KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 白桦抗旱、耐镉基因调控网络解析及反向染色质免疫共沉技术的建立 | |
| Alternative Title | The gene expression regulatory network of birch in response to drought or Cd2+ stress and building a Reverse chromatin immunoprecipitation |
| 文雪静 | |
| Subtype | 博士 |
| Thesis Advisor | 王玉成 |
| 2019-06-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 理学博士 |
| Keyword | 干旱胁迫 镉胁迫 转录因子 长链非编码 RNA 染色质免疫沉淀 drought stress Cadmium stress transcription factors LncRNAs chromatin immunoprecipitation |
| Abstract | 白桦（Betula platyphylla） 是一种在东亚分布广泛的阔叶乔木， 常形成大面积纯林，具有分布面积广，蓄积量大的特点。随着全球范围内气候的变化以及工业化的发展，其生境内土壤干旱和重金属离子胁迫程度日益加重， 但目前对白桦响应干旱胁迫和重金属离子胁迫分子机制的研究较少，本文进行了如下研究：（1） 利用基因表达谱(RNA-seq)技术，在白桦中鉴定到了 2917 个响应干旱胁迫的基因， 包括晚期胚胎丰富蛋白(LEA)家族、热激蛋白(HSP)家族、缺水相关蛋白家族和活性氧(ROS)清除蛋白家族以及许多转录因子(TFs)。在干旱胁迫诱导的转录因子中，乙烯响应因子(ERF)和 MYB 家族基因最为丰富。本研究在干旱胁迫信号诱导的基因中选择强烈诱导表达且本身具有高转录水平的两个转录因子 BpERF2 和 BpMYB102，来建立白桦响应干旱胁迫的基因表达调控网络。首先， 过表达 BpERF2 和 BpMYB102 都能提高白桦的抗旱能力。 进一步通过染色质免疫共沉淀(ChIP)和 qRT-PCR 技术相结合， 研究基因间的调控关系。结果表明， BpERF2 可以调控 LEA 和 HSP 家族的基因，而 BpMYB102 调控病程相关蛋白 1(PRP1)和 4-香豆酸:辅酶 A 连接酶 10 (4CL10)等基因。 此外，有多个基因同时受 BpERF2 和 BpMYB102 的调控。进一步鉴定其中一些基因的功能，发现根原基缺陷蛋白 1 (RPD1)、 PRP1、 4CL10、 LEA1、 SOD5 和 HSPs 等基因都具有明显的抗旱能力。研究结果表明，白桦转录因子 BpERF2 和 BpMYB102 通过直接或间接调控一系列干旱胁迫响应基因的表达，来提高白桦对干旱胁迫的的耐受能力。（2） 利用 RNA-seq 技术，在白桦中鉴定到了 30 个响应镉胁迫的长链非编码 RNA (LncRNAs)。 根据这些 LncRNA 的预测靶基因是否响应镉胁迫，选择了其靶基因明显响应镉胁迫的 9 个 LncRNA， 进行进一步研究。其中，增加 LncRNA28068.1 和 2705.1 的表达量能够提高白桦对镉胁迫的耐受力， 而增加 11415.1和 30505.2 的表达量则使得白桦对镉胁迫敏感。 利用 qRT-PCR 研究了这些LncRNA 的预测靶基因是否真为其所调控，结果表明， LncRNA28068.1 和 2705.1可以分别调控其预测靶基因 YLS9 (LEA 家族) 和 HSP18.1 (HSP 家族)的表达，LncRNA11415.1 可以下调其预测靶基因 LLDA (L-乳酸脱氢酶 A)的表达， 而LncRNA30505.2 对其预测靶基因 HRCS2L (类 H/ACA 核糖核蛋白复合体亚基 2蛋白)的表达无调控作用。进一步鉴定了 4 个靶基因的抗镉功能，发现 HSP18.1 和LLDA 基因可以提高白桦对镉胁迫的耐受性。研究结果表明， 镉胁迫条件下，LncRNA2705.1 表达量上升，进而上调预测靶基因 HSP18.1 的表达量， 增强白桦对镉胁迫的抗性， 而 LncRNA11415.1 表达量下降， 可以上调预测靶基因 LLDA的表达量，进而可以增强白桦对镉胁迫的抗性。通过研究以转录因子或 LncRNA 为中心的白桦响应干旱或镉胁迫的表达调控网络，找到白桦抗干旱或镉胁迫的关键调控基因，从而为白桦的抗逆基因工程育种提供理论基础和基因资源。（3） 蛋白质和 DNA 之间的相互作用几乎涉及所有的生物功能。 因此，鉴定与 DNA 结合的蛋白质，具有十分重要的意义。然而，迄今为止，在植物中，以基因为中心的研究方法非常少见。本研究开发了基因为中心的研究目标 DNA结 合 蛋 白 质 的 技 术 ， 称 为 反 向 染 色 质 免 疫 沉 淀 (Reverse chromatinimmunoprecipitation, R-ChIP)。这项技术使用一系列带有生物素标记的 DNA 探针来分离目的染色质或 DNA 片段，然后用质谱分析法鉴定与目标 DNA 片段结合的蛋白质。 R-ChIP 技术所捕获的与目标 DNA 片段结合的蛋白，其蛋白的数量和纯度均满足质谱鉴定。利用 R-ChIP 技术，我们鉴定了与拟南芥 AtCAT3 基因（编码过氧化氢酶 3）启动子结合的蛋白质组，从中鉴定得到了 AtCAT3 基因上游的调控因子。通过 ChIP 和电泳迁移率实验（EMSA）分析，进一步证实了 R-ChIP鉴定到的 AtCAT3 上游调控因子的可靠性。此外，我们对 R-ChIP 方法进行了改进， 通过在植物中瞬时转入目标 DNA 片段， 从而显著提高了捕获与目标 DNA片段结合的蛋白质的效率。这些结果表明 R-ChIP 技术可能在鉴定染色质组成和分离特定基因的上游调控因子等方面得到广泛应用。 |
| Other Abstract | Betula platyphylla (birch) is a widely distributed tree, but its drought-toleranceand Cadmium-tolerance mechanism have been little studied. Gene expression profilesare powerful tools for investigating mechanisms of plant stress tolerance.（1） Using RNA-Seq, we identified 2917 birch genes involved in its response todrought stress. These drought-responsive genes respectively belonging to the lateembryogenesis abundant (LEA) family, heat shock protein (HSP) family, watershortage-related and ROS-scavenging proteins, and some of them are transcriptionfactors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF)and myeloblastosis oncogene (MYB) families were most abundant. BpERF2 andBpMYB102, which were strongly induced by drought and had high transcriptionlevels, were selected to study their regulatory networks. BpERF2 and BpMYB102both played roles in enhancing drought tolerance in