【作者】 许瑞；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 基因组的表达调控主要受到染色质结构变化的影响，而染色质结构的变化则是由组蛋白的翻译后修饰和DNA甲基化调控等表观遗传修饰所控制的。在真核生物中，DNA甲基化修饰是调控基因表达的一个重要的表观遗传学标志。在类病毒感染的烟草中首先被发现的RNA介导的DNA甲基化（RNA-directed DNA methylation, RdDM），则是植物在非生物胁迫下引起转录水平基因沉默（transcriptional gene silencing, TGS）的一个重要调控机制。小干扰RNA（small interfering RNA, siRNA）的存在与缺失直接影响了其对应的沉默位点的mRNA表达丰度的高低。大约三分之一的DNA甲基化位点是富含siRNA靶向位点的，暗示了siRNA在DNA甲基化修饰中的重要作用。并且越来越多的实验证据证明了siRNA可以被多种外界胁迫所诱导进而影响其靶向的位点染色质结构。虽然非生物胁迫中的脱落酸（abscisic acid,ABA）依赖的盐胁迫信号转导途径已经研究的比较深入，但是DNA甲基化和去甲基化修饰是否参与到这个调控过程中仍不是十分清楚，并且siRNA是如何通过RdDM来响应外界非生物胁迫的作用机理也需要深入研究。本研究通过对AtMYB74基因的系统分析，证明RdDM参与到了植物ABA依赖的盐胁迫调控信号途径，进一步揭示了非生物胁迫下siRNA在RdDM过程中调控基因表达的作用机理。主要研究结果和主要结论如下：（1）AtMYB74基因编码一个R2R3型MYB转录因子通过对GENEVESTIGATOR和Arabidopsis eFP Browser数据库的分析，发现了1286个响应盐胁迫调控的基因。然后利用拟南芥表观遗传学图谱，对它们的启动子DNA甲基化情况进一步分析，鉴定出可能被DNA甲基化修饰调控响应盐胁迫的候选基因，最终选定AtMYB74作为研究对象进行深入探索。对转录物序列分析发现，AtMYB74的mRNA全长包含975个碱基，编码一条324个氨基酸的多肽链。其13位至65位和66位至117位氨基酸编码MYB转录因子家族保守的R2和R3DNA结合结构域，与拟南芥及其他植物物种的MYB家族同源。经过qRT-PCR和GUS染色分析，发现AtMYB74基因在拟南芥各组织器官中特异表达且丰度较低。通过洋葱表皮的瞬时转化实验在细胞核中观察到AtMYB74-GFP融合蛋白的GFP荧光，证明AtMYB74蛋白的亚细胞定位是在细胞核中。（2）超表达AtMYB74转基因植株在种子萌发期对ABA和盐胁迫敏感超表达AtMYB74可以诱导AtRD29B、AtRAB18和AtRD20等盐胁迫响应基因的表达。在NaCl和ABA的处理下，AtMYB74超表达转基因植株的种子萌发率较野生型明显下降；而RNAi转基因植株的种子萌发率则与野生型无差别。当在NaCl处理下添加ABA合成抑制剂钨酸钠时，超表达植株的敏感表型则会部分恢复。由此，萌发率的敏感表型证明了AtMYB74是在种子萌发期间通过ABA依赖的信号途径对盐胁迫进行响应进而行使其生物学功能的。（3）盐胁迫条件下DNA甲基化修饰调控AtMYB74的转录活性在NaCl和ABA处理的野生型中，AtMYB74的基因转录水平有明显的升高，证明AtMYB74是在转录水平上响应盐胁迫。而在RdDM突变体中，AtMYB74的mRNA表达水平变化远不如野生型中明显；同时，DNA去甲基化RNAi转基因植株中，同样处理下也显示出同样的结果，证明RdDM修饰对AtMYB74的转录起调控作用。对AtMYB74转录起始位点上游200个碱基区（-700~-500）域进行亚硫酸氢盐处理测序PCR（bisulfitesequencing PCR, BSP）结果发现，存在NaCl和ABA时，野生型中该区域的5-甲基胞嘧啶含量比对照组有明显升高。值得注意的是，CpHpH位点的胞嘧啶甲基化所占百分比下降了接近一半，而CpG位点只降低了10%。而在RNAi转基因植株中只是在所有位点的胞嘧啶甲基化程度出现轻微下降。通过对不同时间点的AtMYB74表达量及其启动子CpHpH位点DNA甲基化的检测，发现在盐胁迫处理后从0.5小时开始AtMYB74的表达量出现上升，在3小时时达到峰值，从6小时开始有轻微下降但仍维持在明显高于对照组的水平；而对应的CpHpH位点的DNA甲基化水平也是从0.5小时开始下降，在3小时时达到最低值，随后处在低于对照组的水平。同样的结果在ABA处理组中也被检测到，证明盐了胁迫对AtMYB74的表达量诱导升高是通过动态的CpHpH位点DNA甲基化修饰所调控的。（4）靶向AtMYB74启动子的24-nt siRNA的积累影响DNA甲基化序列分析发现，5条24-nt siRNA（ASRP215119、ASRP41948、ASRP27256、ASRP13208、ASRP2423）均可以靶向AtMYB74启动子-603位至-447位区域的相应位点。经过BSP分析后发现，盐胁迫下DNA甲基化的变化位点也是发生在这一区段或临近位置。但不论是在5-azaC处理的野生型中，还是在RdDM突变体以及去甲基化RNAi转基因植株中，都不会因为盐胁迫的存在而使这一区段的DNA甲基化水平发生明显变化，由此证明这一区段的CpHpH位点DNA甲基化修饰具有重要调控作用。通过siRNAqRT-PCR和siRNA Northern Bolt杂交检测实验，也发现靶向这一区段的5条24-nt siRNA的积累量在盐胁迫下会明显下降。而在对照组dcl3突变体中，因为siRNA合成途径被阻断则检测不到24-nt siRNA积累量变化。因此，盐胁迫是通24-nt siRNA的表达量减少来降低DNA甲基化的修饰作用，进而激活的AtMYB74基因表达的。（5）外源表达24-nt siRNA促进AtMYB74启动子DNA甲基化通过pFGC5941表达载体可以产生发夹结构的双链RNA进而生成siRNA的特性，本研究构建了可以分别产生靶向AtMYB74启动子和cDNA的siRNA的表达载体，命名为R1和R2。同样以GUS作为检测对象，构建了35S启动子、AtMYB74启动子和缺失siRNA靶向区段的AtMYB74启动子的pBI121表达载体，命名为P1、P2和P3。通过瞬时共转化实验，发现P1-R1组对应P1组，P2-R2组、P3-R1组对应P2组都没有明显GUS活性变化，而存在siRNA直接靶向的P2-R1组则比对照P2组的GUS活性明显降低。同样经过BSP检测发现，P2-R1组在该靶向区段的DNA甲基化水平升高了43%，而其他组则无明显变化。上述结果证明异源表达人工合成的siRNA可以在瞬时共转化时靶向AtMYB74启动子，同样可以通过RdDM调控对应GUS表达，从另一方面证明了siRNA对于RdDM调控的重要作用。（6）RdDM和ABA响应元件（ABA-responsive cis-elements,ABRE）均调控AtMYB74参与的盐胁迫的信号途径序列分析发现，AtMYB74启动子含有ABRE顺式作用元件，通过CHIP-PCR的方法验证了ABI5转录因子可以结合AtMYB74启动子上的ABRE顺式作用元件。尽管盐胁迫下abi5突变体中AtMYB74的转录本仍然会增加，但增加水平远低于野生型，并且含有P3载体的转基因拟南芥的GUS染色变化也说明ABRE顺式作用元件参与到了AtMYB74的调控中。同样在abi5突变体中检测到了CpHpH位点DNA甲基化水平的降低和靶向启动子的siRNA积累量下降。这些结果证明ABRE顺式作用元件和RdDM途径均参与了AtMYB74响应盐胁迫的调控。更多还原

