侧柏对干旱与自然低温胁迫响应的分子机制研究

发布时间：2018-05-21 10:52

  本文选题：侧柏(Platycladus + orientalis(L.))　； 参考：《西北农林科技大学》2017年博士论文

【摘要】：侧柏(Platycladus orientalis(L.))耐干旱瘠薄、寿命长、抗病虫、具有较强的适应性,并且耗水量低,是干旱半干旱地区的造林先锋树种。侧柏在中国分布较广,历来作为名地古刹的重要栽培树种;在中国许多地区均有树龄超过千年的侧柏,目前对古侧柏的研究还较为滞后,许多科学问题亟待解决,如侧柏生长千年而不衰的分子机制和近年来一些千年古侧柏出现衰退迹象的影响机制问题。作为一个长寿树种,侧柏对环境胁迫的抗性基础可能是其生长千年而不衰的重要因素之一,同时近年来极端不良的环境因素也可能是使古树出现衰退的主要原因。本论文深入研究了水分胁迫条件下侧柏的抗逆分子机制,同时开展了古侧柏低温环境响应的分子机理研究,为揭示侧柏的抗衰老机制、衰退机理奠定基础。主要的研究结果如下:1.侧柏幼苗干旱胁迫根系转录组测序研究与差异基因筛选对侧柏幼苗开展温室模拟干旱实验,经过两个月的干旱胁迫处理,采集侧柏根系建立c DNA文库,得到了53.1-63.4百万clean reads。经过对高质量clean reads进行de novo组装和去除冗余最终得到148,392条unigene。对最终保留的116,716条unigene序列做后续分析,注释结果表明29.9%(34,845/116,716)的基因注释到NCBI nr数据库,20.2%(23,573/116,716)注释到Swiss-Prot蛋白数据库。综合分析,一共有36,900 unigenes(31.62%)至少成功注释到一个数据库中(NCBI nr,Swiss-Prot,KEGG,GO和Pfam),有7998 unigenes(6.85%)同时注释到上述5个数据库。通过GO数据库比对,116716条unigene中有17012条基因被赋予4441个GO条目,划分为60个功能组。对116716条unigene做进一步的KEGG通路分析表明一共有13312(11.41%)条unigene被分配到5个功能通路大类和98个KEGG通路中。通过基因差异分析,得到3930个差异表达基因,其中881个基因发生上调表达,3049个基因发生下调表达。这其中检测到194个转录因子在干旱胁迫下表现出显著表达变化。通过差异基因分析以及基因功能富集分析得到了和侧柏干旱胁迫防御/响应相关的候选基因,这些差异基因中具有信号转导、生物合成、ROS清除、离子/能量转运、蛋白酶、大分子保护等功能。本研究在转录水平得出侧柏干旱分子防御/响应机制是基于众多的基因直接参与侧柏干旱胁迫的保护过程,这包括大量信号转导系统基因、调控基因和功能基因的参与,基于此得到了侧柏干旱胁迫分子响应复杂调控机制:包括,干旱胁迫第一阶段,根系细胞通过膜和/或细胞的变化感受水分胁迫,接着细胞激活Ca2+信号、ROS信号和激素信号(信号感知阶段)引起下游胁迫响应基因的表达,包括转录因子、离子/能量转运蛋白、大分子的防护因子、解毒作用因子、渗透物质合成关键酶和蛋白酶类。总体来说,这些基因可以分成两组:第一组包含的基因(蛋白质)主要参与基因表达的调控(调控蛋白/转录因子),第二组包含基因(蛋白质)主要在胁迫适应性过程起直接防御/修复作用(功能蛋白),通过这些被激活的胁迫响应基因表达最终使细胞重新恢复稳定和平衡,修复和保护被干旱胁迫破坏的细胞膜和蛋白质。2.侧柏根系、叶组织干旱胁迫的生理与蛋白质组学分析基于侧柏转录组测序的完成,为进一步理解侧柏干旱胁迫响应/防御机制,采集四个水分控制条件下(正常水分供应:80±5%的田间持水量;轻度的水分胁迫:70±5%的田间持水量;中等水分胁迫:50±5%的田间持水量;严重干旱胁迫:35±5%的田间持水量)侧柏幼苗根系和叶组织样本开展生理学和蛋白质组学分析。生理研究表明:轻度的水分胁迫不会对侧柏产生显著影响,侧柏根系和叶组织可溶性蛋白、叶片叶绿素含量未见显著变化。中等和严重水分胁迫导致了幼苗在形态上和生理上均有较显著的变化,包括显著减少叶RWC、根系WC和净生长。从净生长来看,20天的严重干旱胁迫和25天的中等干旱胁迫已经对侧柏幼苗生长产生显著的影响。蛋白质组学研究揭示了水分梯度胁迫下根系和叶组织蛋白质表达差异特征。研究分别对根系和叶组织97个和65个具有显著差异表达的蛋白质进行质谱鉴定。从这些蛋白质的功能分类来看这些蛋白质主要参与胁迫响应/防御、碳水化合物代谢过程和氮代谢。从整体的蛋白质变化特征可以得出,侧柏在干旱环境下生存需要许多的策略;如,保持细胞内蛋白质稳定、活性和折叠;ROS解毒过程、调节细胞的渗透势、维持细胞壁完整性、能量代谢和细胞骨架稳定等。综合根系转录组和蛋白质组学结果总体来看,当胁迫发生转录和蛋白质水平调控具有较大的一致性。3.侧柏干旱胁迫响应基因在干旱后复水表达分析验证对得到的干旱胁迫防御/响应相关基因进一步的表达验证,筛选了转录因子WRKY53、NAC72和AP2/ERF;蛋白激酶CDPK34、CDPK16和CDPK1;活性氧清除相关基因Fe-SOD、CAT3、POD3、APX3、APX6;渗透调节相关基因SUS1、SUS3、SUS6、TRE6PS、P5CS2和P5CR;保护因子蛋白s HSP(HSP18.1、HSP23.5和HSP25.3)、HSP70和LEA;14-3-3like蛋白、皮部储藏蛋白(BSP)基因进行了干旱和复水后基因的表达量分析。基因表达分析得出:1.转录因子NAC72参与侧柏干旱胁迫调控,NAC72基因的高表达可能对下游基因的激活有重要调控作用;转录因子AP2/ERF表现出明显的复水补偿特征,可能和侧柏复水后生长、组织损伤修复有关。2.APX3、Tre6PS、LEA、14-3-3蛋白在干旱下显著上调表达可能参与了侧柏干旱响应的相关调控。3.脯氨酸合成关键基因(P5CS2)表达水平分析得出,P5CS2在侧柏干旱胁迫防御/响应过程中可能起到促进渗透调节物质合成的作用。4.s HSP和HSP70在侧柏干旱胁迫不同时期高丰度上调表达,可能参与了干旱不同程度的胁迫修复或响应;研究还发现部分基因在根系和叶组织响应干旱胁迫中具有协同性和不同步性。4.古侧柏叶冬季自然低温响应的生理与差异蛋白质组学研究对树龄20±5,500±100和1200±200年侧柏的叶组织开展自然低温胁迫响应的生理学与蛋白质组学研究,结果证实古树尤其以千年古树受低温的影响更大,叶组织叶绿素、可溶性蛋白质含量的显著下降,MDA含量的升高,SOD、POD活性的下降都成为古树在低温下的重要标志,也预示着古树叶的早衰可能和环境的影响有重要关系。经过对三个树龄叶组织蛋白质电泳分析和差异蛋白质MALDI TOF/TOF MS/MS鉴定,77个差异蛋白质凝胶点得到成功鉴定和注释。这些成功鉴定的蛋白质可以划分为多个功能组,包括防御/胁迫相关、能量和碳水化合物代谢相关、光合作用和激素相关等。从蛋白质组学的对比分析得出成年树和古树间存在较大的蛋白质响应差异,如在低温下参与光合作用、防御/胁迫响应蛋白丰度在古树中(尤其是千年古树中)显著下降。然而,功能分类为能量和碳水化合物代谢相关蛋白在古树中多为高丰度表达。从蛋白质组学分析总体来看,千年古树表现出较为明显的低温下叶衰退迹象;但在古树的叶组织中也筛选到一些高丰度胁迫防御、大分子保护物质、激素合成等相关蛋白,这些蛋白质可能是古侧柏抗衰老的重要因子。生理和蛋白组学分析展示了古树低温响应的一个复杂的生理和分子差异变化特征框架。
