【作者】 李金凤；

【导师】 占新华；

【作者基本信息】 南京农业大学 ， 环境污染控制工程， 2020， 博士

【摘要】 多环芳烃是环境中普遍存在的一类有机污染物,因其不溶于水且易被土壤颗粒吸附,并随植物水分和营养的吸收转运过程进入植物的根、茎、叶和果实或籽粒中,危害人体健康。miRNAs(microRNAs,微小RNA)是一类非编码的长度18-30核苷酸的小RNA,它们在植物生长发育和响应外界环境胁迫过程中发挥重要的调控作用。本论文以六倍体冬小麦和菲(多环芳烃研究的模式化合物)为研究对象,通过水培实验,探讨了菲暴露下小麦根系miRNAs及其靶基因的差异性表达;综合植物学、植物生理学、生物化学、分子生物学和生物信息学的技术和手段,观察并测定了小麦根系形态、内部生理生化活动和基因表达、转录水平等,以阐明miRNAs对小麦根系响应菲暴露的调控机制。论文主要研究结果如下:1、菲暴露(48 h)改变了 18-30 nt各个长度的miRNAs的表达,致使小麦根系多数miRNA家族整体表达丰度明显上调或下调,甚至有些家族的成员增加或减少,其中非保守性家族的变化较保守家族更大。检测出的11个差异性表达miRNAs 为 6 个保守的(miR156、miR164、miR171a、miR398、miR531和miR1121)和 5 个非保守的(miR5048-5p、miR9653b、miR9678-3p、miR9773 和 miR9778),其中 4 个 miRNAs(miR156、miR164、miR171a 和 miR9678-3p)的表达显著上调,7 个 miRNAs(miR398、miR531、miR1121、miR5048-5p、miR9653b、miR9773和miR9778)表达则明显下调。miRNAs荧光定量PCR技术进一步测定了这11个miRNAs在3个菲浓度(0、0.5和1 mgL-1)和4个时间点(0、12、24和48 h)下的表达变化,以确保测序的准确性。2、借助psRobot和psRNATarget两个网站对11个差异性miRNAs的靶基因进行了预测,共250个靶基因被选出,属于6个保守和5个非保守的miRNAs的靶基因分别有152个和98个,其中140个基因被列出,它们涉及到植物生长发育、细胞组分合成和分子调控等功能,并可能在小麦根系菲暴露的过程中发挥着重要作用。对常见且表达量高的5个miRNAs以及它们的靶基因进行了定量验证,建立了小麦根系miRNAs响应菲暴露的调控机制,即当小麦根系暴露于菲中,miR1 56 和 miR164 分别通过靶向 SPL(SQUAMOSA promoter-binding protein-like)和NAC(NAM/ATAF1/CUC)调控小麦的生长发育;miR398、miR1121 和miR9773 分别通过靶向 CSD1/CSD2(Cu/Zn superoxide dismutase 1/2,Cu/Zn-SOD,铜锌超氧化物歧化酶1型/2型)、CAT-1(Catalase 1,过氧化氢酶)和CYP450(Cytochrome P450,细胞色素P450)影响抗氧化系统和新陈代谢。2、miR398通过靶向CSD1/CSD2调控小麦根系的抗氧化过程从而应对菲暴露产生的氧化胁迫,但是其具体的调控过程尚不清楚。为此,通过测定菲处理前后CSD1/CSD2基因表达蛋白/酶产物SOD活性的变化,实施SOD同工酶分离电泳,测定超氧阴离子和过氧化氢的变化(它们作为信号可调控抗氧化酶基因的表达),检测miR398生物合成过程不同阶段的基因产物的表达,最终明确了菲暴露对miR398的具体影响。具体研究结果为:(1)菲处理下的小麦根系总SOD活性明显增强,其中Cu/Zn-SOD活性显著高于对照;SOD同工酶电泳图中,胞质Cu/Zn-SOD的条带在菲暴露下明显变化,此外它的调控基因CSD1也明显上调,这说明胞质Cu/Zn-SOD在小麦根系清除菲胁迫产生活性氧的过程中起主要作用。(2)菲暴露下,小麦根系O2-(超氧阴离子)含量急剧升高,为了清除O2-,CAT活性也随之升高,及时将H2O2(SOD分解O2-的产物)清除掉,这就是H2O2没有明显积累的原因。这说明,菲暴露会首先引起O2-的积累,接着小麦中SOD(主要是铜/锌-SOD)的活性增强,最终来缓解菲污染造成的氧化毒性。(3)菲暴露下,pri-miR398含量减少,pre-miR398升高,miR398成熟体减少,说明菲暴露影响了 miR398调控基因MIR398的转录,阻碍了 pri-miR398向pre-miR398以及向miR398的剪切,导致了 pre-miR398的积累和成熟体miR398的减少,从而减弱了其对CSD1的沉默作用,最终诱导了 SOD酶的合成。(4)在整个调控过程中WRKY74(N端含WRKYGQK氨基酸序列)等转录因子可能发挥了重要的作用。3、miR164可以通过靶向NAC转录因子调节菲暴露下小麦根系生长和新根发生。通过比较菲处理前后小麦根系新老根生长变化,测定根系活力、丙二醛(malonaldehyde,MDA)含量和脂氧合酶(lipoxygenase,LOX)活性,进行 LOX同工酶电泳,并检测调控根系发育的基因,获得以下结果:菲处理会加速小麦老根的衰老或死亡,刺激新根数量增多,但是新根的增长速度较无菲对照组慢,只能在一定程度上弥补老根衰老或死亡带来的损失。营养液中的菲在小麦根系积累,产生的ROS(reactive oxide species,活性氧自由基)作用于根系细胞膜,LOX活性的升高和MDA含量的增加显示了脂质过氧化是造成根损伤的主要原因。菲处理刺激了 miR164的表达,增强了其对NAC1的沉默作用,使得与生长素信号的联系减弱,从而抑制侧根的发生。菲暴露影响了小麦根系中柱鞘细胞周期关键基因CDK(cyclin-dependent kinase,细胞周期蛋白依赖性激酶)和CDC2(cell divisioncycle,细胞分裂周期基因)的表达,从而影响了侧根的发生和生长。此外,基因NAM和NAC23可能也参与了菲暴露下根的生长发育。以上实验结果证实miRNAs参与了小麦根系对菲暴露的响应,miR398和miR164分别通过调控靶基因CSD1和NAC1进而调控菲暴露下小麦根系的抗氧化酶活性和根系生长发育。这些研究结果为理解农作物对多环芳烃积累响应的分子机理提供了理论基础,并为多环芳烃污染土壤或水体植物修复的强化提供科学依据。更多还原

【Abstract】 Polycyclic aromatic hydrocarbons(PAHs)are common organic pollutants in the environment.They are difficult to be removed because of high hydrophobicity and strong combination with soil particles.PAHs can enter the roots,stems,leaves,fruits or grains of plants along with absorption and transport of water and nutrients.They accumulate in human and animal bodies through the food chain,thus threating the health of humans and animals.MiRNAs(microRNAs)are a class of non-coding small RNA with a length of 18-30 nucleotides,which play an important regulatory role in