【作者】 张飞；

【导师】 柳李旺；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2017， 硕士

【摘要】 镉(Cd)是生物毒性极强的重金属之一,其易被植物根部吸收,并可在不同组织部位累积。Cd污染不仅严重影响作物产量和品质,而且可通过食物链危害人类健康。萝卜是以膨大肉质根为食用器官的重要蔬菜作物,其肉质根极易受到Cd等重金属毒害。植物体可通过Cd固定、外排、螯合及在植物组织细胞中的重新分配缓解或消除Cd毒害,提高Cd耐受性。然而,当环境中Cd浓度过高时,植物本身的解毒和抗氧化能力不足以抵抗重金属胁迫的毒害效应。褪黑素(N-乙酰基-5-甲氧基色胺)是一种天然的抗氧化剂,可提高细胞内抗氧化系统酶活性,并能高效清除多种活性氧,其已被证实在植物抵抗重金属胁迫过程中发挥重要作用。但目前褪黑素调控萝卜Cd胁迫响应miRNA和功能基因还未鉴定,褪黑素缓解Cd毒害作用的分子机制尚未解析。鉴于此,本研究采用外源喷施褪黑素的方法,检测镉胁迫下萝卜幼苗系列生理、抗逆指标的变化,分析褪黑素对萝卜幼苗镉毒害的缓解作用;利用Solexa测序技术分离褪黑素处理下萝卜根部Cd胁迫响应miRNA及其靶基因;通过RNA-Seq技术分离Cd胁迫下褪黑素处理的萝卜根部差异表达基因,进行差异功能基因与miRNA关联分析,构建miRNA介导的Cd胁迫响应基因调控网络,以揭示褪黑素缓解萝卜Cd胁迫的分子机制。主要研究结果如下:1.褪黑素可显著增加萝卜幼苗叶片和根中可溶性蛋白含量,增加生物量和干物质含量,有助于维持萝卜幼苗在Cd胁迫下的正常生长发育;褪黑素对Cd胁迫下萝卜幼苗的叶绿素含量、净光合速率(Pn)、胞间CO2浓度(Ci)、气孔导度(Gs)和蒸腾速率(Tr)均有不同程度的影响,说明褪黑素能促进光合色素的积累,增强光合作用,从而增加萝卜幼苗的生长量,提高萝卜幼苗对Cd的耐受性;褪黑素能显著降低Cd胁迫下萝卜幼苗根中丙二醛(MDA)含量、增加叶片和根中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、过氧化物酶(POD)和谷胱甘肽还原酶(GR)的活性。表明褪黑素能有效清除Cd胁迫下萝卜幼苗根和叶片中的活性氧,减轻膜脂质过氧化程度,保护膜的完整性并增强植物的抗氧化能力;最后,褪黑素能降低萝卜叶片和根中Cd的含量,但当其浓度高于50μmol/L时对根中Cd含量的降低程度有限。2.为系统分离鉴定萝卜Cd胁迫下外施褪黑素响应过程中的miRNA及其靶基因,本研究构建了对照组(CK)、镉处理组(Cd)和镉加褪黑素处理组(Cd+MT)萝卜肉质根sRNA文库,利用高通量测序技术鉴定到87个已知miRNA和34个潜在的新型miRNAs。其中,11个已知和6个新型miRNAs被鉴定为褪黑素调控Cd胁迫响应miRNA。功能注释表明,褪黑素调控的miRNA主要靶向一些响应重金属胁迫的转录因子(如热胁迫响应转录因子HsfA)、胁迫防御基因(如谷胱甘肽S-转移酶基因GST)和信号转导途径相关基因(如茉莉酸合成酶基因JAR)以及离子跨膜转运相关基因(如ABC转运蛋白基因)。RT-qPCR结果表明,褪黑素响应miRNA和对应的靶基因在不同的褪黑素浓度下表达模式不同。3.为了在mRNA水平上分离鉴定萝卜Cd胁迫下外施褪黑素响应网络的差异表达基因(DEGs),通过构建对照组(CK)、镉处理组(Cd)和镉加褪黑素处理组(Cd+MT)萝卜肉质根RNA文库,利用RNA-Seq技术进行萝卜Cd胁迫下外施褪黑素的差异基因表达谱分析。共获得343个差异表达基因,包括193个上调表达和150个下调表达基因。功能注释分析表明,许多差异表达基因参与活性氧清除、金属转运和解毒、激素调控、信号识别与转导等过程。褪黑素调控相关响应基因和miRNA的关联分析表明,大部分miRNA与其对应的靶基因(miRNA-mRNA)在表达趋势上呈现出负调控关系,并且部分差异表达基因如谷胱甘肽S-转移酶基因GSTU、漆酶基因IRX12、锌金属肽酶基因PXM16和伴侣蛋白基因TCP-1等基因在增强植物重金属抗性方面发挥重要作用。RT-qPCR分析表明差异基因表达模式与RNA-Seq分析结果一致。更多还原

