褪黑素诱导油菜耐硒性的生理生化、解剖及分子表达谱研究
发布时间：2020-11-03 04:12
硒(Se)是一种普遍存在的元素,土壤中的平均含量为0.1-2 mg kg-1。硒具有丰富的营养价值,是人和动物所必需的非金属元素。较低剂量的硒有利于植物的生长发育,并具有减轻重金属胁迫的能力。但是,过量的硒对植物有毒性,尤其对高等植物的影响还有待商榷。油菜(Brassica napus L.)是主要的食用油料作物,在一定范围内,还具有很强的抵抗重金属、类金属毒性的能力。因此,甘蓝型油菜对抵抗硒毒性具有较高的潜在价值。目前,许多研究专注于使用植物生长调节剂来减轻重金属、类金属的植物毒性。鉴此,本研究旨在通过使用褪黑素(MT)作为生长激素来评估硒的双重特性以及甘蓝型油菜对硒毒性的耐受性。为此,进行了相关实验,所得结果主要如下:(1)探究亚硒酸钠形式的硒(0、25、50和100 μM)对四个甘蓝型油菜品种('浙大619'、'浙大622'、'浙双758'和'浙油50')10日龄幼苗(水培生长)在生理生化、细胞超微结构和基因组变化方面的潜在影响。结果表明,过量的Se(ⅣV)(特别是100 μM)显着降低了植物生长参数、色素含量以及水溶性蛋白质水平,并且产生了过量的H202和MDA。在叶片和根部都检测到抗氧化酶活性和超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)以及还原性谷胱甘肽(GSH)的转录水平均显著增加,而过氧化氢酶(CAT)和根部的氧化型谷胱甘肽(GSSG)水平并没有显著增加。此外,过量的Se(ⅣV)会导致叶肉组织和根尖细胞的超微结构受损。总体而言,在50μ的Se(ⅣV)水平下没有观测到显著的植物毒性作用。在25μ的Se(ⅣV)水平下则表现出边际效应,主要表现为植株生长增强、色素含量和蛋白水平增加,以及限制H202和MDA过量产生。同时,也检测到ROS解毒酶活性(CAT活性除外)边际增加或减少以及GSH和GSSG水平升高。与叶相比,Se(ⅣV)在根中的富集要更高。这种富集在'浙大622'中最高,在'浙双758'中最低,其次是'浙大619'和,浙油50'。总体结果表明,'浙大622'对Se(ⅣV)毒性最敏感,而'浙双758'耐受性最强。(2)研究了Se(ⅣV)(0、25、50和100 μM)对不同甘蓝型油菜品种(即'浙大619'、'浙大622'、'浙油50'和'浙双758')28日龄幼苗(温室田间条件)生理生化、解剖结构和分子变化的双重效应。结果表明,添加硒可通过促进矿物质吸收从而促进植株生长并提高植株生物量,同时还提高了水溶性蛋白质和糖的水平以及与色素和气体交换参数相关的光合效率。较高的硒水平降低了光合效率,阻碍了营养物质吸收,而脯氨酸积累导致的渗透胁迫和根中较高的硒积累导致了植株生长减缓和生物量减少。通过激活参与AsA-GSH循环和ROS-MG解毒的酶,添加硒使ROS(过氧化氢、超氧化物自由基)、丙二醛和甲基乙二醛(MG)的积累最小化。但是,通过降低抗坏血酸水平、过氧化氢酶、谷胱甘肽还原酶和脱氢抗坏血酸还原酶的活性和表达水平,硒水平的升高破坏了抗氧化剂的氧化代谢。次级代谢物基因(PAL、PPO)的上调揭示了硒调控参与氧化应激的转录网络的作用。叶和根超微结构的受损揭示了硒的植物毒性。总之,研究结果揭示了硒(不高于25μM)的保护机制,这体现在硒能增强植物形态、光合作用、渗透保护和氧化还原平衡,以及通过减少ROS和MG组分从而增强ROS-MG解毒相关酶的酶活性。硒过量则会通过削弱上述参数而增强植物毒性,特别是在浓度为100 μM时。在上述甘蓝型油菜品种中,'浙大622'对硒胁迫最敏感,而'浙双758'则表现出最大耐受性。(3)本研究针对生理特征、硫醇生物合成和抗氧化系统,评估了外源性褪黑素(MT)(0、50和100μM)对甘蓝型油菜28日龄幼苗(田间条件)Se(ⅣV)(0、50、100和200 μM)胁迫的防御作用。结果表明,在Se(ⅣV)胁迫条件下,植物生长和生物量积累均呈剂量依赖性抑制,同时,也观测到色素含量、净光合速率(Pn)和PSⅡ光化学效率(Fv/Fm)受到影响,在水培液中添加MT后可有效缓解胁迫。此外,外源施用MT降低了氧化损伤和脂质过氧化,并保护了细胞膜免受硒毒性的负面影响,这主要是通过降低硒诱导的活性氧积累实现的。褪黑素还通过恢复叶片水分和糖水平来缓解渗透压。与单独硒胁迫相比,使用褪黑素和硒(MT + Se)共同处理,以下物质都有更高水平,包括:ROS解毒酶(SOD、APX、GR、CAT)、脯氨酸、游离氨基酸、硫醇组分(GSH、GSSG、NPT、PC和Cys)及其代谢酶(γ-ECS、GST和PCS)。这表明MT在清除硒诱导的氧化损伤方面具有更大的潜力。褪黑素诱导根中螯合剂的积累表明它更有可能在根中对硒进行解毒,从而减少硒转移到叶中。整体研究结果表明,MT诱导渗透保护、抗氧化防御和硫醇配体生物合成的提高,最终通过增强油菜对Se(ⅣV)胁迫的耐受性,有助于硒解毒或隔离。(4)在本研究中,我们采用RNA测序分析深入了解了外源应用褪黑素(MT)时,甘蓝型油菜对硒胁迫的抵抗反应和耐受机制。使用数字基因表达(DGE)和转录组谱分析(TPA)技术揭示了两个甘蓝型油菜品种中硒胁迫和MT诱导的缓解作用所涉及的机制。不同表达基因(DEG)的数据显示,与对照组相比,Se显着减少了DEG的数量(在'浙大622'中有更多DEG下调),并且,施用MT比单独的硒处理增加了DEG的数量。在基因本体论(GO)分析中,在生物过程和细胞组分部分,应激反应光刺激、对karrikin的响应、跨膜受体蛋白酪氨酸激酶信号传导途径、蜡生物合成过程以及脂肪酸生物合成是主要突出过程。添加硒可抑制KEGG途径,这在'浙大622'的脂肪酸代谢、卟啉和叶绿素代谢以及浙双758的光合作用、光合作用-天线生物合成、乙醛酸和二羧酸代谢中被观测到。褪黑素能在硒胁迫下改善或恢复上述特性,并且对'浙双758'耐受性的增强比'浙大622'高。
【学位单位】：浙江大学
【学位级别】：博士
【学位年份】：2019
【中图分类】：S565.4
【文章目录】：
ACKNOWLEDGEMENT
Abbreviations
Abstract
摘要
CHAPTER 1 Introduction and Literature Review
1.1 Significance of oilseed rape
1.2 Selenium occurrence in the environment
