KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 白桦 BpHOX2 基因响应渗透胁迫机制的研究 | |
| Alternative Title | The mechanism of Betula platyphylla BpHOX2 responses to osmotic stress |
| 谭子龙 | |
| Subtype | 博士 |
| Thesis Advisor | 王玉成 |
| 2020-06-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 理学博士 |
| Keyword | 白桦 渗透胁迫 HD-Zip I ChIP-seq Betula platyphylla osmotic stress HD-Zip I ChIP-seq |
| Abstract | 白桦 (Betula platyphylla) 是东北亚地区重要的树种，耐寒喜湿但不耐干旱和盐。 BpHOX2 基因与拟南芥 AtHB6 和 AtHB16 高度同源，该基因具有保守的 HDZip 结构域，属于 HD-Zip I 亚家族成员，可能参与植物逆境胁迫或生长发育过程。由于干旱和盐胁迫都会产生渗透胁迫，所以用 PEG 和 NaCl 处理来研究渗透胁迫。 通过对 BpHOX2 进行表达分析发现它可能与逆境胁迫相关，本实验系统地研究了 BpHOX2 基因在响应渗透胁迫过程中的功能及调控机制，为白桦逆境胁迫应答调控机制方面的研究提供理论基础。首先克隆了 BpHOX2 基因，通过亚细胞定位确定 BpHOX2 蛋白是一种核定位蛋白，通过 qRT-PCR 分析发现 BpHOX2 基因的表达受盐、 PEG 及 ABA 处理的诱导，说明其参与了白桦的非生物胁迫应答。构建 BpHOX2 基因的过表达载体pILC11-BpHOX2 和抑制表达载体 pTZ-BpHOX2，利用农杆菌介导法得到白桦BpHOX2 基因的过表达及表达抑制株系。在 NaCl 和 PEG 胁迫下， BpHOX2 基因抑制表达株系对胁迫更为敏感，而过表达株系则对 NaCl 和 PEG 耐受能力显著提高。进一步研究发现 BpHOX2 基因能够通过调控 SODs 和 PODs 基因的表达来提高 SOD 和 POD 的活性，降低了 ROS 浓度因此减轻了植株的损伤程度；同时，BpHOX2 基因能够提高 BpP5CS1 和 BpP5CS2 的表达水平来增加脯氨酸的含量，从而显著提升了过表达转基因白桦的抗逆能力。通过染色质免疫沉淀测序（ChIP-seq）与 RNA 测序（RNA-seq）的联合分析对 BpHOX2 结合的元件进行预测，之后利用 EMSA 进行体外互作验证。最终发现 BpHOX2 能与脱水反应元件“RCCGAC”、 Myb-p binding BOX “CCWACC”以及两种新的顺式作用元件“AAGAAG”（HBS1）和“TACGTG”（HBS2）结合调节下游基因表达。研究结果表明， BpHOX2 与不同的顺式作用元件结合调节基因表达，最终提高桦树对渗透胁迫的耐受性。对过表达株系样品免疫沉淀（IP）之后发现BpHOX2 本身在胁迫前后没有发生磷酸化的改变，但是免疫共沉淀（Co-IP）的磷酸化检测发现受到胁迫之后 BpHOX2 互作蛋白的磷酸化水平升高，说明 BpHOX2在逆境中对下游基因的调节作用需要其他互作蛋白的参与。综合以上研究结果，最终得到了白桦响应渗透胁迫机制的一种作用模型： 渗透胁迫诱导 BpHOX2 的表达， BpHOX2 蛋白结合不同的顺式作用元件调节下游基因的表达，进而使白桦植株产生一系列的生理生化上的调整， 包括增加 SOD与 POD 的生物合成提升了 ROS 清除能力，提高了脯氨酸的合成量增强了细胞对渗透压的调节能力，最终提高了植株对渗透胁迫的耐受能力。这个模型对我们理解 HD-Zip 家族成员在抗逆过程中的机制具有深远的意义，为通过分子生物学定向培育具有优良抗逆性状的白桦新品系奠定了基础。 |
| Other Abstract | Birch (Betula platyphylla) is an important tree species in Northeast Asia. It isresistant to cold and damp but not tolerant to drought and salt. The BpHOX2 gene inbirch， highly homologous with Arabidopsis AtHB6 and AtHB16， has a conserved HDZip domain and belongs to the HD-Zip I subfamily. Its function may be involved inabiotic stress or growth and development. Because drought and salt stress both produceosmotic stress, PEG and NaCl treatments were used to study osmotic stress. Expressionanalysis revealed that BpHOX2 may be related to stress. In this experiment, wesystematically studied the function and regulatory mechanism of BpHOX2 gene inresponse to osmotic stress, which provided theoretical basis for the stress response ofbirch.The BpHOX2 gene was cloned, and BpHOX2 protein was determined to be anuclear localization protein by subcellular localization. The Real-time quantitative PCR(qRT-PCR) analysis found that the expression of BpHOX2 gene was induced by salt,PEG and ABA treatment, indicating that it participated in the abiotic stress response ofbirch. The overexpression vector pILC11-BpHOX2 and the inhibition expression