plant growth and development and in response to environmental stress.In this paper,wheat is our research material and phenanthrene is the model of PAHs.We studied the differential expression of miRNAs and their target genes in wheat roots exposed to phenanthrene.Changes in wheat root morphology,physiological and biochemical activities,and transcription levels of gene expression were observed and measured.It is helpful to clarify the regulatory mechanism of miRNAs in wheat roots on response to phenanthrene exposure.The main research results are as follows:1.We compared the changes of miRNAs before and after phenanthene treatment(48 h)by high-throughput sequencing technology.Overall,the expression of miRNAs with the length of 18-30 nt changed a lot,in detail:(1)miRNA family in wheat roots obviously changed compared with the control group.The expression of many miRNAs families rose or lowered,and some members of the families increased or decreased.Non-conservative family had more changes then conservative family,because they were sensitive to environmental changes.(2)11 differentially expressed miRNAs in wheat roots were detected under phenanthrene treatment,including 6 conservative miRNAs(miR156,miR164,miR171a,miR398,miR531 and miR1121)and 5 nonconservative miRNAs(miR5048-5p,miR9653b,miR9678-3p,miR9773 and miR9778).In these miRNAs,Four(miR156,miR164,miR171a and miR9678-3p)were significantly up-regulated,and seven miRNAs(miR398,miR531,miR1121,mir50485p,miR9653b,miR9773 and miR9778)were down-regulated markedly.In addition,to ensure the accuracy of sequencing,the expression of these 11 miRNAs at 3 phenanthrene concentrations(0,0.5,and 1 mg L-1)and 4 time points(0,12,24,and 48 h)were further verified by miRNAs fluorescence quantitative PCR.(3)250 target genes of 11 miRNAs were detected with the help of psRobot and psRNATarget,including 152 target genes belonging to 6 conserved miRNAs and 98 target genes belonging to 5 nonconserved miRNAs.140 genes were screened out due to their possible regulation on plant growth and development cell component synthesis and transcription.We surmised that they may play an important role in response to phenanthrene exposure in wheat.(4)Common miRNAs with high expression levels and their target genes were identified.It is found that when wheat root was exposed to phenanthrene,miR156 and miR164 regulated the growth and development of wheat by targeting SPL(SQUAMOSA promoter-binding protein-like)and NAC(NAM/ATAF1/CUC),respectively.miR398,miR1121,and miR9773 affected the antioxidant system and metabolism by targeting CSD1/CSD2(Cu/Zn superoxide dismutase,Cu/Zn-SOD),CAT-1(Catalase 1),and CYP450(Cytochrome P450),respectively.2.miR398 regulated the antioxidase in wheat roots by targeting CSD1/CSD2 to resist the oxidative stress caused by phenanthrenel exposure.However,it is not clear how miRNAs worked in this process.We measured SOD activity and concentration of O2-and H2O2,and isolated SOD isozyme by PAGE.We also determined the expression of miR398 products at different stages of biosynthesis by means of stem ring reverse transcription and fluorescence quantitative PCR to obtain the specific effect of phenanthrene exposure on miR398.The study results were as follows:(1)Total SOD activity of wheat roots was significantly enhanced under phenanthrene treatment,in which the activity of Cu/Zn-SOD was significantly higher than that of the control.SOD isozyme electrophoresis showed that the bands of cytosol Cu/Zn-SOD changed significantly under phenanthrene exposure,and its regulatory gene CSD1 was also significantly up-regulated,indicating that cytosol Cu/Zn-SOD played a major role in removing ROS(reactive oxide species)caused by phenanthrene exposure.