1.3 Selenium uptake, accumulation and metabolism in plants
1.3.1 Selenium uptake and transport
1.3.2 Selenium accumulation in plants
1.3.3 Selenium metabolism in plants
1.4 Dual behavior of selenium in plants
1.4.1 Beneficial effects of selenium and its role in heavy metals tolerance
1.4.2 Selenium phytotoxicity
1.5 Mechanisms of selenium detoxification
1.6 Discovery and biosynthesis of melatonin in plants
1.7 Mechanisms of melatonin-mediated heavy metals/metalloids tolerance and detoxification
1.8 Research objectives
1.9 Overview of the whole study
CHAPTER 2 Comparative response of Brassica napus cultivars against selenium stress by morpho-physiological,and antioxidants system
2.1 Introduction
2.2 Material and methods
2.2.1 Plant materials and experimental conditions
2.2.2 Morphological parameters
2.2.3 Determination of pigment contents and total soluble protein
2.2.4 Analysis of Se content in leaf and root blades
2.2.5 Quantification of lipid peroxidation and hydrogen peroxide content
2.2.6 Biochemical assay of enzyme activities
2.2.7 Determination of non-enzymatic based antioxidants
2.2.8 Ultra-structural observation by transmission electron microscopy
2.2.9 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
2.2.10 Statistical analysis
2.3 Results
2.3.1 Effects of Se （Ⅳ） on plant growth parameters
2.3.2 Effects of Se （Ⅳ） on the Se contents and its translocation in plant tissues
2.3.3 Effects of Se （Ⅳ） on pigment contents and total soluble protein
2.3.4 Variations in oxidative markers and glutathione under Se （Ⅳ） stress
2.3.5 Effect of Se （Ⅳ） on the ROS-detoxifying enzyme activities
2.3.6 Effect of Se （Ⅳ） on the antioxidant genes expression
2.3.7 Ultra-structural alterations under Se （Ⅳ） stress
2.4 Discussion
2.5 Conclusion
CHAPTER 3 Dual behavior of selenium:Insights into physio-biochemical, anatomical and molecular analysesof four Brassica napus cultivars
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Morphological parameters
3.2.3 Estimation of Se contents
3.2.4 Pigment contents and gas exchange parameters
3.2.5 Assessment of micro and macro elements uptake
3.2.6 Total water-soluble protein and total sugar content
3.2.7 Quantification of oxidative markers with histochemical staining
3.2.8 Assays of antioxidants and total ascorbic acid
3.2.9 Measurement of methylglyoxa content
3.2.10 Ultra-structural observations
3.2.11 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
3.2.12 Statistical analysis
3.3 Results
3.3.1 Effects of Se （Ⅳ） on the phenotypical appearance
3.3.2 Dual impacts of Se （Ⅳ） on plant growth and gas exchange parameters
3.3.3 Dual effects of Se （Ⅳ） on the mineral homeostasis and osmotic adjustments
3.3.4 Dual effects of Se （Ⅳ） on oxidative markers and glyoxalase system
