KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 两种极端耐干苔藓抗逆性及其居群遗传分化研究 | |
| 卓露 | |
| Subtype | 博士 |
| Thesis Advisor | 管开云 ; 张道远 |
| 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 | 极端耐干苔藓 适应机制 极端温度 遗传多样性 解剖结构 Desert moss Adaptation mechanism Extreme temperature Genetic diversity Anatomy |
| Abstract | 极端耐干藓类 (Dessication tolerance moss， DT) 具有“干而不死” (配子体可耐受 98 %的组织脱水 (相当于-540 MPa)、“死而复生” (遇水 30 s 内恢复正常生理活动) 的耐干特性，近年来，其特殊的耐干特性越来越受到关注，已成为抗逆生物学研究的热点之一。 齿肋赤藓 (Syntrichia caninervis Mitt.) 和银叶真藓(Bryum argentums) 是沙漠生物结皮的建群种，也是极端耐干苔藓代表种，前人已开展了大量的研究，奠定了坚实的理论基础与系统的研究方法。然而一直以来，有关这 2 种单克隆材料大量扩繁体系尚需进一步完善，其耐干 (干燥-复水循环）过程及耐受极端高温的形态及生理适应性仍需精细刻画，银叶真藓适应不同生境(沙漠、高山、戈壁、 绿洲) 的遗传基础尚未明晰。因此， 本文通过调查 2 种极端耐干苔藓齿肋赤藓 (S. caninervis) 和银叶真藓(B. argenteum) 组织培养的影响因素，探讨无菌培养条件下 2 种苔藓快速扩繁体系和不同年龄阶段齿肋赤藓繁殖体的繁殖潜力，最终确定最优快繁方式。 基于水和温度两个重要生态因素开展调查研究，一方面通过两种荒漠苔藓对失水-复水三次循环的不同响应，调查不同生理指标和配子体再生情况指标，探索两种荒漠苔藓在极端环境中的耐干机制，丰富耐干生物学认识；另一方面通过两种荒漠苔藓对极端高温的响应性变化，探查高温胁迫下耐干植物组织化学染色变化 (DAB(二氨基联苯胺法) 和 NBT 染色 (四唑硝基蓝) )、表观形态及内部细胞器超微结构变化 (扫描电镜 SEM 和透射电镜 TEM)、重要基因高温响应表达变化(RT-qPCR 方法) 以及不同阶段配子体再生能力变化等，进而综合阐述 2 种苔藓植物耐高温响应机制。 此外， 利用石蜡切片解剖学和分子遗传标记研究，探查不同生境下银叶真藓的的解剖结构变化及遗传分化，进而解析其环境适应的形态结构及遗传基础。 总结出以下三点重要结论：1. 极端耐干苔藓齿肋赤藓和银叶真藓具有强大的克隆扩繁能力极端耐干苔藓具有强大的克隆繁殖能力，破碎化的繁殖体能够直接产生原丝体和植物新个体。 不同组织碎片的再生能力不同，茎、茎尖和绿色叶片的碎片组织更迅速繁殖再生，原丝体扩展更长的距离，并产生了更多的新个体。不同组织碎片与原始出芽时间 (P = 0.0033)，原始扩展距离 (P = 0.0002) 和新个体数量 (P <0.0001) 的相互作用呈显著性相关。对银叶真藓组织培养研究， 进一优化了步对消毒方式、培养基筛选、激素及糖源的添加指标等方案。在培养基筛选过程中发现， 1/2MS 培养基适合短期获得鲜绿的个体材料； MS 培养基适合原丝体的获得； Knop 培养基适合银叶真藓长期稳定的组培。2.两种耐干苔藓植物具有极端耐高温，干和盐的能力齿肋赤藓和银叶真藓经过多次脱水-再水化处理，仍可 100 %恢复到完全脱水及完全复水状态，具有超强的耐干性，而小立碗藓作为本实验研究的参照材料，仅能回复到初始状态的 65 %左右， 是一种耐干敏感物种， 此外，小立碗藓只能适应中低度的胁迫，当胁迫浓度过高 (150 mmol/L NaCl， 15 % PEG) 则会抑制本身的生长； 齿肋赤藓对干旱胁迫处理在生理上有一定响应； 5 % PEG 对真藓和小立碗藓的生长有一定促进作用。该结果对进一步利用这些材料进行抗逆分子生物学研究提供数据支持。在极端高温条件处理下， 2 种极端耐干苔藓创造了一个耐热新记录，齿肋赤藓和银叶真藓分别能够在 120° C 20 min 和 120° C 30 min 的极端条件下保持再生能力。本实验从表观结构、生理、细胞、分子等多方面对耐热性进行了研究。结果表明， 120° C 的高温胁迫，苔藓叶片细胞膜和细胞器出现一定程度损害，但能够保持配子体再生能力。随着胁迫温度的升高，细胞的形态完整性遭到破坏，叶绿体解体，但膜结构仍然保持完整。 细胞的颜色随胁迫温度的升高和处理时间的延长而加深，表明细胞受损伤程度增加。 分子水平上， 热相关基因 (HSP70 和HSF) 、 光合相关基因 (ELIP, LHCA 和 LHCB) 及干旱相关功能基因 (ERF,Tr288 和 DHN) 等对极端温度都具有显著的响应。3. 银叶真藓遗传多样性丰富，且生态适应性强银叶真藓遗传多样性非常丰富，居群间具有较大的遗传分化。对银叶真藓的AMOVA 分子方差分析结果显示，居群内部的变异占 23 %，居群间的变异占77 %，表明其叶绿体 DNA 变异主要存在于居群间。此外，基因单倍型分析结果表明，不同的生态环境塑造了特定的基因单倍型，以适应特定环境。通过干旱地区不同区域、不同生境下 18 个居群共计 540 个个体银叶真藓形态及叶片解剖结构进行对比研究，以探查苔藓植物形态结构变异及其对环境的适应机制。测定植株个体高度、芒尖长度、叶片的长和宽、植株横切面直径、叶细胞长、宽，并计算叶片和叶细胞的面积及各指标的变异系数 (CV) 和可塑性指数(PI) 等指标。结果表明：总体上，各个指标之间都存在差异性变化，随着海拔高度的增加，个体株高降低，叶片芒尖增长，叶面积减小。从细胞解剖结构上看茎叶的横切面大体相似，但高海拔地区 (P9-P18 居群) 茎直径和叶片细胞面积的变异系数和可塑性指数较大。说明银叶真藓的个体和叶片解剖结构具有可塑性，且该特性是银叶真藓适应各种环境的重要方式和策略。 |
| Other Abstract | Extreme tolerance moss has drought tolerant characteristic of "dry and not dead"(gametophyte can tolerate 98 % tissue dehydration (equivalent to -540 MPa),"resurrection" (return to normal physiological activity within 30s of water). Thedry-resistance characteristic has been attracted more and more attention by scientistsin recent years, and has become one of the hotspots in anti-biological research.Syntrichia caninervis and Bryum argentum are the constructive species of desertbiological crusts and the representative species to extreme drought resistance. Alot of researches on this field have been carried out and solid theory And basicsystematic research methods have been established. However, there has been a needfor further improvement in the proliferation of these two kinds of monoclonalmaterials. The resistance to dry (dry-rehydration cycle) processes and the extremehigh temperaturetolerance morphology and physiological adaptability still