[Abstract]:Platycladus orientalis (L.) is a forestation pioneer tree in arid and semi-arid areas with drought and barren resistance, long life, strong adaptability and low water consumption. It is widely distributed in China and has always been used as an important planting tree in the famous ancient temples; in many areas there are more than 1000 year old Platycladus orientalis in China. The study of the ancient Platycladus orientalis is still lagging behind, and many scientific problems need to be solved, such as the molecular mechanism of the unfailing growth of the Platycladus orientalis and the mechanism of the decline of some millennial Platycladus orientalis in recent years. As a long-lived tree, the resistance basis of the Platycladus orientalis to environmental stress can be one of the important factors for its growth for a thousand years. At the same time, extreme adverse environmental factors may also be the main reasons for the decline of ancient trees. This paper studies the anti inverse molecular mechanism of Platycladus orientalis under water stress, and studies the molecular mechanism of the environmental response of Platycladus orientalis at low temperature, which lays the foundation for revealing the mechanism of anti aging and the mechanism of decline of the Platycladus orientalis. The results are as follows: 1. the root transcriptional group of Platycladus orientalis seedling drought stress was sequenced and the differential gene screening was used to carry out simulated drought experiment in the greenhouse. After two months of drought stress treatment, the C DNA library was set up by the root system of Platycladus orientalis, and 53.1-63.4 million clean reads. was assembled and gone through the high quality clean reads. In addition to redundancy, 148392 unigene. sequences were eventually obtained for the final 116716 UniGene sequences that were retained. The annotation results showed that 29.9% (34845/116716) gene was annotated to the NCBI NR database, 20.2% (23573/116716) annotated to the Swiss-Prot protein database. A total of 36900 unigenes (31.62%) had been annotated to at least one In the database (NCBI NR, Swiss-Prot, KEGG, GO and Pfam) with 7998 unigenes (6.85%) simultaneously annotated to the above 5 databases. 17012 genes in 116716 UniGene are assigned to 4441 GO entries and 60 functional groups by the GO database alignment. Further KEGG path analysis for 116716 UniGene shows that there are 13312 (11.41%) entries in all. Gene was assigned to 5 functional pathways and 98 KEGG pathways. Through genetic diversity analysis, 3930 differentially expressed genes were obtained, of which 881 genes were up-regulated and 3049 genes were down regulated. Among them, 194 transcriptional factors showed significant changes under drought stress. Gene function enrichment analysis has obtained candidate genes related to the defense / response of Platycladus orientalis drought stress. These genes have signal transduction, biosynthesis, ROS scavenging, ion / energy transport, protease, macromolecule protection and other functions. This study shows that the mechanism of defense / response of Platycladus orientalis drought molecules is based on numerous genes Direct participation in the protection process of Platycladus orientalis drought stress, which includes a large number of signal transduction system genes, regulatory genes and functional genes involved. Based on this, the complex regulation mechanism of the response of droughts stress molecules in the Platycladus orientalis is obtained: the first stage of drought stress, the root cells feel water stress through the changes of membrane and / or cells, and then cell