3.3.5 Regulation of ROS detoxifying enzymes under Se （Ⅳ） stress
3.3.6 Total ascorbic acid,GSH and GSSG levels under Se （Ⅳ） stress
3.3.7 Effects of Se （Ⅳ） on the transcript levels of ASA-GSH cycle enzymes and secondarymetabolites
3.3.8 Ultra-structure changes in response to Se （Ⅳ） stress
3.4 Discussion
3.5 Conclusions
CHAPTER 4 Protective mechanisms of melatonin against selenium toxicity in Brassica napus throughphysiological, thiols and antioxidant machinery
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials and experimental design
4.2.2 Morphological parameters and relative water content （RWC）
4.2.3 Pigment contents, gas exchange and chlorophyll fluorescence measurement
4.2.4 Extraction and quantification of endogenous Se and Me by HPLC
4.2.5 Soluble Sugar, free amino acids and proline contents
4.2.6 Quantification of MDA levels, ROS contents, and relative electrolyte leakage
4.2.7 ROS-detoxifying enzymes
4.2.8 Estimation of thiol compounds and observation of leaf stomata
4.2.9 Extraction of total RNA and quantitative real-time PCR （qRT-PCR） assays
4.2.10 Statistical analysis
4.3 Results
4.3.1 Se-induces the endogenous MT and exogenous MT reduces the Se uptake
4.3.2 MT recovers the plant growth and photosynthesis under Se stress
4.3.3 MT improves the metabolic adjustments and mitigates the oxidative damages underSe stress
4.3.4 MT enhances the antioxidant enzymes and phosphate/silicon transporters
4.3.5 MT stimulates the biosynthesis of chelating compounds under Se stress
4.3.6 Exogenous MT helps in stomata opening
4.4 Discussion
4.5 Conclusions
CHAPTER 5 Transcriptome profiling of Brassica napus against selenium toxicity and its alleviation byexogenous melatonin
5.1 Introduction
5.2 Methods
5.2.1 Plant materials and growth conditions
5.2.2 Estimation of plant biomass,and endogenous MT and Se contents
5.2.3 Establishment of de novo assembly
5.2.4 Screening and expression pattern of differentially expressed genes （DEGs）
5.2.5 Gene Ontology （GO） functional enrichment analysis （WEGO） of DEGs
5.2.6 KEGG pathway enrichment analysis
5.2.7 Statistical analysis
5.3 Results
5.3.1 Exogenous MT minimizes the Se-uptake and improves the plant biomass
5.3.2 de novo assembly and genome mapping
5.3.3 Screening and expression analysis of differentially expressed genes （DEGs）
5.3.4 Gene ontology （GO） functional classification （WEGO） of DEGs
5.3.5 KEGG metabolic pathway enrichment analysis
CHAPTER 6 Major Findings and Future Perspectives
6.1 Major findings
6.2 Novel findings
6.3 Future perspectives
References
List of Publications
本文编号：2868075
【学位单位】：浙江大学
【学位级别】：博士
【学位年份】：2019
【中图分类】：S565.4
【文章目录】：
ACKNOWLEDGEMENT
Abbreviations
Abstract
摘要
CHAPTER 1 Introduction and Literature Review
