【作者】 王燕；

【导师】 柳李旺；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2014， 博士

【摘要】 重金属污染严重影响蔬菜作物产量与品质,通过食物链会严重威胁人类健康。铅(Pb)是最有毒的重金属之一,容易被植物所吸收、并在不同的部位积累。萝卜(Raphanus sativus L.)是一种主要以膨大的肉质根为食用器官的蔬菜作物,其肉质根容易受到Pb等重金属的影响,因此研究萝卜对重金属的胁迫响应机制和遗传调控网络显得极为重要。前人在萝卜Pb吸收累积、转运和胁迫响应生理生化变化等方面已进行相关研究,但对萝卜Pb胁迫响应的分子机制尚不清楚。鉴定出萝卜Pb胁迫响应的功能基因、蛋白或代谢物是阐明其胁迫响应分子机制的重要基础。本研究以萝卜高代自交系’NAU-RG’为试材,采用基于新一代测序技术的RNA测序(RNA-seq)对萝卜肉质根进行转录组de novo测序,并且从转录组、蛋白组与代谢组水平等方面系统研究萝卜重金属Pb胁迫响应的分子机制。主要研究结果如下：1.为了获得较为全面的萝卜肉质根转录组数据,利用Solexa Illumina平台对不同发育阶段(包括幼苗期、破肚期和成熟期)的肉质根进行混样,构建了一个cDNA文库(‘CKA’)进行双末端测序。共获得约66.11M clean reads数据,通过拼接得到73,084条unigenes,其中N50为1,095 bp,总长度为55.73 Mb。对所得到的unigenes与NCBI non-redundant (NR)-. Gene Ontology (GO)、Clusters of Orthologous Group (COG)和Kyoto Encyclopedia of Genes and Genomes (KEGG)等公共蛋白数据库进行相似性比对,共有67,305条unigenes(占总数92.09%)得到了功能注释。这些unigenes主要参与基本生理和代谢过程,次生代谢物的生物合成途径、信号转导和其它一些与细胞成分、分子功能相关的过程。所获得的转录组数据为研究萝卜肉质根中相关性状形成的分子机理和特定响应过程中关键基因分离鉴定提供了重要信息基础。2.为了在mRNA水平上分离鉴定Pb胁迫响应网络中差异表达基因,通过构建对照组(CK)和处理组(Pb1000)萝卜肉质根RNA文库,利用高通量测序技术进行了萝卜Pb胁迫响应的差异基因表达谱分析。共获得4,614个显著的差异表达基因(DEGs),包括2,154个上调表达和2,460个下调表达基因。GO和pathway富集分析表明,Pb胁迫上调表达的差异基因主要参与细胞壁的防御响应、谷胱甘肽代谢相关的生化过程,而下调表达的差异基因则主要参与碳水化合物代谢相关途径。利用qRT-PCR技术对22个差异基因进行表达模式验证,发现与RNA-seq分析结果高度一致。此外,成功鉴定出多个参与防御和解毒机制的候选基因,包括编码信号感知转导蛋白激酶、转录因子、金属转运蛋白及合成螯合物相关酶的基因。3.为系统分离鉴定萝卜肉质根中Pb胁迫响应的miRNA及其靶基因,构建了对照组(CK)和处理组(Pb500)萝卜肉质根sRNA文库。通过应用Solexa测序共鉴定到74个已知miRNA和173个新型miRNA.其中,有25个已知的和9个新型miRNA被鉴定为Pb胁迫响应miRNA.基于萝卜参考基因组序列的降解组分析,共得到1,979个]miRNA-mRNA靶向转录本。GO分析表明,这些靶向转录本主要参与转录调控、防御响应和与结合有关的过程。同时鉴定的Pb胁迫响应miRNA的靶基因主要参与逆境相关的信号感知、转导和相关次级代谢途径,及与金属离子吸收和体内转运机制,包括激活多种金属转运蛋白和调节相关的转录因子。4.运用蛋白组学iTRAQ技术对Pb500和Pb1000胁迫下萝卜肉质根蛋白质组变化进行分析。在95%的置信区间内共鉴定到3,898个蛋白,对2,141个蛋白进行定量分析。2种Pb处理的差异蛋白有部分重叠,共有721个蛋白在至少一种Pb胁迫处理中差异表达,135个蛋白在Pb500与Pb1000处理下均显示差异表达。GO和pathway富集分析表明,135个共同差异表达蛋白主要参与细胞结构组分变化、碳水化合物和能量代谢相关的代谢途径和抗氧化防御。此外,将萝卜Pb胁迫下蛋白水平与转录水平的相关数据进行关联分析,有77.3%可鉴定蛋白和和77.9%的可定量蛋白在转录水平被关联到,但其表达丰度变化却呈现出极低的相关性。5.应用GC-MS技术对Pb胁迫下萝卜肉质根的代谢物组分进行分析。对照组(CK)和处理组(Pb1000)在主成分分析(PCA)和偏最小二乘方判别分析(PLS-DA)中均可明显区分,采用PLS-DA模型第一主成分的VIP (Variable Importance in the Projection)值(阈值>1),并结合学生氏t检验(t-test)(阈值0.05),参照NIST数据库(比较质谱和色谱保留时间)共鉴定出28个代谢物在Pb胁迫处理下具有显著差异性,这些代谢物主要集中于糖类、氨基酸和有机酸。此外,将代谢组分析数据与转录组相关数据进行整合,KEGG pathway分析表明,萝卜Pb胁迫下可影响碳水化合物、能量代谢和谷胱甘肽代谢等相关生化网络。更多还原