excitation Live Ca2+ signal, ROS signal and hormone signal (signal perception stage) cause the expression of downstream stress response gene, including transcription factor, ion / energy transporter, macromolecule protective factor, detoxification factor, osmotic substance synthesis key enzyme and protease. Generally speaking, these genes can be divided into two groups: the first group of genes ( Protein) is mainly involved in the regulation of gene expression (regulatory protein / transcription factor), and the second groups of genes (proteins) mainly play a direct defense / repair function (functional protein) in the process of stress adaptation, which eventually restores and balances the cells through these activated stress response genes, and repairs and protects drought stress. The damaged cell membrane and the protein.2. of Platycladus orientalis root, the physiological and proteomic analysis of leaf tissue drought stress were based on the complete sequence of the Platycladus orientalis transcriptional group. In order to further understand the response / defense mechanism of Platycladus orientalis drought stress, under four water control conditions (normal water supply: 80 + 5% field water holding capacity; mild water stress: 70 + 5%. Field water holding capacity, medium water stress: 50 + 5% field water capacity, severe drought stress: 35 + 5% field water capacity), physiological and proteomics analysis of root and leaf tissue samples of Platycladus orientalis Seedlings. Physiological studies show that mild water stress does not significantly affect the yield of Platycladus orientalis, root and leaf tissue soluble protein of Platycladus orientalis, There was no significant change in leaf chlorophyll content. Medium and severe water stress resulted in significant changes in the morphology and physiology of seedlings, including significantly reducing leaf RWC, root WC and net growth. From the net growth, 20 days of severe drought stress and 25 days of medium drought had a significant effect on the growth of the seedlings of the Platycladus orientalis. Proteomics studies revealed differences in protein expression in root and leaf tissues under water gradient stress. 97 and 65 proteins with significant differences in root and leaf tissues were identified by mass spectrometry. These proteins were mainly involved in stress response / defense and carbon hydration from the functional classification of these proteins. Metabolic processes and nitrogen metabolism. From the overall characteristics of protein change, it can be concluded that the Platycladus orientalis needs a lot of strategies in the drought environment, for example, maintaining protein stability, activity and folding in the cell; ROS detoxification process, regulating cell osmotic potential, maintaining cell wall integrity, energy metabolism and cytoskeleton stability. Integrated root transcription. The results of the group and proteomic analysis showed that when the transcription and protein level of stress were regulated, the response genes for drought stress response genes of.3. in the drought stress were verified to verify the further expression of defense / response related genes of drought stress, and the transcription factor WRKY53, NAC72 and AP2/ERF were screened and the eggs were screened. CDPK34, CDPK16 and CDPK1; active oxygen scavenging related genes Fe-SOD, CAT3, POD3, APX3, APX6; osmotic regulation related