【作者】 王磊；

【导师】 陈鹏；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2020， 博士

【摘要】 铅（Pb）是最具毒性的重金属持久性污染物之一。铅污染已成为世界范围内广泛关注的环境问题。土壤铅污染会严重影响植物（作物）种子的萌发、生长发育及结实等过程,进入食物链会严重损伤人的神经、免疫及生殖系统,且具有累积性。土壤重金属包括铅污染的植物修复被认为是廉价且最具潜力的重金属污染土壤修复技术。现有公认的重金属富集植物普遍存在生物量小、生长周期长等特点,筛选和创制耐受Pb污染的富集植物并解析其内在的富集和耐受机制具有重要的实践和理论价值。前期对多种植物铅耐受性筛选评价中发现,苦荞属于高耐铅的植物。苦荞具有地域适应性强,生育期短、生物量丰富等特点,是潜在可用于铅污染修复的植物。目前尚未有苦荞Pb耐受性及富集的系统研究报道,对其响应铅胁迫的代谢途径及基因表达调控机制尚待揭示。本研究以苦荞“九江苦荞”为材料,在利用透射电镜比较Pb在苦荞不同组织定位及重金属含量测定的基础上,采用Illumina测序技术分析铅胁迫苦荞叶片的高质量转录组数据,鉴定与铅耐受机制相关的关键基因和途径,并利用重金属耐受缺陷的酵母中的过表达以及拟南芥突变体中基因的过表达验证基因的功能,旨在为植物修复提供理论依据。主要研究结果如下:1. 苦荞可耐受最高的硝酸铅（Pb（NO₃）₂）处理浓度为10000 mg/kg土壤,在此浓度下,除株高降低外无其它明显表型变化。同时,在浓度为0-10000 mg/kg胁迫条件下,铅胁迫下叶片中丙二醛、谷胱甘肽、可溶性蛋白和超氧化物歧化酶活性等生理指标无显著变化,说明苦荞对重金属铅具有强的耐受性。铅胁迫处理植株不同组织部位的透射电镜分析显示,铅在苦荞不同组织中均有分布,在苦荞叶片中铅离子主要分布在细胞的液泡中和细胞壁内侧。原子吸收法测定苦荞不同组织部位的铅累积量显示,随着铅处理浓度的升高,苦荞叶片中铅的累积量明显增加,最大累积量为499.126±64.791mg/kg,铅离子转移系数（地上部分含量/地下部分含量）约为0.8,接近超富集植物的转移系数。综合分析显示,苦荞可以作为一种潜在的土壤修复植物。2. 对处理浓度分别为0 mg/kg（CK）、2,000 mg/kg Pb（NO₃）₂（Pb1）和10000 mg/kgPb（NO₃）₂（Pb2）的苦荞叶片进行转录组测序,结果显示CK与Pb1间共鉴定到2400个上调和3413个下调的差异表达基因;CK和Pb2之间鉴定得到2948个上调和3834个下调的差异表达基因。为了鉴定铅胁迫诱导的unigenes,三个文库被分为两组（CK vs Pb1和CK vs Pb2）,并在这两组的比较中共鉴定了4525个差异基因,其中1641个差异基因上调和2884个差异基因发生下调。GO富集分析显示差异基因主要与催化活性、结合、代谢过程和膜组分有关。KEGG富集分析铅胁迫主要影响了苦荞体内的能量代谢、脂质代谢、次生代谢产物、非酶抗氧化剂和氧化磷酸化等途径。3. 研究发现,串联CCCH型的锌指结构蛋白14（Ft TZF14）在铅胁迫下的苦荞叶片中表达量显著上调;利用重金属敏感的酵母突变体Δycf1和拟南芥对Ft TZF14的功能分析表明,Ft TZF14可明显抑制酵母和过表达拟南芥的生长;Ft TZF14在苦荞叶片中瞬时转化显示,Ft ZFP14在苦荞中的过表达可以快速推进Rubisco的降解,蛋白酶体抑制剂MG132可以显著抑制此降解过程。进一步对Ft TZF14的互作蛋白进行筛选和互作验证,确定其可以与泛素连接酶Ft ATL42发生互作。这预示Ft TZF14参与了Rubisco泛素/26S蛋白酶体介导降解的信号转导过程;说明铅胁迫下苦荞植株的生长受抑制可能与Ft ZFP14参与调控的Rubisco泛素化降解有关。4. 对铅胁迫上调的3个差异基因金属抗性蛋白C2（Ft MTPC2）、天然抗性相关巨噬细胞蛋白3（Ft Nramp3）和液泡阳离子交换子1a（Ft VCE1a）,进行实时定量验证基础上,利用重金属敏感的酵母突变体Δycf1进行功能互补验证,结果显示过表达Ft MTPC2、Ft Nramp3和Ft VCE1a的酵母菌株Δycf1对铅的耐受性显著提高。进一步对这些基因在拟南芥中的功能分析显示,Ft MTPC2、Ft Nramp3和Ft VCE1a过表达可提高转基因植株对铅的耐受性;结果表明转运蛋白Ft MTPC2、Ft Nramp3和Ft VCE1a以及结合蛋白在植株对铅的耐受和转运过程中起重要作用。同时,对转运蛋白Ft MTPC2、Ft Nramp3和Ft VCE1a进行互作蛋白的筛选,利用酵母双杂和双分子荧光互补对互作关系进行了验证,并分析了互作蛋白的基因表达量,结果表明,与Ft MTPC2互作的蛋白生长素-丙氨酸抗性蛋白1（Ft IAR1）、镉/锌转运ATPase 1（Ft HMA1）和谷胱甘肽依赖的甲醛脱氢酶（Ft FDH）的基因表达量均上调,说明起到协同转运的功能;与Ft Nramp3互作的蛋白仅镉/锌转运ATPase 3（Ft HMA3）的表达量显著上升,表达模式一致,而热激蛋白70（Ft HSP70）和锌转运体1（Ft ZRT1）两个蛋白的基因表达量下降,可能在铅胁迫下与Ft Nramp3之间存在拮抗作用;此外,与Ft VCE1a互作的蛋白,镁/氢离子交换子（Ft MHX）、还原型谷胱甘肽S7（Ft GRXS7）和天然抗性相关巨噬细胞蛋白6（Ft Nramp6）,经检测铅胁迫前后基因表达量未发生显著变化,表明其在基因表达水平上不受铅胁迫的影响。综上所述,本研究有效证明苦荞对Pb具有高的耐受性。苦荞可以作为土壤修复的植物。利用转录组测序分析了苦荞叶片对铅的耐受和累积机制,同时对重要的功能基因在酵母和拟南芥系统中进行了功能分析,研究结果为苦荞作为潜在的修复植物提供了理论依据和研究基础。更多还原

【Abstract】 Lead（Pb）is one of the most toxic heavy metal persistent pollutants.Lead pollution has become a serious environmental problem in the world.Pb contaminated soil will seriously affect the germination,growth and development of plant（crop）seeds,and will seriously damage the human nervous,immune and reproductive systems after enter the food chain,and also has a cumulative nature.Phytoremediation of heavy metals in soil,including lead contamination,is considered to be the cheapest and most promising technique for remediation of heavy metal contaminated soil.The existing recognized heavy metal enrichment plants are generally characterized by small biomass and long growth cycle,so it is of great practical and theoretical value to screen and create Pb pollution-tolerant enrichment plants and to analyze their intrinsic enrichment and tolerance mechanisms.It is found that Tartary buckwheat is a high lead tolerance plant in the early screening and evaluation of lead tolerance of various plants.Tartary buckwheat has