1.1 Significance of oilseed rape
1.2 Selenium occurrence in the environment
1.3 Selenium uptake, accumulation and metabolism in plants
1.3.1 Selenium uptake and transport
1.3.2 Selenium accumulation in plants
1.3.3 Selenium metabolism in plants
1.4 Dual behavior of selenium in plants
1.4.1 Beneficial effects of selenium and its role in heavy metals tolerance
1.4.2 Selenium phytotoxicity
1.5 Mechanisms of selenium detoxification
1.6 Discovery and biosynthesis of melatonin in plants
1.7 Mechanisms of melatonin-mediated heavy metals/metalloids tolerance and detoxification
1.8 Research objectives
1.9 Overview of the whole study
CHAPTER 2 Comparative response of Brassica napus cultivars against selenium stress by morpho-physiological,and antioxidants system
2.1 Introduction
2.2 Material and methods
2.2.1 Plant materials and experimental conditions
2.2.2 Morphological parameters
2.2.3 Determination of pigment contents and total soluble protein
2.2.4 Analysis of Se content in leaf and root blades
2.2.5 Quantification of lipid peroxidation and hydrogen peroxide content
2.2.6 Biochemical assay of enzyme activities
2.2.7 Determination of non-enzymatic based antioxidants
2.2.8 Ultra-structural observation by transmission electron microscopy
2.2.9 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
2.2.10 Statistical analysis
2.3 Results
2.3.1 Effects of Se （Ⅳ） on plant growth parameters
2.3.2 Effects of Se （Ⅳ） on the Se contents and its translocation in plant tissues
2.3.3 Effects of Se （Ⅳ） on pigment contents and total soluble protein
2.3.4 Variations in oxidative markers and glutathione under Se （Ⅳ） stress
2.3.5 Effect of Se （Ⅳ） on the ROS-detoxifying enzyme activities
2.3.6 Effect of Se （Ⅳ） on the antioxidant genes expression
2.3.7 Ultra-structural alterations under Se （Ⅳ） stress
2.4 Discussion
2.5 Conclusion
CHAPTER 3 Dual behavior of selenium:Insights into physio-biochemical, anatomical and molecular analysesof four Brassica napus cultivars
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Morphological parameters
3.2.3 Estimation of Se contents
3.2.4 Pigment contents and gas exchange parameters
3.2.5 Assessment of micro and macro elements uptake
3.2.6 Total water-soluble protein and total sugar content
3.2.7 Quantification of oxidative markers with histochemical staining
3.2.8 Assays of antioxidants and total ascorbic acid
3.2.9 Measurement of methylglyoxa content
3.2.10 Ultra-structural observations
3.2.11 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
3.2.12 Statistical analysis
3.3 Results
3.3.1 Effects of Se （Ⅳ） on the phenotypical appearance
3.3.2 Dual impacts of Se （Ⅳ） on plant growth and gas exchange parameters
3.3.3 Dual effects of Se （Ⅳ） on the mineral homeostasis and osmotic adjustments