(2)When exposed to phenanthrene,the O2-level in wheat roots increased sharply.Then CAT activity increased to resolve the H2O2.This indicates that phenanthrene exposure first caused the accumulation of O2-and then the increase of SOD(mainly Cu/Zn-SOD)activity in wheat roots,and finally they alleviate the toxicity caused by phenanthrene.(3)Under phenanthrene exposure,the product of pri-miR398 decreased,the premiR398 increased,and the mature miR398 decreased.It indicated that phenanthrene exposure affected the transcription of miR398 regulatory gene-MIR398,hindered the transformation of pri-miR398 to pre-miR398 and then to miR398,resulting in the accumulation of pre-mir398 and the reduction of mature miR398,thus weakening the silencing effect on CSD1 and ultimately inducing the synthesis of SOD.(4)WRKY(The N terminal contains the WRKYGQK amino acid sequence)and/or transcription factors may play an important role in the whole regulation process.3.It is certified that miR164 can regulate root growth and lateral root generation of wheat under phenanthrene exposure by targeting NAC transcription factor.We compared wheat root morphology before and after phenanthrene exposure.Root activity,MDA(malonaldehyde)concentration and LOX(lipoxygenase)activity were measured,LOX isozymes were isolated by electrophoresis,and genes related to root development were detected.The main results are as follows:phenanthrene treatment accelerated the senescence/death of wheat roots,and stimulated the occurrence of new roots.However it is difficult to compensate for the loss caused by old root senescence/death,due to the slower growth of new roots under phenanthrene exposure.The accumulation of phenanthrene in wheat roots caused a lot of ROS,and the increase of LOX activity and MDA concentration,which showed that lipid peroxidation was the main cause of root damage.miR164 was up-related by phenanthrene,enhancing the silence to NAC1,weakening the association with auxin signal,and inhibiting the occurrence of lateral roots.Phenanthrene also affected the expression of CDK(cyclindependent kinase)and CDC2(cell division cycle),the key genes in the cell cycle of pericycle cells,thereby affecting the occurrence and growth of lateral roots.In addition,the genes,NAM and NAC23,may also be involved in the growth and development of wheat roots exposed to phenanthrene.All the above results confirmed that miRNAs in wheat roots are involved in response to phenanthrene exposure.miR398 and miR164 regulate the antioxidant enzyme activity and root growth and development of wheat roots exposed to phenanthrene by targeting genes CSD1 and NAC1,respectively.These results provide not only theoretical basis for understanding the molecular mechanism of crop response to PAHs accumulation,but also strategies for strengthening phytoremediation in PAHcontaminated soil or water.更多还原