the characteristics of strong regional adaptability,short growth period and abundant biomass.At present,there has been no systematic study on Pb tolerance and enrichment of Tartary buckwheat,and its metabolic pathways and gene expression regulation mechanisms in response to lead stress have yet to be revealed.However,none of the genes and metabolic pathways involved in regulating the Pb stress response in tartary buckwheat have been identified.In this study,the Fagopyrum tataricum‘Jiujiang’cultivar was used as the material,and Pb was used to compare the localization of tartary buckwheat in different tissues and the determination of heavy metal content.On the basis of using Illumina sequencing technology the high quality of the buckwheat leaves the transcriptome analysis of lead stress data,identification of key genes associated with lead tolerance mechanism and approaches,and use of defect in yeast expression of heavy metal tolerance and expression of gene in Arabidopsis mutants validation of gene function,aims to provide theoretical basis for phytoremediation.The main results include:1. Lead exposure up to 10,000 mg/kg reduced height but had no other effect on the buckwheat plants.this was the highest concentration assayed in subsequent experiments.Furthermore,lead exposure caused no significant change in MDA content,GSH content,soluble protein and SOD activity indicating the treatments caused less damage to the leaf physiology under the lead treatment concentration range of 0-10000 mg/kg,it indicates that Tartary buckwheat has a strong tolerance to lead.In the present study,Transmission electron microscopy analysis of different tissues under lead stress showed that lead was distributed in different tissues of Tartary buckwheat,and lead ions were mainly distributed in vacuoles and inside cell walls of Tartary buckwheat leaves.The determination of lead accumulation in different tissue parts of tartary buckwheat by atomic absorption spectrometry showed that with the increase of lead concentration,the accumulative amount of lead in Tartary buckwheat leaves also increased significantly,with the maximum accumulative amount of 499.126±64.791 mg/kg,and the lead ion translocation factor（content of overground part/content of underground part）was about 0.8,which was close to the super-enriched plants.Therefore,Tartary buckwheat can be used as a potential phytoremediation of Pb-polluted soil.2. RNA deep sequencing（RNA-Seq）of tartary buckwheat leaves was performed on twoPb-treated samples,including 0 mg/kg（CK）,2000 mg/kg（Pb1）and 10,000 mg/kg Pb（Pb2）.In total,2400 up-regulated and 3413 down-regulated differentially expressed genes（DEGs）were identified between CK and Pb1,and 2948 up-regulated DEGs and 3834downregulated DEGs were generated between CK and Pb2,respectively.To identify unigenes that were induced by Pb stress,the three libraries were divided into two groups（CK vs Pb1 and CK vs Pb2）,and 4525 common DEGs were identified in these comparisons.In total,we identified 1641 upregulated and 2884 downregulated unigenes under Pb stress.GO enrichment analysis identified unigenes in tartary buckwheat under Pb stress were primarily associated with catalytic activity,binding,metabolic processes and membrane components.KEGG enrichment indicate that Pb stress in F.tataricum primarily influences pathways associated with energy metabolism,lipid metabolism,secondary metabolites,nonenzymatic antioxidants and oxidative phosphorylation.3. In this study,we found that the gene