction，reliability assessment and RNA-sequence analyses

6．2．4．Establishment of de-novo assembly

6．2．5．Screening of differentially expressed genes(DEGs) and group differentially expressed genes

6．2．6．Expression pattern analysis of DEGs

6．2．7．Gene Ontology(Go) functional enrichment analysis(WEGO) of DEGs

6．2．8．KEGG pathway enrichment analysis

6．2．9．Statistical analysis

6．3．Results

6．3．1．Plant biomass，Cr and GSH contents determination

6．3．2．RNA sequencing and denovo assembly

6．3．3．Screening，expression pattern analysis and clustering of differentially expressed genes(DEGs)

6．3．4．Gene ontology functional classification(WEGO) of DEGs and cluster of orthologous groups(COGs) classification

6．3．5．KEGG metabolic pathway enrichment analysis

6．3．6．Analysis of top 10 up-regulated stress response-related DEGs

6．4．Discussion

6．5．Conclusions

CHAPTER 7 MAJOR FINDINGS AND FUTURE PERSPECTTVES

7．1．Major findings

7．2．Future perspectives

References

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