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| 蒙古沙冬青响应干旱胁迫的分子机制及转录因子AmNAC1在调控铁营养中的作用研究 | |
| 其他题名 | Molecular mechanisms of drought-resistance in Ammopiptanthus mongolicus and the role of transcription factor AmNAC1 in regulating iron homeostasis |
| 金曼 | |
| 出版年 | 2018 |
| 学位类型 | 博士 |
| 导师 | 苏彦华 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 蒙古沙冬青（Ammopiptanthus mongolicus）是一种古老的豆科灌木，起源于距今200-6500万年的第三纪，主要分布于我国内蒙古阿拉善沙漠地带。该地区年降水量不足100 mm，而年蒸发量却高达3000 mm以上，属于极端干旱气候。本文是在实验室前期研究的基础上，借助高通量转录组测序技术，探究沙冬青响应干旱胁迫的全局性分子机制，并与在拟南芥等模式植物中获得的基本认知相比较，借以揭示沙冬青在长期适应持续干旱胁迫过程中的特殊生存策略及其与维持养分吸收利用之间的关联。本文取得的主要研究结果如下： 1、通过Illumina HiSeq2000TM高通量转录组测序技术，经过无参考序列从头拼接，获得了蒙古沙冬青共44959条基因序列，为后续研究提供了巨大的基因资源。在早期干旱胁迫下，沙冬青地上部鉴定到1035个差异表达基因（DEGs），其中上调和下调的DEGs分别为469个和566个；而根部的1210个差异表达基因中，上调和下调的DEGs分别720个和490个。2、对测序中所得的DEGs进行功能以及代谢通路分析，并对部分DEGs进行了定量PCR验证，发现地上部有8 个DEGs，地下部有12 个DEGs参与了ABA的合成以及信号传递途径。这些基因的变化与该激素在体内的测定含量上升相一致，反映了ABA信号过程也是沙冬青响应干旱胁迫的基本特征之一。然而体内ABA含量的上升及其信号系统的激活，并未导致沙冬青气孔的高效关闭。受到干旱胁迫后，编码ABA信号途径中的PP2Cs基因的表达量被显著诱导，而下游的蛋白激酶SnRK2及其调控的气孔关闭的离子通道基因表达量没有显著变化。气孔观测实验结果表明，与拟南芥相比，沙冬青气孔的关闭过程对ABA的敏感性显著降低；相反，在气孔开放方面，沙冬青则表现出明显的钾浓度依赖性，这与实验室前人关于沙冬青气孔开放通道AmKAT1的钾吸收功能严格受到钾离子浓度控制的报道相吻合。3、实验室前期结果表明，在干旱胁迫时，沙冬青体内的脯氨酸含量急剧上升。该渗透物质的大量积累一方面可维持细胞的持水性，另一方面也有助于清除体内的活性氧；因此对沙冬青抗氧化防御系统关键酶的测定中，并未明显发现其活性受干旱胁迫的强烈诱导。转录组测序中发现14个与脯氨酸合成代谢途径相关的基因，其表达水平的变化符合促进脯氨酸合成、抑制该物质的代谢降解和增强体内运输进而使之大量积累的规律。另外，在地上部和根部分别有19个和36个差异基因分别参与了14 条不同的碳水化合物合成代谢途径，包括促进可溶性糖的合成与积累。编码干旱胁迫保护性二糖的海藻糖的基因表达量显著上升，有利于该物质的合成积累。4、在上述沙冬青的转录组数据中进一步进行了转录因子分析，共发现有49个转录因子家族，包含1575个unigenes。干旱胁迫下，沙冬青地上部有44个转录因子差异表达（33个上调，11个下调）；地下部有57个转录因子差异表达（50个上调，7个下调）。其中NAC转录因子家族中共有11个基因差异表达，为差异表达转录因子个数最多的家族。本文克隆获得了其中上调最为剧烈的一个NAC转录因子，命名为AmNAC1。AmNAC1全长1008bp，包含两个内含子，编码335个氨基酸，具有典型的NAC结构域。通过亚细胞定位实验证明其定位于细胞核，且核定位信号位于N端；通过酵母及显色实验证明其具有转录活性，且活性部位位于C端。克隆获得AmNAC1的部分启动子序列，其中包含一个ABA响应因子结合元件ABRE。定量PCR发现，AmNAC1的表达受到干旱胁迫、盐胁迫、渗透胁迫以及ABA胁迫的响应，是一个典型的响应非生物胁迫的转录因子。5、通过转基因将AmNAC1过表达至拟南芥中，发现过表达株系较野生型拟南芥对非生物胁迫的耐性有显著提高。具体表现为：在NaCl胁迫以及Mannitol模拟的渗透胁迫下，过表达株系种子的萌发率高于野生型；在ABA诱导下，过表达株系气孔的开放度显著低于野生型；离体叶片的失水率实验结果发现，过表达株系水分散失率要低于野生型；控水实验表明，过表达AmNAC1后的拟南芥增强了植物的耐旱能力。通过对拟南芥过表达株系与野生型进行转录组测序，分析两者间差异表达的基因，发现存在355个DEGs，其中差异表达的转录因子为41个，差异表达的蛋白磷酸酶基因为16个，通过GO功能分类发现有86个DEGs属于胁迫响应基因。在蒙古沙冬青转录组和拟南芥转录组中，都发现编码PP2C和OST的基因差异表达，进而通过酵母双杂实验以及双分子荧光互补实验证明了AmNAC1与AmPP2C和AmOST1存在蛋白间相互作用。表明AmNAC1参与到ABA的信号传递途径中，从而调控下游基因的表达，增强了植物的耐干旱能力。6、植株元素含量测定结果发现，过表达AmNAC1株系体内铁含量显著高于野生型。由拟南芥转录组测序得到的差异基因中发现， 调控铁吸收的bHLH转录因子差异表达，与铁吸收相关的基因IRT1以及FRO2差异表达，且定量PCR实验结果与转录组结果相吻合。同时，通过酵母双杂实验以及荧光双分子互补实验证明了AmNAC1与AtbHLH38和AtbHLH100之间存在相互作用。说明在拟南芥中过表达的AmNAC1参与到铁的吸收积累过程中，从而调控下游基因的表达，增强了植物对铁的吸收能力。综上所述，本研究揭示了沙冬青中由干旱胁迫/ABA诱导的气孔关闭效率比拟南芥等正常植物显著降低，该过程与其体内较高的PP2C（type 2C protein phosphatase，ABA诱导气孔关闭的负调控组份）水平有重要关系。这一调控策略可使得沙东青在持续干旱胁迫下保持一定的气孔开度，进而可能维持有限的光合作用，并通过蒸腾拉力驱动水分和矿质养分向地上部的传输。另外，初步证明了沙冬青的干旱胁迫调控转录因子AmNAC1除参与干旱等非生物胁迫响应之外，在拟南芥中过表达还可通过与bHLH转录因子家族成员相互作用，增强植株对铁的吸收和积累。 |
| 英文摘要 | Ammopiptanthus mongolicus, a specific evergreen xerophyte of Leguminosae in Alashan desert and an ancient relic from the Tertiary period, can survive under the extreme drought condition with the annual precipitation less than 100 mm and the annual evaporation over 3000 mm. This study is based on previous research in our lab. By performing Illumina Hiseq2000TM sequencing, we tried to explore the molecular mechanisms of A. mongolicus in response to drought stress. Compared with the current knowledge of Arabidopsis, we attempted to reveal the different survival strategies of A. mongolicus in suffering persistent drought stress and in maintaining the acquisition of mineral nutrients.The main results in this paper are as followed:1. By performing Illumina Hiseq2000TM sequencing and de novo assembly, we obtained a total of 44959 unigenes and these data provide a new gene resource to study drought response of A. mongolicus. The result showed that there are 1035 differentially expressed genes (DEGs) in shoot (566 up-regulated, 469 down-regulated) and 1210 unigenes (490 up-regulated, 720 down-regulated) in root were differentially expressed after 6 hrs 18% PEG stress. 