expression of the tandem CCCH Zinc finger protein14（Ft TZF14）was significantly upregulated in F.tataricum leaves under the Pb stress.We confirmed that the overexpression of Ft TZF14 could seriously inhibit the growth of the Pb sensitive yeast mutantsΔycf1 and Arabidopsis.And the transient transformation of Ft ZFP14showed that it could rapidly promote the degradation of Rubisco in Tartary buckwheat leaves.Meanwhile,the ubiquitination inhibitor MG132 could significantly inhibit this degradation process.Furthermore,we have screened and identified an ubiquitin ligase enzyme Ft ATL42could interact with Ft TZF14.All the results indicate Ft ZFP14 was involved in the signal transduction process of the ubiquitin/26S proteasome mediated degradation of Rubisco.The results suggested that the growth inhibition of Tartary buckwheat under lead stress might be related to the ubiquitination degradation of Rubisco mediated by Ft ZFP14.4. On the basis of real-time quantitative verification of 3 differentially expressed genes（DEGs）（Metal tolerance protein C2（Ft MTPC2）,Natural resistance-associated macrophage protein 3（Ft Nramp3）and Vacuolar cation/proton exchanger 1a（Ft VCE1a））upregulated under lead stress,we verified the gene functional complementarity by using the Pb sensitive yeast mutantsΔycf1.The results suggested that the expression of Ft MTPC2,Ft Nramp3 and Ft VCE1a strongly increased the Pb tolerance in the Pb-sensitive yeast strainΔycf1.Further study indicates that Ft MTPC2,Ft Nramp3 and Ft VCE1a could enhance the tolerance of Arabidopsis thaliana to Pb.The results suggested that Ft MTPC2,Ft Nramp3 and Ft VCE1a are important in the process of Pb transport.We have screened and identified the interaction proteins of the heavy metal transporters（Ft MTPC2,Ft Nramp3 and Ft VCE1a）through the Yeast two-hybrid library of F.tataricum leaves under lead stress,then we have confirmed the interaction through Yeast two-hybrid（Y2H）and Bimolecular Fluorescent Complimentary（Bi FC）assays.Furthermore,we have analyzed the gene expression levels of the interaction proteins,which reflected the gene expression levels of IAA-alanine resistant 1（Ft IAR1）,Cadmium/zinc-transporting ATPase-HMA1（Ft HMA1）and Glutathione-dependent formaldehyde dehydrogenase（Ft FDH）proteins interacting with Ft MTPC2 were all up-regulated,and that indicates these proteins have the co-transport function to Ft MTPC2.For the protein interacting with Ft Nramp3,the gene expression level of Ft HMA3 increased significantly with the same expression pattern to Ft Nramp3;while the gene expression levels of Heat shock protein 70（Ft HSP70）and Zinc transporter 1（Ft ZRT1）decreased,which may be antagonism with Ft Nramp3 under lead stress.In addition,the gene expression of the protein(Mg²⁺/H⁺exchanger（Ft MHX）,Monothiol glutaredoxin-S7（Ft GRXS7）and Natural resistance-associated macrophage protein 6（Ft Nramp6）)interaction with Ft VCE1a did not significantly change,it is speculated that the gene expression are not affected by Pb stress.In summary,this study effectively proved that Tartary buckwheat has a high tolerance to Pb.Tartary buckwheat can be used as a soil remediation plant.We analyzed the tolerance and accumulation mechanism of Tartary buckwheat leaves to lead by transcriptome sequencing;meanwhile,we explored the important functional genes by expressing in yeast and Arabidopsis thaliana system.These results provide theoretical and research basis for Tartary buckwheat as a potential plant for phytoremediation of Pb-polluted soil.更多还原