KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 刚毛柽柳 AP2/ERF 转录因子 ThCRF1 响应盐胁迫的调控机理研究 | |
| 覃利萍 | |
| Subtype | 博士 |
| Thesis Advisor | 王玉成 ; 玉米提·哈力克 |
| 2018-06-05 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 新疆乌鲁木齐 |
| Degree Discipline | 理学博士 |
| Keyword | 细胞分裂素应答因子 乙烯响应因子 刚毛柽柳 盐胁迫 顺式作用元件 Cytokinin response factor Ethylene-Responsive factor Tamarix hispida Salt stress Cis-acting elements |
| Abstract | 乙烯响应因子（ERFs， Ethylene-Responsive factors） 是植物所特有的一类转录因子（TF， Transcription factors）， 涉及多种生物学过程， 尤其是在非生物胁迫方面起重要调控作用。然而，木本盐生植物的 ERFs 很少被研究。本研究中，我们以来自刚毛柽柳（Tamarix hispida）的一个应答于盐胁迫的 ERF 类转录因子成员细胞分裂素应答因子 ThCRF1（CRF， Cytokinin response factor）为研究对象，对其调控耐盐的机制进行了系统研究。（1） ThCRF1 基因的 cDNA 长度 1140 bp，编码 379 个氨基酸的蛋白，该蛋白包含一个典型的 AP2/ERF 保守结构域，且在氨基端存在一个 CRF 保守结构域和一个 TEH 区间，于是我们将这条 ERF 序列命名为 ThCRF1。（2） 利用基因枪、酵母单杂交和双杂交技术，研究发现 ThCRF1 蛋白可以与 TTG、 DRE 和 GCC-box 等顺式作用元件相结合，并具有转录激活结构域的核定位蛋白。（3） 通过瞬时转化的方式获得 ThCRF1 转基因的刚毛柽柳，利用基因获得和功能缺失的方法研究该基因的功能，研究发现 ThCRF1 在刚毛柽柳中过表达（pROKII-ThCRF1-flag）后显著提高了植物对盐胁迫的耐受性；反之， ThCRF1基因的抑制表达（RNAi-ThCRF1） 显著降低了植物对盐胁迫的耐受性。进一步的定量 PCR 实验表明， ThCRF1 蛋白通过诱导脯氨酸合成酶（P5CS，Pyrroline-5-carboxylate synthetase）、海藻糖-6-磷酸合成酶（Trehalose-6-phosphatesynthase， TPS）、海藻糖-6-磷酸磷酸酶（TPP， Trehalose-6-phosphate phosphatase）、超氧化物歧化酶（SOD， Superoxide dismutase）和过氧化物酶（POD， Peroxidase）合成相关基因的表达，导致植物体内脯氨酸和海藻糖含量水平升高， SOD 和POD 活性增加。通过稳定转化获得了过表达的 ThCRF1 转基因拟南芥，对转ThCRF1 基因株系进行了与上述刚毛柽柳抗逆机制类似的研究，结果进一步证实了刚毛柽柳的研究结果。（4） 通过对 ThCRF1 转基因拟南芥株系和野生型拟南芥进行转录组测序（RNAseq） 和免疫共沉淀实验（Chromatin immunoprecipitation assay， ChIPassay） 发现， ThCRF1 转录因子可以通过与 TTG， DRE 和 GCC-box 等顺式作用元件相结合从而诱导抗逆相关基因的表达。综上所述， ThCRF1 转录因子通过增强海藻糖和脯氨酸生物合成来调节渗透势，提高 SOD 和 POD 活性来增强活性氧清除能力，从而提高植物对盐胁迫的耐受性。 |
| Other Abstract | Ethylene-Responsive factors (ERFs) are plant-specific transcription factors(TF), which involve a variety of biological processes, especially in abiotic stress.However, ERFs of woody halophytes that are involved in salt stress are hardlystudied. In this study, we studied ThCRF1, an ERF transcription factor that respondsto salt stress from Tamarix hispida. This study systematically studied the mechanismof ThCRF1 regulation of salt tolerance.(1) ThCRF1 is a protein with a cDNA length of 1140 bp and encoding 379amino acids. The bioinformatics analysis revealed that the ThCRF1 proteincontained a typical AP2/ERF conserved domain and that there was a CRF conserveddomain and a TEH interval at the N-terminus, so we named it ThCRF1.(2) Using the gene gun, yeast one hybrid (Y1H) and yeast two hybrid (Y2H)techniques, it was found that ThCRF1 protein is a nuclear localization protein withtranscriptional activation domain which can be combined with cis-acting elementssuch as TTG, DRE and GCC-box.(3) The transient function of ThCRF1 transgenic T. hispida was used to analyzethe function of the gene by gain and loss of function. It was found that ThCRF1overexpression (pROKII-ThCRF1-flag) in T. hispida significantly improved thetolerance of plants to salt stress, whereas the inhibitory expression of ThCRF1 gene(RNAi-ThCRF1) was significantly reduced the tolerance of plants to salt stress.Further qRT-PCR experiments showed that ThCRF1 induces the expression of genesincluding those encoding pyrroline-5-carboxylate synthetase (P5CS),trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP),superoxide dismutase (SOD) and peroxidase (POD), which lead to enhanced prolineand trehalose levels and increased SOD and POD activities. The overexpressedThCRF1 transgenic Arabidopsis thaliana was obtained by stable transformation, andthe gene function of ThCRF1 transgenic lines with relatively high expression level was studied by quantitative detection. The results further confirmed the results of T.hispida.(4) ThCRF1 transgenic A. thaliana line L2 was used to RNA sequencing andChIP assay the results showed that ThCRF1 could bind to the promoter region of theregulated genes to induce the expression of these stress-related genes.In summary, ThCRF1 improves the tolerance of the plant to salt stress byenhancing the trehalose and proline biosynthesis to regulate the osmotic potential,increase the activity of SOD and POD to enhance the scavenging activity of activeoxygen. |
| Subject Area | 生态学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14938 |
| Collection | 研究系统_荒漠环境研究室 |
| Affiliation | 1.中国科学院新疆生态与地理研究所; 2.新疆大学 |
| Recommended Citation GB/T 7714 | 覃利萍. 刚毛柽柳 AP2/ERF 转录因子 ThCRF1 响应盐胁迫的调控机理研究[D]. 新疆乌鲁木齐. 中国科学院大学,2018. |
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