birch. Chromatinimmunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genessuch as those in the LEA and HSP families, while BpMYB102 regulated genes suchas Pathogenesis-related Protein 1 (PRP1) and 4-Coumarate: Coenzyme A Ligase 10(4CL10). Multiple genes were regulated by both BpERF2 and BpMYB102. Wefurther characterized the function of some of these genes, and the genes that encodeRoot Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs werefound to be involved in drought tolerance. Therefore, our results suggest that BpERF2and BpMYB102 serve as transcription factors that regulate a series ofdrought-tolerance genes in B. platyphylla to improve drought tolerance.（2） Using RNA-Seq, we identified 30 Long non-coding RNAs (LncRNAs)involved in its response to Cadmium stress from birch. Among theseCadmium-responsive LncRNAs, 9 LncRNAs, the target genes of which weredifferentially expressed, were selected to study their regulatory networks. LncRNA28068.1 and 2705.1 both played roles in enhancing Cadmium tolerance in birch.LncRNA 11415.1 and 30505.2 both played roles in reducing Cadmium tolerance inbirch. The result of qRT-PCR indicated that LncRNA 28068.1 and 2705.1 both enhanced the expression of their target genes 2>YLS9 (LEA family) and HSP18.1(HSP family), and LncRNA 11415.1 reduced the expression of its target gene LLDA(L-lactate dehydrogenase A), while LncRNA 30505.2 had no effect on the expressionof its target gene HRCS2L (H/ACA ribonucleoprotein complex subunit 2-like protein).We further characterized the function of the four target genes, HSP18.1 and LLDAwere found to be involved in Cadmium tolerance. Therefore, our results suggest thatLncRNA 2705.1 regulates the expression of its target gene HSP18.1 to improveCadmium tolerance in B. platyphylla, and LncRNA 11415.1 regulates the expressionof its target gene LLDA to play a negative role in Cadmium tolerance.（3） Interactions between proteins and DNA are involved in nearly all biologicalfunctions; therefore, the identification of DNA binding proteins is necessary andimportant. However, until now, few gene-centered methods have been developed forplants. In the present study, we developed a technique to capture DNA-associatedproteins called reverse chromatin immunoprecipitation (R-ChIP). This technologyuses a set of specific DNA probes labeled with biotin to isolate chromatin or DNAfragments, and the DNA associated proteins are then analyzed using massspectrometry. This method can capture DNA associate proteins with sufficientquantity and purity for identification. Using the R-ChIP technology, we identified theproteins that binding to the promoter of the Arabidopsis AtCAT3 gene (encodingcatalase 3), and the upstream regulators of AtCAT3 were identified. The reliability ofR-ChIP in identification of the upstream regulators of AtCAT3 was further confirmedby ChIP and electrophoretic mobility shift assays (EMSA). In addition, we improvedthe R-ChIP method using plants in which the DNA of interest was transientlyintroduced, that improved the efficiency of protein capture significantly. Takentogether, these results suggested that R-ChIP might have a wide application tocharacterize chromatin composition and isolate upstream regulators of a specific gene. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15362 |
| Collection | 中国科学院新疆生态与地理研究所 研究系统 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 文雪静. 白桦抗旱、耐镉基因调控网络解析及反向染色质免疫共沉技术的建立[D]. 北京. 中国科学院大学,2019. |
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