needs to bemeticulously portrayed. The genetic basis for adapting to different habitats (desert,high mountains, Gobi, Oasis) is still unclear.The influencing factors of tissue culture of these two species of S. caninervisand B. argenteum have been investigated. And the propagation potential of B.argenteum and different age stages of S. caninervis breeding under aseptic cultureconditions has been studied. An optimal method of the rapid propagationhas beenestablished. Two moss species are typical stress tolerance species. The gametophytescan tolerate 98% tissue dehydration (equivalent to -540 MPa). Two importantecological factors of water and temperature,different responses to three cycles ofwater-rehydration, different physiological indices and traditional indicators ofregeneration have been studied. The desiccation tolerance responses of two mossspecies in extreme environments, importance for conservation and restoration ofbiological diversity of wild mossy biological crusts were explored. Tissue staining(DAB and NBT), scanning electron microscopy (SEM), transmission electronmicroscopy (TEM) and molecular fluorescence quantification (RT-qPCR) wereperformed through changes in the response of two desert mosses to the extreme hightemperatures. The use of conventional traditional regeneration index assessmenttechniques has opened up new avenues to mosses. At the same time, the differencesbetween B. argenteum in different distribution areas, a theoretical basis for further investigation of the relationship between the anatomical structure of moss and theexternal environment were explored by using paraffin section anatomy andmolecular genetic markers studies. The following three important conclusions aresummarized:1. The extreme dessication tolerance moss of S.carninervis and B. argentumhave very high clonal propagation ability.As the predominant species of biological soil crusts (BSC) in temperate colddeserts, five detached fragments (the stem apex, green leaves, yellow–green leaves,brown leaves and the stem) of S. carnivervis were collected in 2014 and 2015 andcultured on the surface of moistened sand in the lab. Regeneration from the stem apex,the green leaves and the stem could be expected by the 10th day in culture. The stem,the stem apex and the green leaves tended to regenerate more quickly, extend theprotonema farther and produce more shoots than the yellow–green leaves and thebrown leaves. The effect of fragment type to protonema emergence (P = 0.0033),protonema extension (P = 0.0002) and number of shoots (P < 0.0001) was significant.The research on tissue culture of B. argenteum is mainly based on the existedtissue culture technology: relative humidity, light intensity, photoperiod, andtemperature, and on the basis of disinfection methods, medium selection, hormones,and external sugar sources. It was discovered during the screening of different mediathat 1/2MS culture medium was suitable for the individual material of bright green ina short time, MS medium was suitable for obtaining the protofilament; Knop culturemedium was suitable for the long-term stable tissue culture of B. argenteum.2. The moss of S. caninervis and B. argenteum have extreme resistance toheat, dry and salt.In order to further understand the adaptability of the 2 desert mosses in extremestress environments, S. caninervis, B. argenteum and P. patent were used as a materialfor stress resistance studies. Combined with three kinds of mossdehydration-rehydration experiments, the water content was measured and the dry saltresistance of the three