genes SUS1, SUS3, SUS6, TRE6PS, etc. Analysis. Gene expression analysis showed that the 1. transcription factor NAC72 participates in the regulation of drought stress of Platycladus orientalis, the high expression of NAC72 gene may play an important role in regulating the activation of the downstream genes; the transcription factor AP2/ERF shows obvious rehydration characteristics and may grow after the rehydration of the Platycladus orientalis, and the tissue damage repair is related to the.2.APX3, Tre6PS, LEA, 14-3-3 protein. Significant up-regulated expression in drought stress may participate in the related regulation of the response of Platycladus orientalis drought response.3. proline synthesis key gene (P5CS2) expression level analysis, P5CS2 may play the role of promoting the synthesis of osmotic regulators in the process of drought stress and response of Platycladus orientalis,.4.s HSP and HSP70 are high abundance at different periods of drought stress in the Platycladus orientalis. Up-regulated expression may be involved in the drought stress repair or response to different degrees of drought. The study also found that some genes have synergism and asynchrony in response to drought stress in root and leaf tissues. The physiological and differential proteomics of the natural low temperature response of.4. palaeo cypress leaves in winter is 20 + 5500 + 100 and 1200 + 200 years old. The physiological and proteomics studies of the response to natural low temperature stress showed that the ancient trees were more affected by the low temperature, the chlorophyll, the content of soluble protein, the content of MDA and the decrease of the activity of SOD and POD were all the important signs of the ancient trees at low temperature and the ancient trees. The premature senility of leaves may have an important relationship with the environmental impact. After the protein electrophoresis analysis of three tree age leaves and identification of the differential protein MALDI TOF/TOF MS/MS, the 77 differentially identified protein gel points are successfully identified and annotated. The proteins identified successfully can be divided into several functional groups, including defense / stress correlation, and energy. Correlation of carbohydrate metabolism, photosynthesis, and hormone related. Comparative analysis of proteomics from proteomics shows that there are significant differences in protein responses between adult and ancient trees, such as photosynthesis at low temperatures, and the abundances of defense / stress response proteins are significantly decreased in ancient trees (especially in Millennium ancient trees). The proteins associated with energy and carbohydrate metabolism are highly expressed in ancient trees. From the analysis of proteomic analysis, the millennial ancient trees showed obvious signs of lower hypothermia, but in the leaf tissues of the ancient trees, some high abundance stress defenses, large sub protections, hormone synthesis and other related proteins were also screened in the leaf tissues of the ancient trees. Protein may be an important factor in antiaging of Platycladus orientalis. Physiological and proteomics analysis shows a complex framework of physiological and molecular variation in the low temperature response of ancient trees.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S791.38
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本文编号：1918899