【Abstract】 Genome expression is mainly influenced by the chromatin structure, which is governedby processes often associated with epigenetic regulation, including histone post-translationalmodification and DNA methylation. DNA methylation is an important epigenetic mark forthe regulation of gene expression in eukaryotes. RNA-directed DNA methylation (RdDM),which was first discovered in viroid-infected tobacco, is an important regulatory phenomenoninvolved in repressive epigenetic modifications that can trigger transcriptional gene silencing(TGS) under abiotic stress in plants. The gain or loss of DNA methylation is correlated with aconsiderable decrease or increase in the corresponding amount of mRNA abundance and withthe presence or absence of24-nt siRNAs at each silenced epiallele. Approximately one-thirdof methylated DNA loci in Arabidopsis is rich in siRNAs, implying an primary determinantof siRNAs in DNA methylation. Increasing evidence has shown that the activity of siRNAscould be triggered by various environmental stimuli to affect the targeting chromatinstructure.Although the ABA-dependent salt stress signal transduction pathway has beenintensively studied, whether or not DNA methylation/demethylation is involved in thispathway remains unknown. The mechanism of how the siRNA mediates DNA methylation byRdDM to respond to abiotic stress also needs to be elucidated. Our findings provide a novelABA-dependent salt stress regulatory signal pathway and allows for a better understanding ofthe role of siRNAs in controlling the RdDM pathway to regulate gene expression in responseto abiotic stress. The main results and conclusions presented in this thesis are as follows:(1) AtMYB74encodes a R2R3-MYB transcription factor and differentially expresses invarious tissues.By analyzing the database of GENEVESTIGATOR and Arabidopsis eFP Browser withsalt stress treatment,1286genes were found. Promoter analysis of the selected genes was then carried out in Arabidopsis epigenome maps to identify hypermethylated regions. Theresults showed that gene candidates with promoter hypermethylation in response to salt stresswere identified. AtMYB74was chosen for our further study.The full-length cDNA corresponding to the AtMYB74mRNA is975bp and encodes aputative protein of324amino acids. The R2and R3MYB DNA binding repeats (amino acids13to65and66to117, respectively) of AtMYB74are highly conserved with all other MYBproteins in Arabidopsis and in other plant species. AtMYB74is constitutively expressed invarious tissues at low abundance by qRT-PCR analysis and GUS staining detection. Inaddition, onion epidermal cells transformed with an expression plasmid for theAtMYB74-GFP fusion protein exhibited GFP fluorescence in the nucleus.