species of moss was evaluated comprehensively. The resultsshowed that after the dehydration-rehydration treatment, B. argenteum and S.caninervis could still be completely rehydrated and completely rehydrated with 100%,and it has superior resistance to dryness. The reference material (P. patent) for thisexperiment was only able to revert to about 65 % of the initial state and was adry-sensitive species. In addition, P. patens can only adapt to medium and low stress.When the stress concentration is too high (150 mmol/L NaCl, 15 % PEG), it willinhibit their own growth and 5 % PEG has a certain role in promoting the growth of B.argenteum and P. patens. This result provides data supporting further use of thesematerials for the study of stress-resistant molecular biology.Under extreme high temperature conditions, two kinds of desert mosses havecreated a new record. S. caninervis and B. argenteum can survive under extremeconditions of 120 °C for 20 minutes and 120 °C for 30 minutes, respectively. Thisexperiment conducted a comprehensive study of morphology, structure, physiology,cells, and molecules. At 120 °C high temperature stress, the cell membrane andorganelles of mosses were damaged to a certain degree, but they could maintain theirregeneration potential. With the increase of stress temperature, the morphologicalintegrity of cells was destroyed and gradually disintegrated. DAB and NBT stainingturned more dark with increasing temperature and processing time. At the molecularlevel, heat response gene (HSP70 and HSF)、 photosynthetic relative genes(ELIP,LHCA and LHCB) and others (ERF, Tr288 和 DHN) respond significantly to extremetemperatures.3. The desert moss of B. argenteum has genetic diversity and ecologicaladaptabilityOne of the materials in this study is B. argenteum, which is widely distributedthroughout the world and grow in glacier, Gobi, desert, oasis and other ecologicalenvironments in Xinjiang. In this study, three cpDNA gene gaps, atpI-atpH,psbD-trnT and accD-psaI were used to analyze the differences between 21populations and 178 individuals of B. argenteum. Some pedigree geography methodswere used to study the genetic diversity of B. argenteum in order to explore itsdistribution pattern of spread and distribution in arid regions. The ANOVA analysisof B. argenteum revealed that the intra-population variation accounted for 23% andthe inter-population variation accounted for 77%, indicating that the chloroplast DNAvariation was mainly found among populations.The gametophyte morphological and leaf anatomical characteristic of 540individuals from 18 populations in arid region were investigated. Statistical analysisof the indicators including height,awn length,long and wide of leaves, leaf area, stemdiameter, long and wide of cell, cell area, coefficient of variation and plasticity indexwere conducted, the index among different populations were compared and the adaptive mechanism were explored. The cross-sections were similar in terms of cellanatomy, but statistics showed that the coefficient of variation and the plasticity indexof stem diameter and leaf cell area were higher in high-altitude areas (P9-P18population). Therefore, illustrating the plasticity of individual and leaf anatomicalstructures is an important way and strategy to adapt to various ecosystems. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14950 |
| Collection | 研究系统_荒漠环境研究室 |
| 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 | 卓露. 两种极端耐干苔藓抗逆性及其居群遗传分化研究[D]. 新疆乌鲁木齐. 中国科学院大学,2018. |
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