3.3.4 Dual effects of Se （Ⅳ） on oxidative markers and glyoxalase system
3.3.5 Regulation of ROS detoxifying enzymes under Se （Ⅳ） stress
3.3.6 Total ascorbic acid,GSH and GSSG levels under Se （Ⅳ） stress
3.3.7 Effects of Se （Ⅳ） on the transcript levels of ASA-GSH cycle enzymes and secondarymetabolites
3.3.8 Ultra-structure changes in response to Se （Ⅳ） stress
3.4 Discussion
3.5 Conclusions
CHAPTER 4 Protective mechanisms of melatonin against selenium toxicity in Brassica napus throughphysiological, thiols and antioxidant machinery
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials and experimental design
4.2.2 Morphological parameters and relative water content （RWC）
4.2.3 Pigment contents, gas exchange and chlorophyll fluorescence measurement
4.2.4 Extraction and quantification of endogenous Se and Me by HPLC
4.2.5 Soluble Sugar, free amino acids and proline contents
4.2.6 Quantification of MDA levels, ROS contents, and relative electrolyte leakage
4.2.7 ROS-detoxifying enzymes
4.2.8 Estimation of thiol compounds and observation of leaf stomata
4.2.9 Extraction of total RNA and quantitative real-time PCR （qRT-PCR） assays
4.2.10 Statistical analysis
4.3 Results
4.3.1 Se-induces the endogenous MT and exogenous MT reduces the Se uptake
4.3.2 MT recovers the plant growth and photosynthesis under Se stress
4.3.3 MT improves the metabolic adjustments and mitigates the oxidative damages underSe stress
4.3.4 MT enhances the antioxidant enzymes and phosphate/silicon transporters
4.3.5 MT stimulates the biosynthesis of chelating compounds under Se stress
4.3.6 Exogenous MT helps in stomata opening
4.4 Discussion
4.5 Conclusions
CHAPTER 5 Transcriptome profiling of Brassica napus against selenium toxicity and its alleviation byexogenous melatonin
5.1 Introduction
5.2 Methods
5.2.1 Plant materials and growth conditions
5.2.2 Estimation of plant biomass,and endogenous MT and Se contents
5.2.3 Establishment of de novo assembly
5.2.4 Screening and expression pattern of differentially expressed genes （DEGs）
5.2.5 Gene Ontology （GO） functional enrichment analysis （WEGO） of DEGs
5.2.6 KEGG pathway enrichment analysis
5.2.7 Statistical analysis
5.3 Results
5.3.1 Exogenous MT minimizes the Se-uptake and improves the plant biomass
5.3.2 de novo assembly and genome mapping
5.3.3 Screening and expression analysis of differentially expressed genes （DEGs）
5.3.4 Gene ontology （GO） functional classification （WEGO） of DEGs
5.3.5 KEGG metabolic pathway enrichment analysis
CHAPTER 6 Major Findings and Future Perspectives
6.1 Major findings
6.2 Novel findings
6.3 Future perspectives
References
List of Publications
本文编号：2868075
本文链接：https://www.wllwen.com/nykjlw/nzwlw/2868075.html
最近更新
教材专著