(2) Transgenic plants overexpressing AtMYB74show hypersensitivity to ABA and saltstress during seed germination.Overexpression of AtMYB74also induced the expression of salt stress responsive genes,such as AtRD29B, AtRAB18, and AtRD20. However, under NaCl or ABA treatment, thegermination of OE transgenic seeds was inhibited more severely than that of WT plants andthe germination rates of RNAi transgenic seeds were similar to those of WT plants. Moreover,application of ABA biosynthesis inhibitors (tungstate) in the medium suppressed thehypersensitivity of OE transgenic seeds to NaCl. The reduced germination rates of the OElines suggest that AtMYB74is involved in the response to salt stress during seed germinationin an ABA-dependent manner in plants.(3) Dynamic DNA methylation results in AtMYB74activation in response to ABA andsalt stress.The level of AtMYB74transcripts increased significantly by both NaCl and ABAtreatments in WT plants, indicating that AtMYB74responds to salt stress and ABA signals atthe transcriptional level. In addition, in the RdDM mutants, the NaCl and ABA treatmentsdemonstrate much less changes in the accumulation of AtMYB74mRNA than that in WT,suggesting the involvement of RdDM in the transcriptional regulation of AtMYB74.Meanwhile, the deficiency of active DNA demethylation in ROS1-RNAi and ZDP-RNAitransgenic plants resulted in a slight increment after treatment. The200bp promoter regionapproximately500bp upstream of the transcription initiation site of AtMYB74was analyzed by bisulfite sequencing. WT plants treated with NaCl and ABA exhibited the a visiblereduction in total5-meC contents compared with the control. Interestingly, the percentage ofCpHpH methylation was nearly halved in the treated plants, and only around10%reductionin CpG contexts was detected. And the RNAi transgenic plants showed only a slightreduction in all context of DNA methylation.Time-course analysis revealed that the mRNA level under salt stress showed a increasefrom0.5h, with the highest levels occurring at3h, accompanied by the lowest percentage ofCpHpH methylation. After6h, AtMYB74expression under salt stress was appearently higherin treated plants than that in the control; a lower percentage of CpHpH methylation was alsodemonstrated in WT plants. Similar results were obtained in ABA-treated plants. Theseresults indicate that stress increases the expression of AtMYB74by reducing dynamic DNAmethylation in the CpHpH context.