2. We performed function analysis and metabolic pathway analysis of these DEGs, and also performed q-PCR verifications of some selected DEGs. We found that there are 9 DEGs in shoot and 12 DEGs in root involved in ABA synthase and signal pathway. In line with the raised levels of ABA, these DEGs involved in ABA synthesis were up-regulated. In A.mongolicus, ABA signaling is one of mechanisms in response to drought stress. Although as a general consequence, a raised level of ABA is also induced in A. mongolicus following water deficit, this does not accordingly result in efficient stomatal closure. In consistent with this phenomenon, the expression of genes coding for the negative regulators of the ABA signaling cascade—the type 2C protein phosphatases (PP2Cs) are notably induced, whereas the transcription of the downstream SnRK2 protein kinase genes or the destination ion fluxing channel genes remain almost unaffected under water deficit treatments. Stomatal aperture assays showed that，compared to Arabidopsis, the closure of stomata in A.mongolicus was less sensitivity to the induction of ABA; while，the opening of stomata was regulated by external K+ concentrations. This is coincided to the reports of a constrained stomatal opening controlled by extracellular K+ concentrations. 3. Former results showed that proline content was significantly increased under drought stress. Proline is considered to act not only as an osmolyte, but also a ROS scavenger. In consist with the antioxidant enzymes assay in A.mongolicus; the activity of these antioxidant enzymes was not significantly increased. As one of the most important organic osmolytes, there are 14 unigenes involved in proline synthesis and metabolism pathway. The expression pattern of these genes coincided to promoting proline synthesis, inhibiting proline degradation and enhanced proline transport, so that proline accumulated with a high level. And in carbohydrate metabolism pathway, we found 19 DEGs in shoot and 36 DEGs in root participate in 14 carbohydrate metabolism pathways, especially in starch and sucrose metabolic pathways. Unigenes encoded trehalose synthesis were up-regulated significantly, which promoted the accumulation of trehalose (also called survival substance).4. Through transcription factor analysis, there are 1575 unigenes classified into 49 transcription factor families, with 44 transcription factors (33 up-regulated and 11 down-regulated) in shoot and 57 transcription factors (50 up-regulated and 7 down-regulated) in root were differentially expressed in A. mongolicus under 18% PEG stress. We found 11 DEGs in NAC transcription factor family in response to drought stress, which is the most among all the transcription factor families. We obtained a typical NAC transcription factor AmNAC1, with 1008 bp, containing two introns and coding 335 amino acids. Subcellular localization experiment demonstrated that AmNAC1 is located in the nucleus and the nuclear location signal is on the N end. Transcriptional activation assay showed that AmNAC1 possesses transcription activity and the transcription activity is on the C end. We obtained a partial of promoter sequence by performing high-efficiency thermal asymmetric interlaced PCR and found a cis-acting