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题名: 丛枝菌根增强植物抗旱性分子机理研究(博士后)
作者: 李涛
学位类别: 博士后
答辩日期: 2016-03
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 战爱斌
关键词: AM真菌,共生体系建立,基因组对比分析,抗性基因,甘油跨膜通道, ar/R结构域,甘油运输,定点突变,脱落酸,抗旱性,基因调控,玉米 ; AM fungi, symbiotic establishment, comparative genomics, stress-responsive genes, aquaglyceroporin, aromatic/arginine constriction, glycerol transport, point mutation, abscisic acid, drought tolerance, gene regulation, maize
其他题名: Molecular basis for enhancement of plant drought tolerance by arbuscular mycorrhizal symbiosis
学位专业: 环境科学
中文摘要:       诸多研究表明丛枝菌根(arbuscular mycorrhiza,AM)对于植物适应各种逆境胁迫具有重要生态学意义。然而,目前对于AM真菌如何促成菌根共生体建立以及AM真菌和宿主植物间如何进行信号交流的认识极其有限。本论文从AM真菌基因组入手,通过对比分析,旨在挖掘AM共生体系建立及抗性相关关键基因。在此基础上,以抗旱蛋白GintAQPF2为例,通过对其进行功能验证,探讨GintAQPF2结构和功能特征对共生体建成的意义。另一方面,采用分根培养系统,基于植物和AM真菌基因的表达特征,阐明干旱条件下宿主植物和AM真菌间的逆境信号交流机制。本研究的主要内容和结果归纳如下:
1AM共生体系建立及抗性相关基因的筛选
      AM真菌基因组的公布加速了其基础生物学研究的进程。鉴于对比基因组学在获取高质量的基因组注释以及发现新的功能基因等方面的优势,我们分析了四种具不同生存策略的真菌基因组,试图进一步挖掘AM共生体系建立及抗性相关基因,望为今后研究提供新思路。分析结果表明,AM共生体的建立需要磷脂信号途径的介导以及一系列蛋白加工、分配过程。G蛋白介导的信号通路可能在AM真菌应对干旱胁迫的过程中起关键作用。
2AM真菌甘油跨膜通道GintAQPF2ar/R结构域特征促进AM共生体建立
      碳运输对于AM共生体的建立及其功能发挥至关重要。然而,到目前为止,还未见甘油运输是否影响AM共生体建立的报道。甘油经由甘油跨膜输送蛋白通道实现跨膜运输,而我们之前的研究表明AM真菌中的甘油跨膜通道GintAQPF2并不能运输甘油。本研究中,我们以甘油跨膜通道中的决定运输物质种类的蛋白结构域ar/R为研究对象,重点分析了其中两个氨基酸的功能。研究结果表明,当苯丙氨酸-94突变成丙氨酸-94时ar/R结构域的孔径变大,并使得酵母细胞中的甘油含量显著增加,同时也显著增加了酵母细胞在高浓度甘油中的成活率。苯丙氨酸-94突变成色氨酸-94和/或丙氨酸-234突变成甘氨酸-234都使GintAQPF2处于关闭状态,这说明ar/R结构域中的苯丙氨酸对(94和243)和精氨酸-249可能决定着GintAQPF2运输物质的种类。为了确定GintAQPF2的功能是否与AM共生体的建立相关,结合四种以不同方式从植物获取营养物质的真菌的基因组对比分析,我们发现甘油跨膜通道存在于两种兼性真菌中(L. bicolorM. oryzae),而在两种专性真菌中(R. irregularisF. verticillioides)都未发现。这说明甘油运输特征在共生真菌和病原真菌中是相对保守的。总而言之,我们的数据表明AM真菌唯一一个甘油跨膜通道GintAQPF2中ar/R结构域的特殊结构特征使其不能运输甘油,这可能有利于AM共生体建立。
3)肌醇-3-磷酸合酶和14-3-3蛋白在AM共生体协同抗旱过程中的潜在作用
      研究表明AM共生体能够增强植物抗旱性。然而,对于菌根共生体的协同抗旱机制还不是很清楚。本试验采用分根培养系统,通过在每个分室中设置接种与水分两因素处理,结合地上部、根生理特征检测以及AM真菌和根部抗旱基因表达分析,明确不同水分条件下AM真菌和宿主植物间的交互作用。结果表明,当在一个分室或两个分室中进行接种处理时,处于部分干旱或者全部干旱的整个根系统中的脱落酸含量都显著下降,但是表征根生理特征的不同指标以及AM真菌和根部不同抗旱基因表达却受到不同的调控。然而,通过指标间的相关性分析,我们发现当参与脱落酸信号转导的肌醇-3-磷酸合酶和14-3-3基因的表达协同增加时,AM真菌中的14-3-3蛋白和水孔蛋白功能被启动,这可能是AM共生体系统抗旱性增加(表现为地上部生理特征的改善)的原因。这些发现暗示了肌醇-3-磷酸合酶和14-3-3蛋白编码基因的共表达在启动宿主植物和AM真菌间的信号交流以及AM共生体协同抗旱方面发挥着重要作用。
      作为结论,AM共生体的建立需要磷脂信号途径的介导以及一系列蛋白加工、分配过程。共生体建成后,通过G蛋白介导的信号途径,AM真菌中关键抗性蛋白(如GintAQPF2)的活性可能被诱导,这有利于AM共生体系抗旱性的提高。GintAQPF2的功能及其ar/R结构域的特殊结构特征可支持AM共生体系建成。此外,我们证实IPS14-3GF在整个根系统中的共表达能够启动AM真菌对植物抗旱性的系统调控以及宿主植物和AM真菌间的信号交流,这对于实现AM共生体的协同抗旱至关重要。
英文摘要:       Arbuscular mycorrhizal (AM) symbiosis, a ubiquitous symbiotic association established between AM fungi and roots of higher plants in most terrestrial ecosystems, is essentially important for plant adaptation to various environmental stresses, such as nutrient deficiency, environmental pollution and drought, etc. However, very little is known about the basic biology of the fungal partner during AM symbiotic establishment and the cross-talk between the symbiotic partners under drought stress. In this study, comparative genomics was first adopted to excavate AM fungal genes involved in symbiotic establishment and drought tolerance. In addition, combined with the functional verification of the aquaglyceroporin GintAQPF2 from AM fungi, we sought to determine the relationship between the structural and functional features of GintAQPF2 and AM symbiosis development. Furthermore, split-root cultivation experiment was carried out to understand the mechanisms involved in the crosstalk between host plants and AM fungi. The major conclusions are as follows:
 