(4) DNA methylation is controlled by the accumulation of24-nt siRNAs targetingAtMYB74promoter.Five24-nt siRNAs (ASRP215119, ASRP41948, ASRP27256, ASRP13208, andASRP2423) located in a cluster were identified to target at a narrow region (-603bp to-477bp) of the2.9kb promoter of AtMYB74. Moreover, bisulfite sequencing analysis of individualclones showed that DNA methylation considerably changes in or near the siRNA targetregion. However, no obvious changes were detected in the5-azaC-treated WT, ddc, dcl3, rdr2,ROS1-RNAi, and ZDP-RNAi transgenic plants, indicating that CpHpH hypomethylation isdependent on RdDM. The accumulation of five24-nt siRNAs was substantially reducedunder the salt stress and ABA in WT plants compared with those in dcl3plants by siRNAqRT-PCR and Northern Blot analysis, suggesting that decreases in DNA methylation causedby the reductions in24-nt siRNA accumulation lead to the activation of AtMYB74under saltstress.(5) Exogenous24-nt siRNAs direct RdDM in the AtMYB74promoter in Nicotianabenthamiana leaves.Considering that the siRNAs diced from the hairpin RNA could induce transcriptionalsilencing of the target genes, two types of expression cassettes were constructed to generatesiRNAs and express reporter gene (R1and R2). In the reporter gene cassettes of pBI121, gusA was driven by the35S promoter (P1), AtMYB74promoter (P2), and mutated AtMYB74promoter with the deletion of a200bp region where these siRNAs are targeted (P3). GUSactivities between groups P1and P1-R1, P2and P2-R2, and P2and P3-R1did not showobvious changes after transient cotransformation. The GUS activity of group P2-R1wasobviously lower than that of group P2. In addition, bisulfite sequencing analysis revealed thatthe DNA methylation in the200bp promoter region increased43%due to the siRNAsyielded by the hairpin RNA. The constructs P1-R1, P2-R1, and P3-R1could efficientlygenerate siRNAs in the transformed tobacco. Overall, results demonstrate that the ectopicexpression of artificial siRNAs targeting the AtMYB74promoter also can regulate GUSexpression through RdDM.(6) Both RdDM and ABA-responsive cis-elements (ABREs) contribute to the regulationof AtMYB74under salt stress in the ABA-dependent pathway.Four putative ABREs were identified by bioinformatics analysis in the AtMYB74promoter. ChIP-PCR detection found that ABI5occupancy on the AtMYB74promoter viaABREs. Although the transcripts of AtMYB74in abi5increased under stress, they remainedlower than those in WT plants, indicating that ABREs also participate in the regulation ofAtMYB74. GUS staining showed that stress-induced accumulation of AtMYB74transcriptscould be detected in P3transgenic seedlings, suggesting that increasing levels of GUS maybe caused by ABREs’ regulation. Furthermore, in abi5, both the CpHpH DNA methylation ofAtMYB74and the accumulation of five24-nt siRNAs remarkably decreased. Theseobservations reveal that both ABREs and RdDM participate in the regulation of the AtMYB74promoter activity to respond to salt stress.更多还原