【Abstract】 Cadmium(Cd)is one of the most highly toxic heavy metals(HMs),which easily absorbed by plants and accumulated in different tissues and organs.Cadmium(Cd)pollution not only affects the crop yield and quality,but also harm human health through the food chain.Radish(Raphanus sativus L.)is an important vegetable crop,and its fleshy taproot is highly susceptible to HMs such as Cd.Plants has complex molecular mechanism of Cd detoxification and resistance,including immobilization,exclusion,chelation,and redistribution of Cd in plant cells.However,when Cd concentration is too high in the environment,the detoxification and antioxidant capacity of the plant is not sufficient to counteract the damage of HM stress.The application of exogenous plant hormones can enhance the ability of plants to resist HM stress.In recent years,melatonin(N-acetyl-5-methoxytryptamine)has been extensively studied as a plant hormone.Melatonin is a natural antioxidant,which can remove a variety of reactive oxygen species and improve the activity of intracellular antioxidant enzymes efficiently.Melatonin can enhance plant tolerance to high/low temperature,high salt,drought and UV exfoliation,as well as other abiotic stress such as chemical or metal contaminants in water and soil.In this study,the effects of melatonin on the radish seedlings were studied using the exogenous spray of melatonin method.The changes of physiological indexes of radish seedlings under Cd stress were studied.The differentially expressed miRNAs and genes of radish seedlings treated with melatonin under Cd stress were analyzed by high-throughput sequencing.These results would provide a theoretical basis for revealing the molecular mechanism of melatonin-alleviated cadmium stress in radish.The main achievements obtained were as follows.1.Melatonin treatment significantly reduced the content of malondialdehyde(MDA)in radish roots under Cd stress and increased the activities of superoxide dismutase(SOD),catalase(CAT),ascorbate peroxidase(APX),peroxidase(POD)and glutathione reductase(GR).The results showed that melatonin can effectively remove the production of reactive oxygen species in roots and leaves of radish seedlings under Cd stress,reduce the degree of membrane lipid peroxidation,protect the integrity of the membrane and enhance the antioxidant capacity of plants.Melatonin treatment can significantly increase the content of soluble protein in radish leaves and roots,increase the biomass and dry matter content,and maintain radish normal growth and development under Cd stress;The chlorophyll content,net photosynthetic rate(Pn),intercellular CO2 concentration(Ci),stomatal conductance(Gs)and transpiration rate(Tr)were significantly affected by melatonin treatment under Cd stress.Melanin can promote the accumulation of photo synthetic pigments,enhance photosynthesis,and therefore increase the radish growth and improve the Cd tolerance.Finally,melatonin can reduce the Cd content in radish leaves and roots;however,when the melatonin concentration is too high,the degree of Cd reduction is limited.in root.2.In this study,three sRNA of radish fleshy root were constructed from the control(CK),Cd-treated(Cd)and Cd plus melatonin treated group(Cd+MT),which was used for identification of miRNA and its target genes responsive to melatonin under Cd stress.A total of 87 known and 34 potential novel miRNAs were identified by high throughput sequencing.Among them,11 known and 6 novel miRNAs were identified as melatonin responsive miRNAs under Cd stress.Functional annotation indicated that the melatonin-regulated miRNAs are primarily targeted by some HM-responsive transcription factors(e.g.heat stress response transcription factor HsfA),stress-defense genes(e.g.glutathione S-transferase gene GST)and signal transduction pathway-related genes(e.g.jasmonate synthase gene JAR),and ion transport-related genes(e.g.ABC transporter gene).RT-qPCR results showed that melatonin-responsive miRNA and corresponding target genes were differentially expressed under different melatonin concentrations treatments.3.In order to identify the differentially expressed genes(DEGs)responsive for melatonin under the Cd stress,three RNA libraries of radish fleshy root were constructed using the RNA-Seq technique in control(CK),Cd group(Cd)and Cd plus melatonin treatment group(Cd+MT).Gene expression analysis showed that 343 differentially expressed genes(193 up-and 150 down-regulated genes)were obtained.Functional annotation analysis showed that many differentially expressed genes were involved in the process of reactive oxygen scavenging,metal transport and detoxification,hormone regulation,signal recognition and transduction and so on.The correlation analysis between melatonin-related response genes and miRNAs showed that most of the miRNAs and their corresponding target genes(miRNA-mRNA)showed a negative regulatory relationship in the expression trend.Some differentially expressed genes such as glutathione S-transferase GSTU,laccase gene IRX12,zinc metallopeptidase gene PXM16 and chaperone protein gene TCP-1 were involved in heavy metal response process in plants.RT-qPCR technique showed that the expression pattern of differentially expressed genes were consistent with that from the RNA-Seq analysis.更多还原