element ABRE, involved in the abscisic acid responsiveness. By q-PCR analysis, we found that AmNAC1 is an abiotic transcription factor which is in response to ABA, drought stress, salt stress and osmotic stress.5. Overexpressed AmNAC1 in Arabidopsis enhanced plants tolerance to abiotic stress. The germination rate of the seeds in overexpressed lines (OE) was higher than the wild type (WT) under NaCl and mannitol stress. The stomatal aperture index of OE plants was smaller than WT plants under ABA condition, indicated that in OE plants stoma close faster and more sensitive. The water loss rate of detached leaves from AmNAC1 OE plants was significantly lower than that of WT plants during dehydration stress. Consistent with these results, most WT plants wilted while OE plants remained turgid and green after withheld water. We found 355 DEGs between 35S:AmNAC1 transgenic Arabidopsis and wild type plants under normal condition by transcriptomic analysis. Among these DEGs, there are 41 transcription factors and 16 protein phosphatases differentially expressed. GO function analysis showed that 86 DEGs were classified into stress response gene. We found several unigenes encoded PP2C and OST both differentially expressed in A. mongolicus transcriptome data and Arabidopsis transcriptome data. The interaction between AmNAC1 and AmPP2C, AmNAC1and AmOST was demonstrated by both yeast two-hybrid experiment and bimolecular fluorescence complementation (BiFC) experiment. These results indicated that AmNAC1 is involved in ABA signal pathway and regulated downstream genes expression, thus enhanced plant tolerance to abiotic stress.6. The element content of AmNAC1 OE plants and WT plants was measured by HPLC-ICP-MS. The results show that iron content in AmNAC1 OE lines was significantly higher than that in WT plants, while other elements show no significant difference. According to the Arabidopsis transcriptome data, we found IRT1, FRO2 and several bHLHs, which related to iron up-take pathway differentially expressed. These results were consistent with q-PCR analysis. Furthermore, by performing yeast two-hybrid experiment and bimolecular fluorescence complementation (BiFC) experiment, the interaction between AmNAC1 and AtbHLH38, AmNAC1 and AtbHLH100 was demonstrated. These results implied that AmNAC1 participate in iron up-take pathway, regulating downstream genes expression and enhance iron absorption in Arabidopsis.In summary, this study revealed that the stomatal movement in A. mongolicus is less sensitive to drought stress/ABA than in Arabidopsis, which is related to the high expression level of PP2Cs (type 2C phosphatase, a negative regulator of ABA signaling cascade). This strategy can make A. mongolicus survive under persistent drought stress by maintaining the necessary stomatal required for the entrance of photosynthetic and acquisition of mineral nutrients through a transpiration driven force. Furthermore, we found AmNAC1, a typical NAC transcription factor, which is in response to abiotic stress. In the overexpressing AmNAC1 Arabidopsis, AmNAC1 is participated in iron uptake through the interaction with bHLHs transcription factors. |
| 中文关键词 | 蒙古沙冬青 ; 干旱胁迫响应 ; 分子机制 ; NAC转录因子 ; 铁营养 |
| 英文关键词 | Ammopiptanthus mongolicus drought stress response molecular mechanisms NAC transcription factors iron nutrition |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 植物营养学 |
| 来源机构 | 中国科学院南京土壤研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/288104 |
| 推荐引用方式 GB/T 7714 | 金曼. 蒙古沙冬青响应干旱胁迫的分子机制及转录因子AmNAC1在调控铁营养中的作用研究[D]. 中国科学院大学,2018. |
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