【Abstract】 The problem of heavy metal pollution is becoming a major worldwide ecological concern due to its potential adverse impacts on human health through the food chain. Lead (Pb) is one of the most toxic heavy metals and can be easily taken up by plants and accumulated in different parts. Radish{Raphamis sativus L.), is an economically important vegetable crop with an edible taproot. Because the root was considered as the vulnerable part that is affected by the heavy metal Pb, it has become of vital importance to investigate the Pb response mechanisms and explore the regulatory network of tolerance and homeostasis in radishes. Considerable efforts have been invested into investigating Pb stress in radishes, especially its accumulation, translocation, physiological and metabolic variations. However, little is known about the mechanism of radish in response to Pb stress at the molecular level. It is a fundamental step to isolate the functional-genes,proteins and metabolites for revealing the molecular mechanisms invlolved in reponse to Pb stress of radish. In this study, Next-Generation Sequencing (NGS)-based RNA-sequencing (RNA-seq) technology was employed to characterize the de novo transcriptome of radish roots taking an advanced inbred line ’NAU-RG’as the plant material, and the regulatory networks at different molecular levels including transcriptomic, proteomic and metabolomic studies in response to Pb stress of radish roots were revealed. The main achievements obtained were as follows.1. To develop a comprehensive overview of the radish root transcriptome, a cDNA library named’CKA’, prepared from three mixed RNA of taproots at different developmental stages including seedling, taproot thickening, and mature stages was subjected to pair-end read (PE) sequencing with the Solexa Illumina platform. Approximately 66.11 million paired-end clean reads representing 73,084 unigenes with a N50 length of 1,095 bp, and a total length of 55.73 Mb were obtained. A total of 67,305 (about 92.09%of the assembled unigenes) unigenes were successfully annotated using the publically available protein database. The functional annotation and classification including NCBI non-redundant protein (Nr), Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the main activated genes of radish taproots were predominately involved in basic physiological and metabolic processes, biosynthesis of secondary metabolite pathways, signal transduction mechanisms and other cellular components and molecular function related terms. Our transcriptome dataset will serve as an important public information platform to improve the understanding of molecular mechanisms involved in taproot formation and the responses to various stresses.2. To isolate the differentially-regulated genes involved in Pb stress networks at mRNA level, two RNA libraries of untreated (CK) and Pb-treated (Pb10000) roots were constructed and sequenced using RNA-Seq approach. A total of 4,614 significantly differentially-expressed genes (DEGs) were detected consisted of 2,154 up- and 2,460 down-regulated genes between the two libraries. GO and KEGG pathway enrichment analysis revealed that upregulated DEGs under Pb stress were predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by qRT-PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to Pb stress of radish.3. To systematically isolate and dissect Pb-responsive miRNAs and their targets at the global level in radish roots, we constructed two small RNA libraries from one untreated control (CK) and one Pb-stressed (Pb500) of radish roots. Using Solexa sequencing technology, a total of 74 known and 173 novel candidate miRNAs were successfully identified from two libraries. Of these,25 known and nine novel miRNAs were significantly differentially expressed and identified as Pb-responsive miRNAs. Based on the reference sequences,1,979 miRNA-mRNA target transcripts could potentially be cleaved with degradome analysis. GO analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms.4. The changes in the protein profile of radish roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) respectively upon Pb500 and Pb1000 exposure. A total of 3,898 proteins were successfully detected at a 95% confidence limit, and 2,141 were reliably quantified. A subset of 721 proteins was differently regulated upon at least one Pb treatment, and 135 proteins showed abundance changes during both two Pb-stressed conditions. GO and KEGG pathway enrichment analysis revealed that the 135 co-differently expressed proteins were strongly enriched in the categories of cell structure, carbohydrate and energy metabolism-related pathways and antioxidative defense. Furthermore, the changes of protein levels were compared with transcript levels of radish in response to Pb stress. Although the transcripts could be detected 77.3%and 77.9% respectively for the identified and quantified proteins, the abundance ratios showed large discrepancies between the levels of mRNA and protein expression in radish roots response to Pb stress.5. The metabolite profiling analysis of radish roots exposed to Pb stress has been performed using gas chromatography-mass spectrometry (GC-MS). The score plots of principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed clear discrimination between control and Pb-treated samples. The altered metabolites were found from the line plots of the X-loadings of the first component of the PLS-DA pairwise comparison models. On the basis of the parameter VIP>1 using student’s T test (T-test),28 Pb-metabolites with significant changes were identified (P<0.05), which mainly on saccharides, amino acids, and organic acids. Furthermore, an integrated analysis of the effects of Pb stress was performed on the levels of metabolites and gene transcripts in radish roots, and the integrative biochemical networks of the radish in response to Pb stress was characterized. KEGG pathway analysis of integration data demonstrated that exposure of radish to Pb stress resulted in profound biochemical changes including carbohydrate metabolism, energy metabolism, and glutathione metabolism.更多还原