(1) Screening of AM fungal genes involved in symbiotic establishment and drought tolerance
 
      The recently published genome sequence of an AM fungus Rhizophagus irregularis has accelerated the progress of AM fungal cellular programs. In view of the potential roles of comparative genomics in obtaining high quality genome annotation and driving new discoveries, comparative genomic analyses of four representative fungi with different strategies for accessing host nutrients were performed. The results showed that phospholipid-mediated signaling pathways and a series of protein processing and sorting in AM fungi favored AM symbiotic establishment. G protein-mediated signaling pathways potentially played fundamental roles in AM fungal drought tolerance. The study is expected to lay foundations for future research into AM fungal basic biology.
 
(2) Structural features of the aromatic/arginine constriction in the aquaglyceroporin GintAQPF2 from AM fungi are favorable for establishing AM symbiosis
 
      Carbon transport in AM symbiosis is of fundamental importance. However, relationship between glycerol transport and AM symbiotic establishment has not yet been resolved. Glycerol transport is mediated by aquaglyceroporins (AQGPs) across cell membranes, while our previous study inferred that the AQGP GintAQPF2 from AM fungi disfavored glycerol transport. Here, we analyzed functions of two residues in the aromatic/arginine (ar/R) constriction that is a major selectivity filter in AQGPs. Replacement of phenylalanine-94 (Phe-94) by alanine (Ala) enlarged the diameter of the ar/R constriction and ensured yeast cells to significantly increase intracellular glycerol accumulation and survival rate at high glycerol levels, while individual or joint replacement of Phe-94 and Ala-234 by tryptophan and glycine made GintAQPF2 in a closed state, suggesting that the potential new double gates (Phe94-Phe243 and arginine-249) of the ar/R constriction also likely determined solute permeability. To figure out whether GintAQPF2 functions were potentially favorable for establishing AM symbiosis, genomic analyses of four representative fungi with different strategies for accessing host nutrients were performed. We found that glycerol facilitators existed in the facultative fungi (the ectomycorrhizal fungus Laccaria bicolor and hemibiotrophic pathogen Magnaporthe oryzae), but not in the obligatory fungi (the AM fungus Rhizophagus irregularis and necrotrophic pathogen Fusarium verticillioides), revealing a conserved pattern of glycerol transport between symbionts and pathogens. Collectively, our data imply that glycerol blocks due to the special structural features of the ar/R constriction in the only one AM fungal AQGP may favor AM symbiotic establishment.
 
(3) Potential role of D-myo-inositol-3-phosphate synthase and 14-3-3 genes in crosstalk between host plants and AM fungi and systemic drought tolerance of AM associations
 
      AM symbiosis is known to stimulate plant drought tolerance. However, the mechanisms underlying the synergetic resistance of the symbionts are largely unknown. A split-root experiment was performed to obtain maize plants with whole or only half root system colonized in the presence of localized or non-localized drought. Combining analyses of shoot and root properties, and expression of drought-tolerance related genes in both roots and AM fungi (AMF), we could investigate the interactions between the symbiotic partners. Although mycorrhizal inoculation in either one or both compartments systemically decreased abscisic acid (ABA) content in the whole root system subjected to total or partial drought, both root physiological state and expression of the genes in both roots and AMF were differently regulated. However, the simultaneous increase in the expression of plant genes encoding D-myo-inositol-3-phosphate synthase (IPS) and 14-3-3-like protein GF14 (14-3GF), which were involved in ABA signal transduction, was involved in activation of AMF 14-3-3 protein and aquaporins, and potentially led to systemic actions through improved physiological state in shoots. These findings suggest that co-expression of IPS and 14-3GF could play fundamental roles in initiating the crosstalk between host plants and AMF and thus inducing synergetic drought-tolerance of the AM association.
 
      In conclusion, phospholipid-mediated signaling pathways and a series of protein processing and sorting in AM fungi favored AM symbiotic establishment. G protein-mediated signaling pathways potentially induced downstream stress-responsive genes such as GintAQPF2, which played crucial roles in enhancing drought tolerance of AM symbiosis. In addition, we figured out that GintAQPF2 functions and the structural features of the ar/R constriction in GintAQPF2 were potentially favorable for establishing AM symbiosis. Furthermore, we proposed that coexpression of IPS and 14-3GF in the whole root system potentially activated the systemic regulation of plant drought tolerance by AM fungi and the crosstalk between host plants and AM fungi, which was crucial to the synergetic stress resistance of AM symbiosis.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36887
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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Recommended Citation:
李涛. 丛枝菌根增强植物抗旱性分子机理研究(博士后)[D]. 北京. 中国科学院研究生院. 2016.
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