vectorpTZ-BpHOX2 of BpHOX2gene were constructed, and transgenic lines of birch wereobtained by Agrobacterium-mediated method. Under NaCl and PEG stress, wild-typebirch as a control, BpHOX2 gene suppression expression lines are more sensitive tostress, while overexpression lines are more resistant to stress phenotypes andphysiological indicators. BpHOX2 gene can increase the activity of SOD (superoxidedismutase) and POD (peroxidase) by regulating the expression of SODs and PODsgenes, reduce the concentration of ROS and thus reduce the damage of plants; increasethe expression level of BpP5CS1 and BpP5CS2 to increase the content of proline, thussignificantly improves overexpression lines stress resistance.Through the combined analysis of chromatin immunoprecipitation sequencing(ChIP-seq) and RNA sequencing (RNA-seq), the interaction elements of BpHOX2were predicted, and then EMSA (electrophoretic mobility shift assay) was used to verify the interaction in vitro. Finally, it was found that BpHOX2 could be bound with thedehydration reaction elements RCCGAC, Myb-p binding BOX CCWACC and two newcis-acting elements AAGAAG (HBS1) and TACGTG (HBS2) bind to regulatedownstream gene expression. The results show that BpHOX2 is a transcription factorthat interacts with different cis-acting elements to regulate gene expression andultimately improve the dehydration tolerance of birch. After immunoprecipitation (IP)of overexpressed lines, it was found that BpHOX2 itself did not undergophosphorylation before and after stress, but the phosphorylation level of BpHOX2interacting protein increased after Co-Immunoprecipitation (Co-IP) was stressed,indicating that the regulation of BpHOX2 on downstream genes in adversity requiresthe participation of other interacting proteins.Finally based on the above research results, an action model of Betula alba inresponse to osmotic stress was obtained. Osmotic stress induces the expression ofBpHOX2, and BpHOX2 protein binds to different cis-acting elements to regulate theexpression of downstream genes, which in turn makes the birch plant produce a seriesof physiological and biochemical adjustments, such as increasing the biosynthesis ofSOD and POD and enhancing the ROS scavenging ability It increases the synthesis ofproline, enhances the ability of cells to regulate osmotic pressure, and ultimatelyimproves the tolerance of plants to osmotic stress. This model has profound significancefor us to understand the mechanism of HD ZIP family members in the process of stressresistance, and laid a foundation for the cultivation of new birch strains with excellentstress resistance through molecular biology. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15405 |
| 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 | 谭子龙. 白桦 BpHOX2 基因响应渗透胁迫机制的研究[D]. 北京. 中国科学院大学,2020. |
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